U.S. patent application number 11/552710 was filed with the patent office on 2007-03-01 for cytokine receptor zcytor17 multimers.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Maria M. Dasovich, Stacey R. Dillon, Zeren Gao, Francis J. Grant, Jane A. Gross, Angela K. Hammond, Joseph L. Kuijper, Julia E. Novak, Scott R. Presnell, Cindy A. Sprecher, Theodore E. Whitmore.
Application Number | 20070048834 11/552710 |
Document ID | / |
Family ID | 30003762 |
Filed Date | 2007-03-01 |
United States Patent
Application |
20070048834 |
Kind Code |
A1 |
Sprecher; Cindy A. ; et
al. |
March 1, 2007 |
CYTOKINE RECEPTOR ZCYTOR17 MULTIMERS
Abstract
Novel polypeptide combinations, polynucleotides encoding the
polypeptides, and related compositions and methods are disclosed
for zcytor17-containing multimeric or heterodimer cytokine
receptors that may be used as novel cytokine antagonists, and
within methods for detecting ligands that stimulate the
proliferation and/or development of hematopoietic, lymphoid and
myeloid cells in vitro and in vivo. The present invention also
includes methods for producing the multimeric or heterodimeric
cytokine receptor, uses therefor and antibodies thereto.
Inventors: |
Sprecher; Cindy A.; (Sierra
Vista, AZ) ; Gao; Zeren; (Redmond, WA) ;
Kuijper; Joseph L.; (Kenmore, WA) ; Dasovich; Maria
M.; (Seattle, WA) ; Grant; Francis J.;
(Seattle, WA) ; Presnell; Scott R.; (Tacoma,
WA) ; Whitmore; Theodore E.; (Redmond, WA) ;
Hammond; Angela K.; (Maple Valley, WA) ; Novak; Julia
E.; (Bainbridge Island, WA) ; Gross; Jane A.;
(Seattle, WA) ; Dillon; Stacey R.; (Seattle,
WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
30003762 |
Appl. No.: |
11/552710 |
Filed: |
October 25, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10351157 |
Jan 21, 2003 |
|
|
|
11552710 |
Oct 25, 2006 |
|
|
|
60435361 |
Dec 19, 2002 |
|
|
|
60389108 |
Jun 14, 2002 |
|
|
|
60350325 |
Jan 18, 2002 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 1/18 20180101; A61P
13/08 20180101; A61P 17/06 20180101; A61P 35/00 20180101; C07K
2317/24 20130101; A61P 31/00 20180101; C07K 14/52 20130101; A61P
1/16 20180101; A61P 15/00 20180101; A61P 19/02 20180101; A61P 17/04
20180101; A61P 1/04 20180101; A61P 29/00 20180101; A61P 37/00
20180101; A61P 11/06 20180101; C07K 16/2866 20130101; A61P 37/08
20180101; C07K 14/715 20130101; A61K 38/00 20130101; A61P 1/02
20180101; A61P 31/04 20180101; C07K 2317/76 20130101; A61P 37/06
20180101; A01K 2217/05 20130101; A61P 17/00 20180101; A61P 11/00
20180101; A61P 39/02 20180101; A61P 43/00 20180101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07H 21/04 20060101 C07H021/04; C07K 14/715 20070101
C07K014/715 |
Claims
1. An isolated polynucleotide that encodes a cytokine receptor
polypeptide comprising an amino acid sequence having at least 95%
sequence identity with amino acid residues 33-662 of SEQ ID NO:5,
wherein the cytokine receptor polypeptide forms a multimeric or
heterodimeric cytokine receptor with a second cytokine receptor
polypeptide comprising amino acid residues 28-979 of SEQ ID NO:7,
and wherein the multimeric or heterodimeric cytokine receptor binds
a ligand comprising amino acid residues 27-164 of SEQ ID NO:2.
2. The isolated polynucleotide of claim 1 wherein the cytokine
receptor polypeptide comprises amino acid residues 33-662 of SEQ ID
NO:5.
3. The isolated polynucleotide of claim 1 wherein the isolated
polynucleotide comprises nucleotides 593-2482 of SEQ ID NO:4.
4. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment encoding a
cytokine receptor polypeptide comprising an amino acid sequence
having at least 95% sequence identity with amino acid residues
33-662 of SEQ ID NO:5; and a transcription terminator; wherein the
cytokine receptor polypeptide forms a multimeric or heterodimeric
cytokine receptor with a second cytokine receptor polypeptide
comprising amino acid residues 28-979 of SEQ ID NO:7, and wherein
the multimeric or heterodimeric cytokine receptor binds a ligand
comprising amino acid residues 27-164 of SEQ ID NO:2.
5. The expression vector of claim 4 wherein the cytokine receptor
polypeptide comprises amino acid residues 33-662 of SEQ ID
NO:5.
6. The expression vector of claim 4 wherein the DNA segment
comprises nucleotides 593-2482 of SEQ ID NO:4.
7. An expression vector comprising the following operably linked
elements: a) a first transcription promoter; a first DNA segment
encoding a first cytokine receptor polypeptide comprising an amino
acid sequence having at least 95% sequence identity with amino acid
residues 33-662 of SEQ ID NO:5; and a first transcription
terminator; and b) a second transcription promoter; a second DNA
segment encoding a second cytokine receptor polypeptide comprising
amino acid residues 28-979 of SEQ ID NO:7; and a second
transcription terminator; wherein the first cytokine receptor
polypeptide and the second cytokine receptor polypeptide form a
multimeric or heterodimeric cytokine receptor; and wherein the
multimeric or heterodimeric cytokine receptor binds to a ligand
comprising amino acid residues 27-164 of SEQ ID NO:2.
8. The expression vector of claim 7 wherein the cytokine receptor
polypeptide comprises amino acid residues 33-662 of SEQ ID
NO:5.
9. The expression vector of claim 7 wherein the first DNA segment
comprises nucleotides 593-2482 of SEQ ID NO:4.
10. A cultured cell comprising the expression vector of claim 4,
wherein the cell expresses the cytokine receptor polypeptide
encoded by the DNA segment.
11. A cultured cell comprising the expression vector of claim 7,
wherein the cell expresses the first cytokine receptor polypeptide
and the second cytokine receptor polypeptide encoded by the DNA
segments.
12. A cultured cell comprising: a first expression vector
comprising: a) a first transcription promoter; b) a first DNA
segment encoding a first cytokine receptor polypeptide comprising
an amino acid sequence having at least 95% sequence identity with
amino acid residues 33-662 of SEQ ID NO:5; and c) a first
transcription terminator; and a second expression vector
comprising: a) a second transcription promoter; b) a second DNA
segment encoding a second cytokine receptor polypeptide comprising
amino acid residues 28-979 of SEQ ID NO:7; and c) a second
transcription terminator; wherein the first cytokine receptor
polypeptide and the second cytokine receptor polypeptide form a
multimeric or heterodimeric cytokine receptor; and wherein the
multimeric or heterodimeric cytokine receptor binds to a ligand
comprising amino acid residues 27-164 of SEQ ID NO:2.
13. A method of producing an antibody to a soluble multimeric or
heterodimeric cytokine receptor comprising a first cytokine
receptor polypeptide consisting of an amino acid sequence having at
least 95% sequence identity with amino acid residue 33 to amino
acid residue 532 of SEQ ID NO:5 and a second cytokine receptor
polypeptide consisting of amino acid residues 28-739 of SEQ ID
NO:7, the method comprising: inoculating an animal with the soluble
multimeric or heterodimeric cytokine receptor, wherein the soluble
multimeric or heterodimeric cytokine receptor elicits an immune
response in the animal to produce an antibody that specifically
binds the soluble multimeric or heterodimeric cytokine receptor;
and isolating the antibody from the animal.
14. A method of producing a multimeric or heterodimeric cytokine
receptor comprising: culturing a cell according to claim 11; and
isolating the multimeric or heterodimeric cytokine receptor
produced by the cell.
15. A method of producing a multimeric or heterodimeric cytokine
receptor comprising: culturing a cell according to claim 12; and
isolating the multimeric or heterodimeric cytokine receptor
produced by the cell.
16. An isolated polynucleotide that encodes a soluble cytokine
receptor polypeptide comprising an amino acid sequence having at
least 95% sequence identity with amino acid residues 33-532 of SEQ
ID NO:5 wherein the soluble cytokine receptor polypeptide forms a
soluble multimeric or heterodimeric cytokine receptor with a second
soluble cytokine receptor comprising amino acid residues 28-739 of
SEQ ID NO:7, and wherein the soluble multimeric or heterodimeric
cytokine receptor binds a ligand comprising amino acid residues
27-164 of SEQ ID NO:2.
17. The isolated polynucleotide of claim 16 wherein the soluble
multimeric or heterodimeric cytokine receptor antagonizes an
activity of the ligand comprising amino acid residues 27-164 of SEQ
ID NO:2.
18. The isolated polynucleotide of claim 17 wherein the soluble
multimeric or heterodimeric cytokine receptor inhibits
proliferation of immune cells, inhibits proliferation of
inflammatory cells, or inhibits an inflammatory response.
19. The isolated polynucleotide of claim 16 wherein the soluble
heterodimeric or multimeric receptor further comprises an
immunoglobulin heavy chain constant region.
20. The isolated polynucleotide of claim 19 wherein the
immunoglobulin heavy chain constant region is an F.sub.c
fragment.
21. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment encoding a
soluble cytokine receptor polypeptide comprising an amino acid
sequence having at least 95% sequence identity with amino acid
residues 33-532 of SEQ ID NO:5; and a transcription terminator;
wherein the cytokine receptor polypeptide forms a soluble
multimeric or heterodimeric cytokine receptor with a second soluble
cytokine receptor polypeptide comprising amino acid residues 28-739
of SEQ ID NO:7, and wherein the soluble multimeric or heterodimeric
cytokine receptor binds a ligand comprising amino acid residues
27-164 of SEQ ID NO:2.
22. A cultured cell comprising the expression vector of claim 21
and a second expression vector comprising: a second transcription
promoter; a second DNA segment encoding a second soluble cytokine
receptor polypeptide comprising amino acid residues 28-739 of SEQ
ID NO:7; and a second transcription terminator; wherein the cell
expresses the soluble cytokine receptor polypeptide encoded by the
DNA segment and the second soluble cytokine receptor polypeptide
encoded by the second DNA segment to form a soluble multimeric or
heterodimeric cytokine receptor.
23. A cultured cell comprising the expression vector of claim 21,
wherein the cell expresses the soluble cytokine receptor
polypeptide encoded by the DNA segment.
24. A method of producing a soluble multimeric or heterodimeric
cytokine receptor comprising: culturing a cell according to claim
23; and isolating the soluble multimeric or heterodimeric cytokine
receptor produced by the cell.
25. A composition comprising: an effective amount of a soluble
multimeric or heterodimeric cytokine receptor comprising a first
soluble cytokine receptor polypeptide comprising an amino acid
sequence having at least 95% sequence identity with amino acid
residues 33-532 of SEQ ID NO:5 and a second soluble cytokine
receptor comprising amino acid residues 28-739 of SEQ ID NO:7; and
a pharmaceutically acceptable vehicle.
26. The composition of claim 25 wherein the soluble multimeric or
heterodimeric cytokine receptor further comprises an immunoglobulin
heavy chain constant region.
27. The composition of claim 26 wherein the immunoglobulin heavy
chain constant region is an F.sub.c fragment.
Description
[0001] The present application is a divisional of U.S. patent
application Ser. No. 10/351,157, filed Jan. 21, 2003, which claims
the benefit of U.S. Patent Application Ser. No. 60/435,361, filed
Dec. 19, 2002, U.S. Patent Application Ser. No. 60/389,108, filed
Jun. 14, 2002, and U.S. Patent Application Ser. No. 60/350,325,
filed Jan. 18, 2002, all of which are herein incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] Proliferation and differentiation of cells of multicellular
organisms are controlled by hormones and polypeptide growth
factors. These diffusable molecules allow cells to communicate with
each other and act in concert to form cells, tissues and organs,
and to repair damaged tissue. Examples of hormones and growth
factors include the steroid hormones (e.g., estrogen,
testosterone), parathyroid hormone, follicle stimulating hormone,
the interleukins, platelet derived growth factor (PDGF), epidermal
growth factor (EGF), granulocyte-macrophage colony stimulating
factor (GM-CSF), erythropoietin (EPO) and calcitonin.
[0003] Hormones and growth factors influence cellular metabolism by
binding to receptors. Receptors may be integral membrane proteins
that are linked to signaling pathways within the cell, such as
second messenger systems. Other classes of receptors are soluble
molecules, such as the transcription factors.
[0004] Cytokines generally stimulate proliferation or
differentiation of cells of the hematopoietic lineage or
participate in the immune and inflammatory response mechanisms of
the body. Examples of cytokines which affect hematopoiesis are
erythropoietin (EPO), which stimulates the development of red blood
cells; thrombopoietin (TPO), which stimulates development of cells
of the megakaryocyte lineage; and granulocyte-colony stimulating
factor (G-CSF), which stimulates development of neutrophils. These
cytokines are useful in restoring normal blood cell levels in
patients suffering from anemia, thrombocytopenia, and neutropenia
or receiving chemotherapy for cancer.
[0005] The interleukins are a family of cytokines that mediate
immunological responses, including inflammation. The interleukins
mediate a variety of inflammatory pathologies. Central to an immune
response are T cells, which produce many cytokines and adaptive
immunity to antigens. Cytokines produced by T cells have been
classified as type 1 and type 2 (Kelso, A. Immun. Cell Biol.
76:300-317, 1998). Type 1 cytokines include IL-2, IFN-.gamma.,
LT-.alpha., and are involved in inflammatory responses, viral
immunity, intracellular parasite immunity and allograft rejection.
Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and are
involved in humoral responses, helminth immunity and allergic
response. Shared cytokines between Type 1 and 2 include IL-3,
GM-CSF and TNF-.alpha.. There is some evidence to suggest that Type
1 and Type 2 producing T cell populations preferentially migrate
into different types of inflamed tissue.
[0006] Mature T cells may be activated, i.e., by an antigen or
other stimulus, to produce, for example, cytokines, biochemical
signaling molecules, or receptors that further influence the fate
of the T cell population.
[0007] B cells can be activated via receptors on their cell surface
including B cell receptor and other accessory molecules to perform
accessory cell functions, such as production of cytokines.
[0008] Monocytes/macrophages and T-cells can be activated by
receptors on their cell surface and play a central role in the
immune response by presenting antigen to lymphocytes and also act
as accessory cells to lymphocytes by secreting numerous
cytokines.
[0009] Natural killer (K) cells have a common progenitor cell with
T cells and B cells, and play a role in immune surveillance. NK
cells, which comprise up to 15% of blood lymphocytes, do not
express antigen receptors, and therefore do not use MHC recognition
as requirement for binding to a target cell. NK cells are involved
in the recognition and killing of certain tumor cells and virally
infected cells. In vivo, NK cells are believed to require
activation, however, in vitro, NK cells have been shown to kill
some types of tumor cells without activation.
[0010] The demonstrated in vivo activities of these cytokines
illustrate the enormous clinical potential of, and need for, other
cytokines, cytokine agonists, and cytokine antagonists or binding
partners. The present invention addresses these needs by providing
a new hematopoietic multimeric cytokine receptor, as well as
related compositions and methods.
[0011] The present invention provides such polypeptides for these
and other uses that should be apparent to those skilled in the art
from the teachings herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is an illustration of a multiple alignment of human
zcytor17lig (SEQ ID NO:2) (zcytor17lig), and mouse zcytor17lig (SEQ
ID NO:11) (mzcytor17lig), mouse IL-3 (mIL-3) (SEQ ID NO:100), and
human IL-3 (hIL-3) (SEQ ID NO:102).
[0013] FIG. 2 is an illustration of a multiple alignment of human
zcytor17lig (SEQ ID NO:2) (zcytor17lig), and mouse zcytor17lig (SEQ
ID NO:11) (mzcytor17lig).
[0014] FIG. 3 is a Hopp/Woods hydrophilicity plot of human
zcytor17lig (SEQ ID NO:2).
[0015] FIG. 4 is a multiple alignment of zcytor17 polynucleotide
sequences SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:5, SEQ ID NO:111,
and SEQ ID NO:115.
[0016] FIG. 5 is an alignment of human zcytor17 (ZCYTOR) (SEQ ID
NO:5) and mouse zcytor17 (M17R-O) (SEQ ID NO:117). Between the two
sequences, identical residues (:), Conserved residues (.) and gaps
(-) are indicated.
SUMMARY OF THE INVENTION
[0017] The present invention provides an isolated multimeric or
heterodimeric cytokine receptor comprising at least one polypeptide
having at least 90 percent sequence identity with SEQ ID NO:111 or
SEQ ID NO:109; and wherein the multimeric or heterodimeric cytokine
receptor binds a ligand comprising SEQ ID NO:2. Optionally, the
isolated multimeric or heterodimeric cytokine receptor may further
comprise a cytokine-binding domain of a class I cytokine receptor.
The cytokine-binding domain of the class I cytokine receptor may
comprise amino acid residue 28 to amino acid residue 429 of SEQ ID
NO:7, amino acid residue 28 to amino acid residue 739 of SEQ ID
NO:7, amino acid residue 1 to amino acid residue 429 of SEQ ID
NO:7, amino acid residue 1 to amino acid residue 739 of SEQ ID
NO:7, amino acid residue 1 to amino acid residue 761 of SEQ ID
NO:7, amino acid residue 28 to amino acid residue 761 of SEQ ID
NO:7, amino acid residue 28 to amino acid residue 979 of SEQ ID
NO:7, or amino acid residue 1 to amino acid residue 979 of SEQ ID
NO:7. The isolated multimeric or heterodimeric cytokine receptor
may antagonize an activity of SEQ ID NO:2. The isolated multimeric
or heterodimeric cytokine receptor may inhibit proliferation of
hematopoietic cells, inhibit proliferation of immune cells, inhibit
proliferation of inflammatory cells, inhibit an immune response,
inhibit an inflammatory response, or inhibit proliferation of tumor
cells of epithelial origin. The isolated multimeric or
heterodimeric cytokine receptor may be soluble. The isolated
multimeric or heterodimeric cytokine receptor may further comprises
an affinity tag, such as, for instance, polyhistidine, protein A,
glutathione S transferase, Glu-Glu, substance P, Flag.TM. peptide,
streptavidin binding peptide, and immunoglobulin F.sub.c
polypeptide, or cytotoxic molecule, such as, for instance, a toxin
or radionuclide. The isolated multimeric or heterodimeric cytokine
receptor wherein the polypeptide having at least 90 percent
identity with SEQ ID NO:111 may comprise amino acid residue 20 to
amino acid residue 227 of SEQ ID NO:111, amino acid residue 20 to
amino acid residue 519 of SEQ ID NO:111, amino acid residue 20 to
amino acid residue 543 of SEQ ID NO:111, amino acid residue 20 to
amino acid residue 732 of SEQ ID NO:111, amino acid residue 1 to
amino acid residue 227, amino acid residue 1 to amino acid residue
519, amino acid residue 1 to amino acid residue 543, or amino acid
residue 1 to amino acid residue 732. The isolated multimeric or
heterodimeric cytokine receptor wherein the polypeptide having at
least 90 percent identity with SEQ ID NO:109 may comprise amino
acid residue 1 to amino acid residue 649 of SEQ ID NO:109, or amino
acid residue 20 to amino acid residue 649 of SEQ ID NO:109.
[0018] The present invention also provides an isolated multimeric
or heterodimeric cytokine receptor comprising at least one
polypeptide comprising amino acid residue 20 to amino acid residue
227 of SEQ ID NO:111. The at least one polypeptide may comprise
amino acid residue 1 to amino acid residue 227 of SEQ ID NO: III,
amino acid residue 20 to amino acid residue 519 of SEQ ID NO:11,
amino acid residue 1 to amino acid residue 519 of SEQ ID NO:111,
amino acid residue 1 to amino acid residue 543 of SEQ ID NO:111,
amino acid residue 20 to amino acid residue 543 of SEQ ID NO:111,
amino acid residue 1 to amino acid residue 732 of SEQ ID NO:111, or
amino acid residue 20 to amino acid residue 732 of SEQ ID NO:111.
The isolated multimeric or heterodimeric cytokine receptor may
further comprise a cytokine-binding domain of a class I cytokine
receptor, for instance, amino acid residue 28 to amino acid residue
429 of SEQ ID NO:7, amino acid residue 1 to amino acid residue 429
of SEQ ID NO:7, amino acid residue 28 to amino acid residue 739 of
SEQ ID NO:7, amino acid residue 1 to amino acid residue 739 of SEQ
ID NO:7, amino acid residue 28 to amino acid residue 761 of SEQ ID
NO:7, amino acid residue 1 to amino acid residue 761 of SEQ ID
NO:7, amino acid residue 28 to amino acid residue 979 of SEQ ID
NO:7, or amino acid residue 1 to amino acid residue 979 of SEQ ID
NO:7. The isolated multimeric or heterodimeric cytokine receptor
may antagonize an activity of a ligand comprising SEQ ID NO:2. The
isolated multimeric or heterodimeric cytokine receptor may inhibit
proliferation of hematopoietic cells, inhibit proliferation of
immune cells, inhibit proliferation of inflammatory cells, inhibit
an immune response, inhibit an inflammatory response, or inhibit
proliferation of tumor cells of epithelial origin. Optionally, the
isolated multimeric or heterodimeric cytokine receptor may be is
soluble. The isolated multimeric or heterodimeric cytokine receptor
may further comprise an affinity tag, such as, for instance,
polyhistidine, protein A, glutathione S transferase, Glu-Glu,
substance P, Flag.TM. peptide, streptavidin binding peptide, and
immunoglobulin F.sub.c polypeptide, or cytotoxic molecule, such as,
for instance, a toxin or radionuclide.
[0019] The present invention also provides a soluble multimeric or
heterodimeric cytokine receptor comprising amino acid residue 20 to
amino acid residue 227 of SEQ ID NO:111 and amino acid residue 28
to amino acid residue 429 of SEQ ID NO:7.
[0020] The present invention also provides an isolated
polynucleotide that encodes a cytokine receptor polypeptide
comprising an amino acid sequence having at least 90 percent
sequence identity with SEQ ID NO:111 or SEQ ID NO:109, wherein the
cytokine receptor polypeptide forms a multimeric or heterodimeric
cytokine receptor, and wherein the multimeric or heterodimeric
cytokine receptor binds a ligand comprising SEQ ID NO:2. The
multimeric or heterodimeric cytokine receptor may further comprise
a cytokine-binding domain of a class I cytokine receptor, such as,
for instance, amino acid residue 28 to amino acid residue 429 of
SEQ ID NO:7, amino acid residue 28 to amino acid residue 739 of SEQ
ID NO:7, amino acid residue 1 to amino acid residue 429 of SEQ ID
NO:7, amino acid residue 1 to amino acid residue 739 of SEQ ID
NO:7, amino acid residue 1 to amino acid residue 761 of SEQ ID
NO:7, amino acid residue 28 to amino acid residue 761 of SEQ ID
NO:7, amino acid residue 28 to amino acid residue 979 of SEQ ID
NO:7, or amino acid residue 1 to amino acid residue 979 of SEQ ID
NO:7. The multimeric or heterodimeric cytokine receptor may
antagonize an activity of SEQ ID NO:2. The multimeric or
heterodimeric cytokine receptor may inhibit proliferation of
hematopoietic cells, inhibit proliferation of immune cells, inhibit
proliferation of inflammatory cells, inhibit an immune response,
inhibit an inflammatory response, or inhibit proliferation of tumor
cells of epithelial origin. Optionally, the multimeric or
heterodimeric cytokine receptor may be soluble. The multimeric or
heterodimeric cytokine receptor may further comprise an affinity
tag, such as, for instance, polyhistidine, protein A, glutathione S
transferase, Glu-Glu, substance P, Flag.TM. peptide, streptavidin
binding peptide, and immunoglobulin F.sub.c polypeptide, or
cytotoxic molecule, such as, for instance, a toxin or radionuclide.
The encoded cytokine receptor polypeptide having at least 90
percent identity with SEQ ID NO:111 may comprise amino acid residue
20 to amino acid residue 227 of SEQ ID NO:111, amino acid residue
20 to amino acid residue 519 of SEQ ID NO:111, amino acid residue
20 to amino acid residue 543 of SEQ ID NO:111, amino acid residue
20 to amino acid residue 732 of SEQ ID NO:111, amino acid residue 1
to amino acid residue 227 of SEQ ID NO:1, amino acid residue 1 to
amino acid residue 519 of SEQ ID NO:111, amino acid residue 1 to
amino acid residue 543 of SEQ ID NO:111, or amino acid residue 1 to
amino acid residue 732 of SEQ ID NO:1. The encoded cytokine
receptor polypeptide having at least 90 percent identity with SEQ
ID NO:109 may comprise amino acid residue 1 to amino acid residue
649 of SEQ ID NO:109, or amino acid residue 20 to amino acid
residue 649 of SEQ ID NO:109.
[0021] The present invention also provides an isolated
polynucleotide that encodes a cytokine receptor polypeptide
comprising amino acid residue 20 to amino acid residue 227 of SEQ
ID NO:111, wherein the cytokine receptor polypeptide forms a
multimeric or heterodimeric cytokine receptor. The cytokine
receptor polypeptide may comprise amino acid residue 1 to amino
acid residue 227 of SEQ ID NO:111, amino acid residue 20 to amino
acid residue 519 of SEQ ID NO:111, amino acid residue 1 to amino
acid residue 519 of SEQ ID NO:111, amino acid residue 1 to amino
acid residue 543 of SEQ ID NO:111, amino acid residue 20 to amino
acid residue 543 of SEQ ID NO:111, amino acid residue 1 to amino
acid residue 732 of SEQ ID NO:1, or amino acid residue 20 to amino
acid residue 732 of SEQ ID NO:1. The multimeric or heterodimeric
cytokine receptor may further comprise a cytokine-binding domain of
a class I cytokine receptor, such as, for instance, amino acid
residue 28 to amino acid residue 429 of SEQ ID NO:7, amino acid
residue 1 to amino acid residue 429 of SEQ ID NO:7, amino acid
residue 28 to amino acid residue 739 of SEQ ID NO:7, amino acid
residue 1 to amino acid residue 739 of SEQ ID NO:7, amino acid
residue 28 to amino acid residue 761 of SEQ ID NO:7, amino acid
residue 1 to amino acid residue 761 of SEQ ID NO:7, amino acid
residue 28 to amino acid residue 979 of SEQ ID NO:7, or amino acid
residue 1 to amino acid residue 979 of SEQ ID NO:7. The multimeric
or heterodimeric cytokine receptor may antagonize an activity of a
ligand comprising SEQ ID NO:2. The multimeric or heterodimeric
cytokine receptor may inhibit proliferation of hematopoietic cells,
inhibit proliferation of immune cells, inhibit proliferation of
inflammatory cells, inhibit an immune response, inhibit an
inflammatory response, or inhibit proliferation of tumor cells of
epithelial origin. Optionally, the multimeric or heterodimeric
cytokine receptor may be soluble. The multimeric or heterodimeric
cytokine receptor may further comprise an affinity tag or cytotoxic
molecule as described herein.
[0022] The present invention also provides an expression vector
that comprises the following operably linked elements: a
transcription promoter; a DNA segment encoding a cytokine receptor
polypeptide having at least 90 percent sequence identity with SEQ
ID NO:111; and a transcription terminator; wherein the cytokine
receptor polypeptide forms a multimeric or heterodimeric cytokine
receptor, and wherein the multimeric or heterodimeric cytokine
receptor binds a ligand comprising SEQ ID NO:2.
[0023] Alternatively, the present invention also provides an
expression vector that comprises the following operably linked
elements: a) a first transcription promoter; a first DNA segment
encoding a cytokine receptor polypeptide having at least 90 percent
sequence identity with SEQ ID NO:111; and a first transcription
terminator; and b) a second transcription promoter; a second DNA
segment encoding a cytokine-binding domain of a class I cytokine
receptor; and a second transcription terminator; wherein the
cytokine receptor polypeptide and the class I cytokine receptor
form a multimeric or heterodimeric cytokine receptor; and wherein
the multimeric or heterodimeric cytokine receptor binds to a ligand
comprising SEQ ID NO:2.
[0024] Alternatively, the present invention also provides an
expression vector that comprises the following operably linked
elements: a) a first transcription promoter; a first DNA segment
encoding a polypeptide having at least 90 percent sequence identity
with SEQ ID NO:111; and a first transcription terminator; and b) a
second transcription promoter; a second DNA segment encoding at
least a portion of a class I cytokine receptor; and a second
transcription terminator; wherein the polypeptide and the class I
cytokine receptor form a multimeric cytokine receptor; and wherein
the multimeric cytokine receptor binds to at least a portion of SEQ
ID NO:2.
[0025] The expression vectors of the present invention may further
include a secretory signal sequence linked to the first and second
DNA segments. The multimeric or heterodimeric cytokine receptor may
be soluble, membrane-bound, or attached to a solid support. The
multimeric or heterodimeric cytokine receptor may antagonize an
activity of a ligand comprising SEQ ID NO:2. The multimeric or
heterodimeric cytokine receptor may inhibit proliferation of
hematopoietic cells, inhibit proliferation of immune cells, inhibit
proliferation of inflammatory cells, inhibit an immune response,
inhibit an inflammatory response, or inhibit proliferation of tumor
cells of epithelial origin. Optionally, the multimeric or
heterodimeric cytokine receptor may be soluble. The multimeric or
heterodimeric cytokine receptor may further comprise an affinity
tag or cytotoxic molecule as described herein.
[0026] The present invention also provides a cultured cell
including an expression vector as described herein, wherein the
cell expresses the polypeptide or polypeptides encoded by the DNA
segment or segments. The cell may secrete the multimeric or
heterodimeric cytokine receptor. The multimeric cytokine receptor
may bind and/or antagonize an activity of SEQ ID NO:2 as further
described herein.
[0027] The present invention also provides a cultured cell which
includes a first expression vector comprising: a) a transcription
promoter; b) a DNA segment encoding a cytokine receptor polypeptide
having at least 90 percent sequence identity with SEQ ID NO:111;
and c) a transcription terminator; and a second expression vector
comprising: a) a transcription promoter; b) a DNA segment encoding
a cytokine-binding domain of a class I cytokine receptor; and c) a
transcription terminator; wherein the cytokine receptor polypeptide
and the class I cytokine receptor form a multimeric or
heterodimeric cytokine receptor, and wherein the multimeric or
heterodimeric cytokine receptor binds to a ligand that comprises
SEQ ID NO:2. The first and second expression vectors may include a
secretory signal sequence operably linked to the first and second
DNA segments. The cultured cell may further comprise a third
expression vector which includes a) a transcription promoter; b) a
DNA segment encoding a cytokine-binding domain of a second class I
cytokine receptor; and c) a transcription terminator; wherein the
cytokine receptor polypeptide, the first class I cytokine receptor,
and the second class I cytokine receptor form a multimeric cytokine
receptor. The cytokine-binding domain of a class I cytokine
receptor may be of SEQ ID NO:7 and/or SEQ ID NO:9. Optionally, the
multimeric or heterodimeric cytokine receptor may be soluble. The
multimeric or heterodimeric cytokine receptor may further include
an affinity tag as described herein. The multimeric or
heterodimeric cytokine receptor may bind to at least a portion of
SEQ ID NO:2 and/or antagonize an activity of SEQ ID NO:2 as
described herein.
[0028] The present invention also provides a method of producing an
antibody to a multimeric or heterodimeric cytokine receptor
comprising amino acid residue 20 to amino acid residue 227 of SEQ
ID NO:111 and a cytokine-binding domain of a class I cytokine
receptor. The method includes inoculating an animal with the
multimeric or heterodimeric cytokine receptor, wherein the
multimeric or heterodimeric cytokine receptor elicits an immune
response in the animal to produce an antibody that specifically
binds the multimeric or heterodimeric cytokine receptor; and
isolating the antibody from the animal. The antibody may optionally
be a monoclonal antibody. The antibody may optionally be a
neutralizing antibody. The antibody may specifically bind to a
multimeric or heterodimeric cytokine receptor as described
herein.
[0029] The present invention also provides a composition which
includes an effective amount of a soluble multimeric or
heterodimeric cytokine receptor comprising amino acid residue 20 to
amino acid residue 227 of SEQ ID NO:111 and a cytokine-binding
domain of a class I cytokine receptor; and a pharmaceutically
acceptable vehicle. The binding domain of the class I cytokine
receptor may include amino acid residue 28 to amino acid residue
429 of SEQ ID NO:7. The soluble multimeric or heterodimeric
cytokine receptor may bind to a ligand comprising SEQ ID NO:2. The
soluble multimeric or heterodimeric cytokine receptor may further
include an affinity tag or cytotoxic molecule as described herein.
The composition may antagonize an activity of a ligand comprising
SEQ ID NO:2. The composition may inhibit proliferation of
hematopoietic cells, inhibit proliferation of immune cells, inhibit
proliferation of inflammatory cells, inhibit an immune response,
inhibit an inflammatory response, or inhibit proliferation of tumor
cells of epithelial origin
[0030] The present invention also provides a method of producing a
multimeric or heterodimeric cytokine receptor comprising culturing
a cell as described herein, and isolating the multimeric or
heterodimeric cytokine receptor produced by the cell.
[0031] The present invention also provides an immune cell
inhibiting composition which includes an effective amount of a
soluble multimeric or heterodimeric cytokine receptor comprising
amino acid residue 20 to amino acid residue 227 of SEQ ID NO:111
and a cytokine-binding domain of a class I cytokine receptor; and a
pharmaceutically acceptable vehicle; wherein the soluble multimeric
or heterodimeric cytokine receptor inhibits the proliferation of
immune cells.
[0032] The present invention also provides an immune response
inhibiting composition which includes an effective amount of a
soluble multimeric or heterodimeric cytokine receptor comprising
amino acid residue 20 to amino acid residue 227 of SEQ ID NO:111
and a cytokine-binding domain of a class I cytokine receptor; and a
pharmaceutically acceptable vehicle; wherein the soluble multimeric
or heterodimeric cytokine receptor inhibits an immune response.
[0033] The present invention also provides an inflammatory cell
inhibiting composition which includes an effective amount of a
soluble multimeric or heterodimeric cytokine receptor comprising
amino acid residue 20 to amino acid residue 227 of SEQ ID NO:111
and a cytokine-binding domain of a class I cytokine receptor; and a
pharmaceutically acceptable vehicle; wherein the soluble multimeric
or heterodimeric cytokine receptor inhibits the proliferation of
inflammatory cells.
[0034] The present invention also provides an inflammatory response
inhibiting composition which includes an effective amount of a
soluble multimeric or heterodimeric cytokine receptor comprising
amino acid residue 20 to amino acid residue 227 of SEQ ID NO:111
and a cytokine-binding domain of a class I cytokine receptor; and a
pharmaceutically acceptable vehicle; wherein the soluble multimeric
or heterodimeric cytokine receptor inhibits an inflammatory
response.
[0035] The present invention also provides a method of inhibiting
an immune response in a mammal exposed to an antigen or pathogen.
The method includes (a) determining directly or indirectly the
level of antigen or pathogen present in the mammal; (b)
administering a composition comprising a soluble multimeric or
heterodimeric cytokine receptor in a pharmaceutically acceptable
vehicle; (c) determining directly or indirectly the level of
antigen or pathogen in the mammal; and (d) comparing the level of
the antigen or pathogen in step (a) to the antigen or pathogen
level in step (c), wherein a change in the level is indicative of
inhibiting an immune response. The method may further comprise (e)
re-administering a composition comprising a multimeric cytokine
receptor in a pharmaceutically acceptable vehicle; (f) determining
directly or indirectly the level of antigen or pathogen in the
mammal; and (g) comparing the number of the antigen or pathogen
level in step (a) to the antigen level in step (f), wherein a
change in the level is indicative of inhibiting an immune
response.
[0036] The present invention also provides a method for reducing
hematopoietic cells and/or hematopoietic progenitors cells in a
mammal. The method includes culturing bone marrow or peripheral
blood cells with a composition comprising an effective amount of a
soluble multimeric or heterodimeric cytokine receptor to produce a
decrease in the number of lymphoid cells in the bone marrow or
peripheral blood cells as compared to bone marrow or peripheral
blood cells cultured in the absence of the multimeric cytokine
receptor. The hematopoietic cells and hematopoietic cell
progenitors may be lymphoid, which can be monocytic cells,
macrophages, or T cells.
[0037] The present invention also provides a method of detecting
the presence of a multimeric or heterodimeric cytokine receptor in
a biological sample. The method includes contacting the biological
sample with an antibody, or an antibody fragment, as described
herein, wherein the contacting is performed under conditions that
allow the binding of the antibody or antibody fragment to the
biological sample; and detecting any of the bound antibody or bound
antibody fragment.
[0038] The present invention also provides a method of a method of
killing cancer cells. The method includes obtaining ex vivo a
tissue or biological sample containing cancer cells from a patient,
or identifying cancer cells in vivo; producing a multimeric or
heterodimeric cytokine receptor by a method as described herein;
formulating the multimeric or heterodimeric cytokine receptor in a
pharmaceutically acceptable vehicle; and administering to the
patient or exposing the cancer cells to the multimeric or
heterodimeric cytokine receptor formulation; wherein the multimeric
or heterodimeric cytokine receptor kills the cells. The multimeric
or heterodimeric cytokine receptor may be further conjugated to a
toxin.
[0039] The present invention also provides an antibody that
specifically binds to a multimeric or heterodimeric cytokine
receptor as described herein. The antibody may be a polyclonal
antibody, a murine monoclonal antibody, a humanized antibody
derived from a murine monoclonal antibody, an antibody fragment, a
neutralizing antibody, or a human monoclonal antibody. The antibody
or antibody fragment may specifically bind to a multimeric or
heterodimeric cytokine receptor of the present invention which may
comprise a cytokine receptor polypeptide comprising amino acid
residue 20 to amino acid residue 227 of SEQ ID NO:111 and a
cytokine-binding domain of a class I cytokine receptor. The
antibody may further include a radionuclide, enzyme, substrate,
cofactor, fluorescent marker, chemiluminescent marker, peptide tag,
magnetic particle, drug, or toxin.
[0040] The present invention also provides a method for inhibiting
zcytor17lig-induced proliferation or differentiation of
hematopoietic cells and hematopoietic progenitor cells. The method
includes culturing bone marrow or peripheral blood cells with a
composition comprising an amount of a soluble multimeric or
heterodimeric cytokine receptor comprising a cytokine receptor
polypeptide comprising amino acid residue 20 to amino acid residue
227 of SEQ ID NO:111 and a cytokine-binding domain of a class I
cytokine receptor 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 the soluble multimeric or
heterodimeric cytokine receptor. The hematopoietic cells and
hematopoietic progenitor cells may be lymphoid cells, such as
macrophages or T cells.
[0041] The present invention also provides a method of reducing
zcytor17lig-induced induced inflammation. The method includes
administering to a mammal with inflammation an amount of a
composition comprising amino acid residue 20 to amino acid residue
227 of SEQ ID NO: 111 and a cytokine-binding domain of a class I
cytokine receptor sufficient to reduce inflammation.
[0042] The present invention also provides a method of suppressing
an inflammatory response in a mammal with inflammation. The method
includes (1) determining a level of an inflammatory molecule; (2)
administering a composition comprising amino acid residue 20 to
amino acid residue 227 of SEQ ID NO:111 and a cytokine-binding
domain of a class I cytokine receptor in a pharmaceutically
acceptable vehicle; (3) determining a post administration level of
the inflammatory molecule; (4) comparing the level of the
inflammatory molecule in step (1) to the level of the inflammatory
molecule in step (3), wherein a lack of increase or a decrease the
inflammatory molecule level is indicative of suppressing an
inflammatory response.
[0043] The present invention also provides a method for inhibiting
zcytor17lig-induced proliferation or differentiation of
hematopoietic cells and hematopoietic progenitor cells. The method
includes culturing bone marrow or peripheral blood cells with a
composition comprising amino acid residue 20 to amino acid residue
227 of SEQ ID NO:111 and a cytokine-binding domain of a class I
cytokine receptor in a pharmaceutically acceptable vehicle
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 multimeric or heterodimeric cytokine receptor.
The hematopoietic cells and hematopoietic progenitor cells may be
lymphoid cells, such as macrophages or T cells.
[0044] The present invention also provides a method of reducing
zcytor17lig-induced induced inflammation. The method includes
administering to a mammal with inflammation an amount of a
composition comprising amino acid residue 20 to amino acid residue
227 of SEQ ID NO: 111 and a cytokine-binding domain of a class I
cytokine receptor in a pharmaceutically acceptable vehicle
sufficient to reduce inflammation.
[0045] The present invention also provides a method of suppressing
an inflammatory response in a mammal with inflammation. The method
includes (1) determining a level of an inflammatory molecule; (2)
administering a composition comprising a multimeric or
heterodimeric cytokine receptor which comprises amino acid residue
20 to amino acid residue 227 of SEQ ID NO: 111 in a
pharmaceutically acceptable vehicle; (3) determining a post
administration level of the inflammatory molecule; (4) comparing
the level of the inflammatory molecule in step (1) to the level of
the inflammatory molecule in step (3), wherein a lack of increase
or a decrease in the inflammatory molecule level is indicative of
suppressing an inflammatory response.
[0046] The present invention also provides a method of treating a
mammal afflicted with an inflammatory disease in which zcytor17lig
plays a role. The method includes administering an antagonist of
zcytor17lig to the mammal such that the inflammation is reduced,
wherein the antagonist is a soluble multimeric or heterodimeric
cytokine receptor comprising amino acid residue 20 to amino acid
residue 227 of SEQ ID NO:111 and a cytokine-binding domain of a
class I cytokine receptor in a pharmaceutically acceptable vehicle.
The inflammatory disease may be a chronic inflammatory disease,
such as, for instance, inflammatory bowel disease, ulcerative
colitis, Crohn's disease, atopic dermatitis, eczema, or psoriasis.
The inflammatory disease may be an acute inflammatory disease, such
as, for instance, endotoxemia, septicemia, toxic shock syndrome, or
infectious disease. Optionally, the soluble multimeric or
heterodimeric cytokine receptor may further comprise a
radionuclide, enzyme, substrate, cofactor, fluorescent marker,
chemiluminescent marker, peptide tag, magnetic particle, drug, or
toxin.
[0047] The present invention also provides a method for detecting
inflammation in a patient. The method includes obtaining a tissue
or biological sample from a patient; incubating the tissue or
biological sample with a soluble multimeric or heterodimeric
cytokine receptor comprising amino acid residue 20 to amino acid
residue 227 of SEQ ID NO:111 and a cytokine-binding domain of a
class I cytokine receptor under conditions wherein the soluble
multimeric or heterodimeric cytokine receptor binds to its
complementary polypeptide in the tissue or biological sample;
visualizing the soluble multimeric or heterodimeric cytokine
receptor bound in the tissue or biological sample; and comparing
levels of soluble multimeric or heterodimeric cytokine receptor
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 soluble multimeric or heterodimeric cytokine receptor
bound to the patient tissue or biological sample relative to the
normal control tissue or biological sample is indicative of
inflammation in the patient.
[0048] The present invention also provides a method for detecting a
multiple cytokine receptor ligand from a test sample. The method
includes contacting the test sample with a multimeric or
heterodimeric cytokine receptor comprising a cytokine receptor
polypeptide comprising amino acid residue 20 to amino acid residue
227 of SEQ ID NO:111 and a cytokine-binding domain of a class I
cytokine receptor; and detecting the binding of the multimeric or
heterodimeric cytokine receptor to the ligand in the test
sample.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0049] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0050] Unless otherwise specified, "a," "an," "the," and "at least
one" are used interchangeably and mean one or more than one.
[0051] 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 82:7952-4, 1985), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-10, 1988),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2: 95-107, 1991. DNAs encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0052] 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.
[0053] 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.
[0054] 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 <10.sup.9 M.sup.-1.
[0055] The term "complements of a polynucleotide molecule" denotes
a polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to 5'
CCCGTGCAT 3'.
[0056] The term "contig" denotes a polynucleotide that has a
contiguous stretch of identical or complementary sequence to
another polynucleotide. Contiguous sequences are said to "overlap"
a given stretch of polynucleotide sequence either in their entirety
or along a partial stretch of the polynucleotide. For example,
representative contigs to the polynucleotide sequence
5'-ATGGCTTAGCTT-3' are 5'-TAGCTTgagtct-3' and
3'-gtcgacTACCGA-5'.
[0057] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide 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).
[0058] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0059] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0060] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, 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.
[0061] The term "neoplastic", when referring to cells, indicates
cells undergoing new and abnormal proliferation, particularly in a
tissue where in the proliferation is uncontrolled and progressive,
resulting in a neoplasm. The neoplastic cells can be either
malignant, i.e., invasive and metastatic, or benign.
[0062] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0063] 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 speciation.
[0064] "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.
[0065] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired.
[0066] 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".
[0067] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0068] 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.
[0069] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-peptide structure comprising an
extracellular ligand-binding domain and an intracellular effector
domain that is typically involved in signal transduction. 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. This interaction in turn leads to an
alteration in the metabolism of the cell. Metabolic events that are
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. In general, 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).
[0070] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (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.
[0071] 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. 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. Soluble
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.
[0072] 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 protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0073] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
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%.
[0074] The present invention is based in part upon the discovery of
a novel multimeric cytokine receptor protein having the structure
of a class I cytokine receptor, referred to herein as "multimeric
cytokine receptor," or "zcytor17 multimeric cytokine receptor." The
multimeric cytokine receptor includes at least a portion of a
zcytor17 receptor subunit, disclosed in the commonly owned U.S.
patent application Ser. No. 09/892,949. Another receptor subunit
polypeptide that may be included in the multimeric cytokine
receptor of the present invention includes at least a portion of at
least one polypeptide of a class I cytokine receptor, such as
OSMRbeta and/or WSX-1. For example, the deduced amino acid sequence
indicated that zcytor17 belongs to the receptor subfamily that
includes gp130, LIF, IL-12, oncostatinM receptor beta (OSMRbeta)
(SEQ ID NO:7), WSX-1 receptors (SEQ ID NO:9) (Sprecher, Calif. et
al., Biochem. Biophys. Res. Comm., 246:81-90 (1998); and U.S. Pat.
No. 5,925,735), DCRS2 (WIPO Publication No. WO 00/73451), the IL-2
receptor .beta.-subunit and the .beta.-common receptor (i.e., IL-3,
IL-5, and GM-CSF receptor subunits). A further example of class I
cytokine receptor subunit polypeptides that may be included in the
multimeric cytokine receptor are the receptors for IL-2, IL-4,
IL-7, Lif, IL-12, IL-15, EPO, TPO, GM-CSF and G-CSF (Cosman,
Cytokine, 5(2):95-106 (1993)).
[0075] Cytokine receptor subunits are characterized by a
multi-domain structure comprising an extracellular domain, a
transmembrane domain that anchors the polypeptide in the cell
membrane, and an intracellular domain. The extracellular domain may
be a ligand-binding domain, and the intracellular domain may be an
effector domain involved in signal transduction, although
ligand-binding and effector functions may reside on separate
subunits of a multimeric receptor. The ligand-binding domain may
itself be a multi-domain structure. Multimeric receptors include
homodimers (e.g., PDGF receptor .alpha..alpha. and .beta..beta.
isoforms, erythropoietin receptor, MPL, and G-CSF receptor),
heterodimers whose subunits each have ligand-binding and effector
domains (e.g., PDGF receptor .alpha..beta. isoform), and multimers
having component subunits with disparate functions (e.g., IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor
subunits are common to a plurality of receptors. For example, the
AIC2B subunit, which cannot bind ligand on its own but includes an
intracellular signal transduction domain, is a component of IL-3
and GM-CSF receptors. Many cytokine receptors can be placed into
one of four related families on the basis of the structure and
function. Hematopoietic receptors, for example, are characterized
by the presence of a domain containing conserved cysteine residues
and the WSXWS motif (SEQ ID NO:3). Cytokine receptor structure has
been reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228, 1991
and Cosman, Cytokine 5:95-106, 1993. Under selective pressure for
organisms to acquire new biological functions, new receptor family
members likely arise from duplication of existing receptor genes
leading to the existence of multi-gene families. Family members
thus contain vestiges of the ancestral gene, and these
characteristic features can be exploited in the isolation and
identification of additional family members. Thus, the cytokine
receptor superfamily is subdivided into several families, for
example, the immunoglobulin family (including CSF-1, MGF, IL-1, and
PDGF receptors); the hematopoietin family (including IL-2 receptor
.beta.-subunit, GM-CSF receptor .alpha.-subunit, GM-CSF receptor
.beta.-subunit; and G-CSF, EPO, IL-3, IL-4, IL-5, IL-6, IL-7, and
IL-9 receptors); TNF receptor family (including TNF (p80) TNF (p60)
receptors, CD27, CD30, CD40, Fas, and NGF receptor).
[0076] Analysis of the zcytor17 sequence suggests that it is a
member of the same receptor subfamily as the gp130, LIF, IL-12,
WSX-1, IL-2 receptor .beta.-subunit, IL-3, IL-4, and IL-6
receptors. Certain receptors in this subfamily (e.g., G-CSF)
associate to form homodimers that transduce a signal. Other members
of the subfamily (e.g., gp130, IL-6, IL-11, and LIF receptors)
combine with a second subunit (termed a .beta.-subunit) to bind
ligand and transduce a signal. Specific .beta.-subunits associate
with a plurality of specific cytokine receptor subunits. For
example, the .beta.-subunit gp130 (Hibi et al., Cell 63:1149-1157,
1990) associates with receptor subunits specific for IL-6, IL-11,
and LIF (Gearing et al., EMBO J. 10:2839-2848, 1991; Gearing et
al., U.S. Pat. No. 5,284,755). Oncostatin M binds to a heterodimer
of LIF receptor and gp130. CNTF binds to trimeric receptors
comprising CNTF receptor, LIF receptor, and gp 130 subunits.
[0077] A multimeric cytokine receptor of the present invention can
be a heterodimer, trimer, tetramer, pentamer, and the like,
comprising at least a portion of zcytor17 and at least a portion of
a class I cytokine receptor. In addition, a multimeric cytokine
receptor can be soluble, membrane-bound, or attached to a solid
support. Analysis of the tissue distribution of the mRNA of the
zcytor17 receptor revealed expression in activated CD4+ and CD8+
T-cell subsets, CD14+ monocytes, and weaker expression in CD19+
B-cells. Moreover, the mRNA was present in both resting or
activated monocytic cell lines THP-1 (ATCC No. TIB-202), U937 (ATCC
No. CRL-1593.2) and HL60 (ATCC No. CCL-240).
[0078] Nucleotide sequences of representative zcytor17-encoding DNA
are described in SEQ ID NO:110 (from nucleotide 171 to 2366), with
its deduced 732 amino acid sequence described in SEQ ID NO:11; SEQ
ID NO:108 (from nucleotide 162 to 2108), with its deduced 649 amino
acid sequence described in SEQ ID NO:109; and in SEQ ID NO:4 (from
nucleotide 497 to 2482), with its deduced 662 amino acid sequence
described in SEQ ID NO:5. In its entirety, the zcytor17 polypeptide
(SEQ ID NO:111, SEQ ID NO:109 or SEQ ID NO:5) represents a
full-length polypeptide segment (residue 1 (Met) to residue 732
(Val) of SEQ ID NO:111; residue 1 (Met) to residue 649 (Ile) of SEQ
ID NO:109; residue 1 (Met) to residue 662 (Ile) of SEQ ID NO:5).
The domains and structural features of the zcytor17 polypeptides
are further described below.
[0079] Analysis of the zcytor17 polypeptide encoded by the DNA
sequence of SEQ ID NO: 110 revealed an open reading frame encoding
732 amino acids (SEQ ID NO:111) comprising a predicted secretory
signal peptide of 19 amino acid residues (residue 1 (Met) to
residue 19 (Ala) of SEQ ID NO:111), and a mature polypeptide of 713
amino acids (residue 20 (Ala) to residue 732 (Val) of SEQ ID
NO:111). Analysis of the zcytor17 polypeptide encoded by the DNA
sequence of SEQ ID NO: 108 revealed an open reading frame encoding
649 amino acids (SEQ ID NO:109) comprising a predicted secretory
signal peptide of 19 amino acid residues (residue 1 (Met) to
residue 19 (Ala) of SEQ ID NO:109), and a mature polypeptide of 630
amino acids (residue 20 (Ala) to residue 649 (Ile) of SEQ ID
NO:109). Analysis of the zcytor17 polypeptide encoded by the DNA
sequence of SEQ ID NO:4 revealed an open reading frame encoding 662
amino acids (SEQ ID NO:5) comprising a predicted secretory signal
peptide of 32 amino acid residues (residue 1 (Met) to residue 32
(Ala) of SEQ ID NO:5), and a mature polypeptide of 630 amino acids
(residue 33 (Ala) to residue 662 (Ile) of SEQ ID NO:5). In addition
to the WSXWS motif (SEQ ID NO:3) (corresponding to residues 211 to
215 of SEQ ID NO:111 and SEQ ID NO:109; and residues 224 to 228 of
SEQ ID NO:5), the receptor comprises an extracellular domain
(residues 20 (Ala) to 519 (Glu) of SEQ ID NO:111 and SEQ ID NO:
109; residues 33 (Ala) to 532 (Glu) of SEQ ID NO:5) which includes
a cytokine-binding domain of approximately 200 amino acid residues
(residues 20 (Ala) to 227 (Pro) of SEQ ID NO:111 and SEQ ID NO:109;
residues 33 (Ala) to 240 (Pro) of SEQ ID NO:5); a domain linker
(residues 122 (Thr) to 125 (Pro) of SEQ ID NO:111 and SEQ ID
NO:109; residues 135 (Thr) to 138 (Pro) of SEQ ID NO:111); a
penultimate strand region (residues 194 (Phe) to 202 (Arg) of SEQ
ID NO:111 and SEQ ID NO:109; residues 207 (Phe) to 215 (Arg) of SEQ
ID NO:5); a fibronectin type III domain (residues 228 (Cys) to 519
(Glu) of SEQ ID NO:111 and SEQ ID NO:109; residues 241 (Cys) to 532
(Glu) of SEQ ID NO:5); a transmembrane domain (residues 520 (Ile)
to 543 (Leu) of SEQ ID NO:111 and SEQ ID NO:109; residues 533 (Ile)
to 556 (Leu) of SEQ ID NO:5); complete intracellular signaling
domain (residues 544 (Lys) to 732 (Val) of SEQ ID NO:111; residues
544 (Lys) to 649 (Ile) of SEQ ID NO:109; and residues 557 (Lys) to
662 (Ile) of SEQ ID NO:5) which contains a "Box I" signaling site
(residues 554 (Trp) to 560 (Pro) of SEQ ID NO:111 and SEQ ID
NO:109; residues 567 (Trp) to 573 (Pro) of SEQ ID NO:5), and a "Box
II" signaling site (residues 617 (Gln) to 620 (Phe) of SEQ ID
NO:111 and SEQ ID NO:109; residues 630 (Gln) to 633 (Phe) of SEQ ID
NO:5). Those skilled in the art will recognize that these domain
boundaries are approximate, and are based on alignments with known
proteins and predictions of protein folding. In addition to these
domains, conserved receptor features in the encoded receptor
include (as shown in SEQ ID NO:111 and SEQ ID NO:109) a conserved
Cys residue at position 30 (position 43 as shown in SEQ ID NO:5),
CXW motif (wherein X is any amino acid) at positions 40-42
(positions 53-55 as shown in SEQ ID NO:5), Trp residue at position
170 (position 183 as shown in SEQ ID NO:5), and a conserved Arg
residue at position 202 (position 215 as shown in SEQ ID NO:5). The
corresponding polynucleotides encoding the zcytor17 polypeptide
regions, domains, motifs, residues and sequences described above
are as shown in SEQ ID NO:110, SEQ ID NO:108, and SEQ ID NO:4.
[0080] Moreover, truncated forms of the zcytor17 polypeptide appear
to be naturally expressed. Both forms encode soluble zcytor17
receptors. A polynucleotide encoding a "long-form" of the soluble
zcytor17 receptor, truncated within the fibronectin type III
domain, is shown in SEQ ID NO:112 and the corresponding polypeptide
is shown in SEQ ID NO:113. This truncated form encodes residues 1
(Met) through 324 (Lys) of SEQ ID NO:111 and SEQ ID NO:109), and
thus comprises an intact signal sequence, WSXWS (SEQ ID NO:3)
motif, linker, cytokine binding domain, penultimate strand, and
conserved, Cys, CXW motif, Trp and Arg residues as described above.
A polynucleotide encoding a "short-form" of the soluble zcytor17
receptor, truncated at the end of the cytokine binding domain is
shown in SEQ ID NO:114 and the corresponding polypeptide is shown
in SEQ ID NO:115. This truncated form encodes a 239 residue
polypeptide that is identical to residues 1 (Met) through 225 (Glu)
of SEQ ID NO:111 and SEQ ID NO:109 and then diverges, and thus
comprises an intact signal sequence, WSXWS (SEQ ID NO:3) motif,
linker, cytokine binding domain, penultimate strand, and conserved,
Cys, CXW motif, Trp and Arg residues as described above. A multiple
alignment of the truncated forms compared to the full-length forms
of zcytor17 is shown in FIG. 1.
[0081] Moreover, the zcytor17 cDNA of SEQ ID NO:110, SEQ ID NO:108,
SEQ ID NO: 112, and SEQ ID NO:114 encode polypeptides that may use
an alternative initiating methionine (at nucleotide 75 of SEQ ID
NO:110, at nucleotide 66 of SEQ ID NO:108, at nucleotide 66 of SEQ
ID NO:112, and at nucleotide 66 of SEQ ID NO:114) that would encode
a polypeptide in the same open reading frame (ORF) as the zcytor17
polypeptides of SEQ ID NO:111, SEQ ID NO:109, SEQ ID NO:113, and
SEQ ID NO:115. Use of the alternative initiating methionine would
add 32 amino acids (shown in SEQ ID NO:48) in-frame to the
N-terminus of SEQ ID NO:111, SEQ ID NO:109, SEQ ID NO:113, and SEQ
ID NO:111. In addition, nucleotide 536 of SEQ ID NO:4 may serve as
an alternative initiating methionine, thus generating the same
N-terminus (starting at amino acid 14 (Met) of SEQ ID NO:5) and
signal polypeptide sequence, as SEQ ID NO:111, SEQ ID NO:109, SEQ
ID NO: 113, and SEQ ID NO:115. Moreover, the second Met at amino
acid number 2 in the SEQ ID NO: 111, SEQ ID NO:109, SEQ ID NO:113,
and SEQ ID NO:115 sequences (similarly at amino acid number 15
(Met) in SEQ ID NO:5) may also serve as an alternative starting
methionine for the polypeptides.
[0082] Nucleotide sequences of representative OSMRbeta-encoding DNA
are described in SEQ ID NO:6 (from nucleotide 368 to 3304), with
its deduced 979 amino acid sequence described in SEQ ID NO:7. In
its entirety, the OSMRbeta polypeptide (SEQ ID NO:7) represents a
full-length polypeptide segment (residue 1 (Met) to residue 979
(Cys) of SEQ ID NO:7. The domains and structural features of the
OSMRbeta polypeptides are further described below.
[0083] Analysis of the OSMRbeta polypeptide encoded by the DNA
sequence of SEQ ID NO:6 revealed an open reading frame encoding 979
amino acids (SEQ ID NO:7) comprising a predicted secretory signal
peptide of 27 amino acid residues (residue 1 (Met) to residue 27
(Ala) of SEQ ID NO:7), and a mature polypeptide of 952 amino acids
(residue 28 (Glu) to residue 979 (Cys) of SEQ ID NO:7. In addition
to the two WSXWS motifs (SEQ ID NO:3) (corresponding to residues
129 to 133 and residues 415 to 419 of SEQ ID NO:7), the receptor
comprises an extracellular domain (residues 28 (Glu) to 739 (Ser)
of SEQ ID NO:7); which includes a cytokine-binding domain of
approximately 400 amino acid residues (residues 28 (Glu) to 429
(Ala) of SEQ ID NO:7, which includes two linker domains (residues
31 (Pro) to 34 (Pro) and residues 343 (Asn) to 347 (Thr)), three
regions of cytokine binding (residues 35 (Val) to 137 (Glu),
residues 240 (Pro) to 342 (Glu), and residues 348 (Asn) to 429
(Ala), an immuglobulin domain (residues 138 (Val) to 239 (Glu), two
penultimate strand regions (residues 106 (His) to 115 (Lys) and
residues 398 (Thr) to 405 (Arg) of SEQ ID NO:7), and a fibronectin
type III domain (residues 430 (Pro) to 739 (Ser) of SEQ ID NO:7); a
transmembrane domain (residues 740 (Met) to 761 (Leu) of SEQ ID
NO:7); complete intracellular signaling domain (residues 762 (Lys)
to 979 (Cys) of SEQ ID NO:7) which contains a "Box I" signaling
site (residues 771 (Tyr) to 777 (Pro) of SEQ ID NO:7), and a "Box
II" signaling site (residues 829 (Glu) to 832 (Leu) of SEQ ID
NO:7). Those skilled in the art will recognize that these domain
boundaries are approximate, and are based on alignments with known
proteins and predictions of protein folding. In addition to these
domains, conserved receptor features in the encoded receptor
include (as shown in SEQ ID NO:7) conserved Trp residues at
positions 52 and 353, a conserved Cys residue at position 288, CXW
motif (wherein X is any amino acid) at positions 294-296, and a
conserved Arg residue at position 405. The corresponding
polynucleotides encoding the OSMRbeta polypeptide regions, domains,
motifs, residues and sequences described above are as shown in SEQ
ID NO:6.
[0084] The presence of transmembrane regions, and conserved and low
variance motifs generally correlates with or defines important
structural regions in proteins. Regions of low variance (e.g.,
hydrophobic clusters) are generally present in regions of
structural importance (Sheppard, P. et al., supra.). Such regions
of low variance often contain rare or infrequent amino acids, such
as Tryptophan. The regions flanking and between such conserved and
low variance motifs may be more variable, but are often
functionally significant because they may relate to or define
important structures and activities such as binding domains,
biological and enzymatic activity, signal transduction, cell-cell
interaction, tissue localization domains and the like.
[0085] The regions of conserved amino acid residues in zcytor17,
described above, can be used as tools to identify new family
members. For instance, reverse transcription-polymerase chain
reaction (RT-PCR) can be used to amplify sequences encoding the
conserved regions from RNA obtained from a variety of tissue
sources or cell lines. In particular, highly degenerate primers
designed from the zcytor17 sequences are useful for this purpose.
Designing and using such degenerate primers may be readily
performed by one of skill in the art.
[0086] The present invention also contemplates a multimeric
zcytor17 receptor, as detailed herein, which is capable of
intracellular signaling. Such receptors may include at least a
portion of at least one extracellular domain of a zcytor17
receptor, and an intracellular domain from a zcytor17 receptor or
another class I cytokine receptor. In addition to the extracellular
domain of zcytor17, the multimeric cytokine receptor can also
include the extracellular domain of at least a portion of class I
cytokine receptor, for instance, the ligand binding domains of
OSMRbeta receptor and/or WSX-1 receptor. Alternatively, the
multimeric cytokine receptor may include the extracellular domain
of another receptor, such as another class I cytokine receptor, and
the intracellular domain of zcytor17 to effect intracellular
signaling.
[0087] The present invention further contemplates a multimeric
cytokine receptor that is soluble. For example, a multimeric
cytokine receptor may be, for instance, a heterodimer which
includes, for example, a portion of the extracellular domain of
zcytor17 and a portion of the extracellular domain of a class I
cytokine receptor, such as OSMRbeta (SEQ ID NO:7) and/or WSX-1 (SEQ
ID NO:9). Additionally, a soluble multimeric cytokine receptor may
also include an affinity tag, such as an immuglobulin F.sub.c
polypeptide. The soluble multimeric cytokine receptor can be
expressed as a fusion with an immunoglobulin heavy chain constant
region, such as an F.sub.c fragment, which contains two constant
region domains and lacks the variable region. Such fusions are
typically secreted as multimeric molecules wherein the F.sub.c
portions are disulfide bonded to each other and two non-Ig
polypeptides are arrayed in closed proximity to each other. Fusions
of this type can be used for example, for dimerization, increasing
stability and in vivo half-life, to affinity purify ligand, as in
vitro assay tool or antagonist.
[0088] Through processes of cloning, and proliferation assays
described in detail herein, a multimeric cytokine receptor of the
present invention has been shown to bind a novel ligand polypeptide
(zcytor17lig) (SEQ ID NO:2), disclosed in commonly owned U.S.
Patent Application Ser. No. 60/350,325 and commonly owned U.S.
Patent Application Ser. No. 60/375,323, with high specificity.
Zcytor17lig was isolated from a cDNA library generated from
activated human peripheral blood cells (hPBCs), which were selected
for CD3. CD3 is a cell surface marker unique to cells of lymphoid
origin, particularly T cells.
[0089] A zcytor17lig positive clone was isolated, and sequence
analysis revealed that the polynucleotide sequence contained within
the plasmid DNA was novel. The secretory signal sequence is
comprised of amino acid residues 1 (Met) to 23 (Ala), and the
mature polypeptide is comprised of amino acid residues 24 (Ser) to
164 (Thr) (as shown in SEQ ID NO:2). Further, N-Terminal sequencing
analysis of purified zcytor17lig from 293T cells showed an
N-terminus at residue 27 (Leu) as shown in SEQ ID NO:2, with the
mature polypeptide comprised of amino acid residues 27 (Leu) to 164
(Thr) (as shown in SEQ ID NO:2).
[0090] In general, cytokines are predicted to have a four-alpha
helix structure, with helices A, C and D being most important in
ligand-receptor interactions, and are more highly conserved among
members of the family. Referring to the human zcytor17lig amino
acid sequence shown in SEQ ID NO:2, alignment of human zcytor17lig,
human IL-3, and human cytokine amino acid sequences it is predicted
that zcytor17lig helix A is defined by amino acid residues 38-52;
helix B by amino acid residues 83-98; helix C by amino acid
residues 104-117; and helix D by amino acid residues 137-152; as
shown in SEQ ID NO:2. Structural analysis suggests that the A/B
loop is long, the B/C loop is short and the C/D loop is long. This
loop structure results in an up-up-down-down helical organization.
Based on 4-helix bundle structure, the cysteine residues within
zcytor17lig that are conserved correspond to amino acid residues
72, 133, and 147 of SEQ ID NO:2; and 74, 137, and 151 of SEQ ID
NO:11 described herein. Consistent cysteine placement is further
confirmation of the four-helical-bundle structure. Also highly
conserved in the zcytor17lig is the Glu residue as shown in SEQ ID
NO:2 at residue 43.
[0091] Moreover, the predicted amino acid sequence of murine
zcytor17lig shows 31% identity to the predicted human protein over
the entire length of the sequences (SEQ ID NO:2 and SEQ ID NO:11).
Based on comparison between sequences of human and murine
zcytor17lig conserved residues were found in the regions predicted
to encode alpha helices C and D. The corresponding polynucleotides
encoding the human zcytor17lig polypeptide regions, domains,
motifs, residues and sequences described herein are as shown in SEQ
ID NO:1.
[0092] While helix D is relatively conserved between human and
murine zcytor17lig, helix C is the most conserved. While both
species have predominant acidic amino acids in this region, the
differences may account for species specificity in interaction
between zcytor17lig and its receptor, zcytor17, comprising
monomeric, heterodimeric (e.g., zcytor17/OSMRbeta, WSX-1/OSMRbeta,
zcytor17/WSX-1) or multimeric (e.g., zcytor17/OSMRbeta/WSX-1)
receptors. Loop A/B and helix B of zcytor17lig are marginally
conserved, and helix C through Loop C/D into helix D is most
conserved between species; conservation through this region
suggests that it is functionally significant. The D helices of
human and murine zcytor17lig are also conserved. Zcytor17 receptor
antagonists may be designed through mutations within zcytor17lig
helix D. These may include truncation of the protein from residue
Thr156 (SEQ ID NO:2), or conservation of residues that confer
binding of the ligand to the receptor, but diminish signaling
activity.
[0093] Four-helical bundle cytokines are also grouped by the length
of their component helices. "Long-helix" form cytokines generally
consist of between 24-30 residue helices, and include IL-6, ciliary
neutrotrophic factor (CNTF), leukemia inhibitory factor (LIF) and
human growth hormone (hGH). "Short-helix" form cytokines generally
consist of between 18-21 residue helices and include IL-2, IL-4 and
GM-CSF. Zcytor17lig is believed to be a new member of the
short-helix form cytokine group. Studies using CNTF and IL-6
demonstrated that a CNTF helix can be exchanged for the equivalent
helix in IL-6, conferring CTNF-binding properties to the chimera.
Thus, it appears that functional domains of four-helical cytokines
are determined on the basis of structural homology, irrespective of
sequence identity, and can maintain functional integrity in a
chimera (Kallen et al., J. Biol. Chem. 274:11859-11867, 1999).
Therefore, the helical domains of zcytor17lig may be useful for
preparing chimeric fusion molecules, particularly with other
short-helix form cytokines to determine and modulate receptor
binding specificity. The present invention also envisions fusion
proteins engineered with helix A and/or helix D, and fusion
proteins that combine helical and loop domains from other
short-form cytokines such as IL-2, IL-4, IL-15, Lif, IL-12, IL-3
and GM-CSF.
[0094] The polynucleotide sequence for human IL-2 is shown in SEQ
ID NO:176 and the corresponding amino acid sequence is shown in SEQ
ID NO:177. The secretory signal sequence is comprised of amino acid
residues 1 (Met) to 20 (Ser) of SEQ ID NO:177; nucleotides 48 to
107 of SEQ ID NO:176. The mature polypeptide is comprised of amino
acid residues 21 (Ala) to 156 (Thr) of SEQ ID NO:177; nucleotides
108 to 515 of SEQ ID NO:176. Helix A of human IL-2 is comprised of
amino acid residues 27 (Thr) to 48 (Leu) of SEQ ID NO:177;
nucleotides 126 to 191 of SEQ ID NO:176. Helix B of human IL-2
comprises Helix B1 and Helix B2. Helix B1 of human IL-2 is
comprised of amino acid residues 73 (Ala) to 80 (Gln) of SEQ ID
NO:177; nucleotides 264 to 287 of SEQ ID NO:176. Helix B2 of human
IL-2 is comprised of amino acid residues 83 (Glu) to 92 (Val) of
SEQ ID NO:177; nucleotides 294 to 323 of SEQ ID NO:176. Thus, Helix
B (comprising Helices B 1 and B2) of IL-2 is represented by the
amino acid sequence of SEQ ID NO:183 (nucleotide sequence of SEQ ID
NO:182) wherein amino acid residues 9 and 10 can be any amino acid.
SEQ ID NO: 183 is identical to amino acids 73 (Ala) to 92 (Val) of
SEQ ID NO:177 wherein amino acids 81 and 82 are any amino acid. In
a preferred form, Helix B of IL-2 comprises amino acids 73 (Ala) to
92 (Val) of SEQ ID NO:177; nucleotides 264 to 323 of SEQ ID NO:176.
Helix C of human IL-2 is comprised of amino acid residues 102 (His)
to 116 (Val) of SEQ ID NO:177 nucleotides 351 to 395 of SEQ ID
NO:176. Helix D of human IL-2 is comprised of amino acid residues
134 (Thr) to 149 (Gln) of SEQ ID NO:177; nucleotides 447 to 494 of
SEQ ID NO:176.
[0095] The polynucleotide sequence for human IL-4 is shown in SEQ
ID NO:178 and the corresponding amino acid sequence is shown in SEQ
ID NO:179. The secretory signal sequence is comprised of amino acid
residues 1 (Met) to 24 (Gly) of SEQ ID NO:179; nucleotides 64 to
135 of SEQ ID NO:178. The mature polypeptide is comprised of amino
acid residues 25 (His) to 153 (Ser) of SEQ ID NO:179; nucleotides
136 to 522 of SEQ ID NO:178. Helix A of human IL-4 is comprised of
amino acid residues 30 (Thr) to 42 (Thr) of SEQ ID NO:179;
nucleotides 151 to 189 of SEQ ID NO: 178. Helix B of human IL-4 is
comprised of amino acid residues 65 (Glu) to 83 (His) of SEQ ID
NO:179; nucleotides 256 to 312 of SEQ ID NO:178. Helix C of human
IL-4 is comprised of amino acid residues 94 (Ala) to 118 (Ala) of
SEQ ID NO:179; nucleotides 343 to 417 of SEQ ID NO:178. Helix D of
human IL-4 is comprised of amino acid residues 133 (Leu) to 151
(Cys) of SEQ ID NO:179; nucleotides 460 to 516 of SEQ ID
NO:178.
[0096] The polynucleotide sequence for human GM-CSF is shown in SEQ
ID NO:180 and the corresponding amino acid sequence is shown in SEQ
ID NO:181. The secretory signal sequence is comprised of amino acid
residues 1 (Met) to 17 (Ser) of SEQ ID NO:181; nucleotides 9 to 59
of SEQ ID NO:180. The mature polypeptide is comprised of amino acid
residues 18 (Ala) to 144 (Glu) of SEQ ID NO:181; nucleotides 60 to
440 of SEQ ID NO:180. Helix A of human GM-CSF is comprised of amino
acid residues 30 (Trp) to 44 (Asn) of SEQ ID NO:181; nucleotides 96
to 140 of SEQ ID NO:180. Helix B of human GM-CSF is comprised of
amino acid residues 72 (Leu) to 81 (Gln) of SEQ ID NO:181;
nucleotides 222 to 251 of SEQ ID NO:180. Helix C of human GM-CSF is
comprised of amino acid residues 85 (Gly) to 103 (Gln) of SEQ ID
NO:181; nucleotides 261 to 317 of SEQ ID NO:180. Helix D of human
GM-CSF is comprised of amino acid residues 120 (Phe) to 131 (Leu)
of SEQ ID NO:181; nucleotides 366 to 401 of SEQ ID NO:180.
[0097] The amino acid residues comprising helices A, B, C, and D,
for human zcytor17lig, IL-3, IL-2, IL-4, and GM-CSF are shown in
Table 1. TABLE-US-00001 TABLE 1 Helix A Helix B Helix C Helix D
zcytor17lig 38-52 83-98 104-117 137-152 of SEQ ID NO: 2 IL-3 35-45
73-86 91-103 123-141 of SEQ ID NO: 102 IL-2 27-48 73-92 102-116
134-149 of SEQ ID NO: 177 or Helix B as described in SEQ ID NO: 183
IL-4 30-42 65-83 94-118 133-151 of SEQ ID NO: 179 GM-CSF 30-44
72-81 85-103 120-131 of SEQ ID NO: 181
[0098] The present invention provides polynucleotide molecules,
including DNA and RNA molecules that encode the zcytor17
polypeptides disclosed herein that can be included in the
multimeric cytokine receptor. Those skilled in the art will
recognize that, in view of the degeneracy of the genetic code,
considerable sequence variation is possible among these
polynucleotide molecules. SEQ ID NO:120, SEQ ID NO:121 and SEQ ID
NO:122 are degenerate DNA sequences that encompass all DNAs that
encode the zcytor17 polypeptide of SEQ ID NO:111, SEQ ID NO:109 and
SEQ ID NO:5, respectively, and fragments thereof. Those skilled in
the art will recognize that the degenerate sequences of SEQ ID
NO:120, SEQ ID NO:121 and SEQ ID NO:122 also provide all RNA
sequences encoding SEQ ID NO:111, SEQ ID NO:109 and SEQ ID NO:5 by
substituting U for T. Thus, zcytor17 polypeptide-encoding
polynucleotides comprising nucleotide 1 to nucleotide 2196 of SEQ
ID NO:120, nucleotide 1 to nucleotide 1947 of SEQ ID NO:121, and
nucleotide 1 to nucleotide 1986 of SEQ ID NO:122 and their RNA
equivalents are contemplated by the present invention. Table 2 sets
forth the one-letter codes used within SEQ ID NO:120, SEQ ID NO:121
and SEQ ID NO:122 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-00002 TABLE 2 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
[0099] The degenerate codons used in SEQ ID NO:120, SEQ ID NO:121
and SEQ ID NO: 122, encompassing all possible codons for a given
amino acid, are set forth in Table 3. TABLE-US-00003 TABLE 3 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 . TAA TAG TGA TRR Asn|Asp B RAY
Glu|Gln Z SAR Any X NNN
[0100] 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 each 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:111, SEQ ID NO:109 and SEQ ID NO:5; or SEQ ID NO:117 and SEQ ID
NO:119. Variant sequences can be readily tested for functionality
as described herein.
[0101] One of ordinary skill in the art will also appreciate that
different species can exhibit "preferential codon usage." In
general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980;
Haas, et al. Curr. Biol 6:315-24, 1996; Wain-Hobson et al., Gene
13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm,
Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol 158:573-97,
1982. 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 3). 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 in SEQ ID NO:120, SEQ ID NO:121 and SEQ ID NO:122 serve
as templates for optimizing expression of zcytor17 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.
[0102] As previously noted, the isolated polynucleotides of the
present invention include DNA and RNA. Methods for preparing DNA
and RNA are well known in the art. In general, RNA is isolated from
a tissue or cell that produces large amounts of zcytor17 RNA. Such
tissues and cells are identified by Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include PBLs,
spleen, thymus, bone marrow, prostate, and lymph tissues, human
erythroleukemia cell lines, acute monocytic leukemia cell lines,
other lymphoid and hematopoietic cell lines, and the like. Total
RNA can be prepared using guanidinium isothiocyanate extraction
followed by isolation by centrifugation in a CsCl gradient
(Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A).sup.+ RNA
is prepared from total RNA using the method of Aviv and Leder
(Proc. Natl. Acad. Sci. USA 69:1408-12, 1972). Complementary DNA
(cDNA) is prepared from poly(A).sup.+ RNA using known methods. In
the alternative, genomic DNA can be isolated. Polynucleotides
encoding zcytor17 polypeptides are then identified and isolated by,
for example, hybridization or polymerase chain reaction (PCR)
(Mullis, U.S. Pat. No. 4,683,202).
[0103] A full-length clone encoding zcytor17 can be obtained by
conventional cloning procedures. Complementary DNA (cDNA) clones
are preferred, although for some applications (e.g., expression in
transgenic animals) it may be preferable to use a genomic clone, or
to modify a cDNA clone to include at least one genomic intron.
Methods for preparing cDNA and genomic clones are well known and
within the level of ordinary skill in the art, and include the use
of the sequence disclosed herein, or parts thereof, for probing or
priming a library. Expression libraries can be probed with
antibodies to zcytor17, receptor fragments, or other specific
binding partners.
[0104] The polynucleotides of the present invention can also be
synthesized using DNA synthesis machines. Currently the method of
choice is 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 polynucleotides
(60 to 80 bp) is technically straightforward and can be
accomplished by synthesizing the complementary strands and then
annealing them. However, for producing longer polynucleotides
(>300 bp), special strategies are usually employed, 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.
[0105] An alternative way to prepare a full-length gene is to
synthesize a specified set of overlapping oligonucleotides (40 to
100 nucleotides). After the 3' and 5' short overlapping
complementary regions (6 to 10 nucleotides) are annealed, large
gaps still remain, but the short base-paired regions are both long
enough and stable enough to hold the structure together. The gaps
are filled and the DNA duplex is completed via enzymatic DNA
synthesis by E. coli DNA polymerase I. After the enzymatic
synthesis is completed, the nicks are sealed with T4 DNA ligase.
Double-stranded constructs are sequentially linked to one another
to form the entire gene sequence which is verified by DNA sequence
analysis. See Glick and Pasternak, Molecular Biotechnology,
Principles & Applications of Recombinant DNA, (ASM Press,
Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53:
323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA
87:633-7, 1990. Moreover, other sequences are generally added that
contain signals for proper initiation and termination of
transcription and translation.
[0106] The present invention also provides reagents which will find
use in diagnostic applications. For example, the zcytor17lig gene,
a probe comprising zcytor17lig DNA or RNA or a subsequence thereof,
can be used to determine if the zcytor17lig gene is present on a
human chromosome, such as chromosome 12, or if a gene mutation has
occurred. Zcytor17lig is located at the 12q24.31 region of
chromosome 12 (Example 13). Detectable chromosomal aberrations at
the zcytor17lig gene locus include, but are not limited to,
aneuploidy, gene copy number changes, loss of heterozygosity (LOH),
translocations, insertions, deletions, restriction site changes and
rearrangements. Such aberrations can be detected using
polynucleotides of the present invention by employing molecular
genetic techniques, such as restriction fragment length
polymorphism (RFLP) analysis, short tandem repeat (STR) analysis
employing PCR techniques, and other genetic linkage analysis
techniques known in the art (Sambrook et al., ibid.; Ausubel et.
al., ibid.; Marian, Chest 108:255-65, 1995).
[0107] The precise knowledge of a gene's position can be useful for
a number of purposes, including: 1) determining if a sequence is
part of an existing contig and obtaining additional surrounding
genetic sequences in various forms, such as YACs, BACs or cDNA
clones; 2) providing a possible candidate gene for an inheritable
disease which shows linkage to the same chromosomal region; and 3)
cross-referencing model organisms, such as mouse, which may aid in
determining what function a particular gene might have.
[0108] One of skill in the art would recognize that the 12q24
region is frequently involved in gross genomic rearrangements,
including translocations, deletions, inversions, and duplications,
that are associated with various cancers. The Mitelman Database of
Chromosomal Aberrations in Cancer, at the Cancer Genome Anatomy
Project, National Institutes of Health, Bethesda, Md. located on
the Internet lists 199 cases of cancers with genomic rearrangements
involving 12q24. Of these, most are part of complex karyotypes with
other rearrangements; however, in some cases the rearrangement
involving 12q24 is the only genomic alteration. Given the
expression of the receptor for zcytor17lig on cells of lymphoid and
myeloid lineages, it is particularly significant to note that there
are at least 4 cases of myeloid leukemia reported in the literature
in which either translocation (2 cases: Yamagata et al, Cancer
Genet Cytogenet 97:90-93, 1997; Dunphy and Batanian, Cancer Genet
Cytogenet 114:51-57, 1999) or duplication (2 cases: Bonomi et al,
Cancer Genet Cytogenet 108:75-78, 1999) are the sole genomic
alteration. This suggests that a gene or genes residing within
12q24 could be directly involved in the malignant transformation of
these patients' cells. Inappropriate over expression of zcytor17lig
could contribute to malignant transformation by promoting aberrant
proliferation of receptor-bearing cells, through either autocrine
or paracrine mechanisms. Inhibition of zcytor17lig activity could
thus inhibit growth of such cells. Alternatively, a genomic
rearrangement resulting in inactivation of the zcytor17lig gene may
promote malignant transformation and/or metastasis by removing
zcytor17lig immunoregulatory functions. Indeed, a gene suppressing
metastasis in prostate cancer has been mapped to 12q24-qter
(Ichikawa et al, Asian J Androl 2:167-171, 2000). If zcytor17lig is
the gene within this region responsible for the suppression of
metastasis, then zcytor17lig itself may have therapeutic value in
the treatment of cancer.
[0109] A diagnostic could assist physicians in determining the type
of disease and appropriate associated therapy, or assistance in
genetic counseling. As such, the inventive anti-zcytor17lig
antibodies, polynucleotides, and polypeptides can be used for the
detection of zcytor17lig polypeptide, mRNA or anti-zcytor17lig
antibodies, thus serving as markers and be directly used for
detecting or genetic diseases or cancers, as described herein,
using methods known in the art and described herein. Further,
zcytor17lig polynucleotide probes can be used to detect
abnormalities or genotypes associated with chromosome 12q24.3
deletions and translocations associated with human diseases, or
other translocations involved with malignant progression of tumors
or other 12q24.3 mutations, which are expected to be involved in
chromosome rearrangements in malignancy; or in other cancers.
Similarly, zcytor17lig polynucleotide probes can be used to detect
abnormalities or genotypes associated with chromosome 12 trisomy
and chromosome loss associated with human diseases or spontaneous
abortion. Thus, zcytor17lig polynucleotide probes can be used to
detect abnormalities or genotypes associated with these
defects.
[0110] One of skill in the art would recognize that zcytor17lig
polynucleotide probes are particularly useful for diagnosis of
gross chromosomal abnormalities associated with loss of
heterogeneity (LOH), chromosome gain (e.g., trisomy),
translocation, DNA amplification, and the like. Translocations
within chromosomal locus 12q24.3 wherein the zcytor17lig gene is
located are known to be associated with human disease. For example,
12q24 deletions and translocations, duplications and trisomy are
associated with cancers as discussed above. Thus, since the
zcytor17lig gene maps to this critical region, zcytor17lig
polynucleotide probes of the present invention can be used to
detect abnormalities or genotypes associated with 12q24
translocation, deletion and trisomy, and the like, described
above.
[0111] As discussed above, defects in the zcytor17lig gene itself
may result in a heritable human disease state. Molecules of the
present invention, such as the polypeptides, antagonists, agonists,
polynucleotides and antibodies of the present invention would aid
in the detection, diagnosis prevention, and treatment associated
with a zcytor17lig genetic defect. In addition, zcytor17lig
polynucleotide probes can be used to detect allelic differences
between diseased or non-diseased individuals at the zcytor17lig
chromosomal locus. As such, the zcytor17lig sequences can be used
as diagnostics in forensic DNA profiling.
[0112] In general, the diagnostic methods used in genetic linkage
analysis, to detect a genetic abnormality or aberration in a
patient, are known in the art. Analytical probes will be generally
at least 20 nt in length, although somewhat shorter probes can be
used (e.g., 14-17 nt). PCR primers are at least 5 nt in length,
preferably 15 or more, more preferably 20-30 nt. For gross analysis
of genes, or chromosomal DNA, a zcytor17lig polynucleotide probe
may comprise an entire exon or more. Exons are readily determined
by one of skill in the art by comparing zcytor17lig sequences (SEQ
ID NO:1) with the genomic DNA for mouse zcytor17lig (SEQ ID NO:76).
In general, the diagnostic methods used in genetic linkage
analysis, to detect a genetic abnormality or aberration in a
patient, are known in the art. Most diagnostic methods comprise the
steps of (a) obtaining a genetic sample from a potentially diseased
patient, diseased patient or potential non-diseased carrier of a
recessive disease allele; (b) producing a first reaction product by
incubating the genetic sample with a zcytor17lig polynucleotide
probe wherein the polynucleotide will hybridize to complementary
polynucleotide sequence, such as in RFLP analysis or by incubating
the genetic sample with sense and antisense primers in a PCR
reaction under appropriate PCR reaction conditions; (iii)
visualizing the first reaction product by gel electrophoresis
and/or other known methods such as visualizing the first reaction
product with a zcytor17lig polynucleotide probe wherein the
polynucleotide will hybridize to the complementary polynucleotide
sequence of the first reaction; and (iv) comparing the visualized
first reaction product to a second control reaction product of a
genetic sample from wild type patient, or a normal or control
individual. A difference between the first reaction product and the
control reaction product is indicative of a genetic abnormality in
the diseased or potentially diseased patient, or the presence of a
heterozygous recessive carrier phenotype for a non-diseased
patient, or the presence of a genetic defect in a tumor from a
diseased patient, or the presence of a genetic abnormality in a
fetus or pre-implantation embryo. For example, a difference in
restriction fragment pattern, length of PCR products, length of
repetitive sequences at the zcytor17lig genetic locus, and the
like, are indicative of a genetic abnormality, genetic aberration,
or allelic difference in comparison to the normal wild type
control. Controls can be from unaffected family members, or
unrelated individuals, depending on the test and availability of
samples. Genetic samples for use within the present invention
include genomic DNA, mRNA, and cDNA isolated from any tissue or
other biological sample from a patient, which includes, but is not
limited to, blood, saliva, semen, embryonic cells, amniotic fluid,
and the like. The polynucleotide probe or primer can be RNA or DNA,
and will comprise a portion of SEQ ID NO:1, the complement of SEQ
ID NO:1, or an RNA equivalent thereof. Such methods of showing
genetic linkage analysis to human disease phenotypes are well known
in the art. For reference to PCR based methods in diagnostics 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).
[0113] Mutations associated with the zcytor17lig locus can be
detected using nucleic acid molecules of the present invention by
employing standard methods for direct mutation analysis, such as
restriction fragment length polymorphism analysis, short tandem
repeat analysis employing PCR techniques, amplification-refractory
mutation system analysis, single-strand conformation polymorphism
detection, RNase cleavage methods, denaturing gradient gel
electrophoresis, fluorescence-assisted mismatch analysis, and other
genetic analysis techniques known in the art (see, for example,
Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press,
Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis,
Molecular Diagnostics (Human Press, Inc. 1996), Elles (ed.)
Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996),
Landegren (ed.), Laboratory Protocols for Mutation Detection
(Oxford University Press 1996), Birren et al (eds.), Genome
Analysis, Vol 2: Detecting Genes (Cold Spring Harbor Laboratory
Press 1998), Dracopoli et at. (eds.), Current Protocols in Human
Genetics (John Wiley & Sons 1998), and Richards and Ward,
"Molecular Diagnostic Testing," in Principles of Molecular
Medicine, pages 83-88 (Humana Press, Inc. 1998). Direct analysis of
an zcytor17lig gene for a mutation can be performed using a
subject's genomic DNA. Methods for amplifying genomic DNA, obtained
for example from peripheral blood lymphocytes, are well-known to
those of skill in the art (see, for example, Dracopoli et at.
(eds.), Current Protocols in Human Genetics, at pages 7.1.6 to
7.1.7 (John Wiley & Sons 1998)).
[0114] The present invention further provides counterpart
polypeptides and polynucleotides 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 zcytor17
polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human zcytor17 can be cloned using
information and compositions provided by the present invention in
combination with conventional cloning techniques. For example, a
cDNA can be cloned using mRNA obtained from a tissue or cell type
that expresses zcytor17 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. A
zcytor17-encoding cDNA can then be isolated by a variety of
methods, such as by probing with a complete or partial human cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using PCR (Mullis,
supra.), using primers designed from the representative human
zcytor17 sequence disclosed herein. Within an additional method,
the 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 zcytor17 polypeptide. Similar techniques can also be
applied to the isolation of genomic clones.
[0115] A polynucleotide sequence for the mouse ortholog of human
zcytor17 has been identified and is shown in SEQ ID NO:116 and the
corresponding amino acid sequence shown in SEQ ID NO:117. Analysis
of the mouse zcytor17 polypeptide encoded by the DNA sequence of
SEQ ID NO: 116 revealed an open reading frame encoding 662 amino
acids (SEQ ID NO:117) comprising a predicted secretory signal
peptide of 45 amino acid residues (residue 1 (Met) to residue 45
(Ala) of SEQ ID NO:117), and a mature polypeptide of 617 amino
acids (residue46 (Val) to residue 662 (Cys) of SEQ ID NO:117).
Moreover, an additional Met residue, Met (28) can be used as a
starting methionine; comprising a second predicted secretory signal
peptide of 18 amino acid residues (residue 28 (Met) to residue 45
(Ala) of SEQ ID NO:117), and the same mature polypeptide of 617
amino acids (residue46 (Val) to residue 662 (Cys) of SEQ ID NO:117.
In addition to the WSXWS motif (SEQ ID NO:3) corresponding to
residues 224-228 of SEQ ID NO:117, the receptor comprises an
extracellular domain from residues 46 (Val) to 533 (Glu) of SEQ ID
NO:117) that includes a cytokine-binding domain of approximately
200 amino acid residues (residues 46 (Val) to 240 (Pro) of SEQ ID
NO:117) and a fibronectin III domain (residues 241 (His) to 533
(Glu) of SEQ ID NO:117); a CXW motif (residues 66 (Cys) to 68 (Trp)
of SEQ ID NO:117); a domain linker (residues 142 (Thr) to 145 (Pro)
of SEQ ID NO:117); a penultimate strand region (residues 207 (Phe)
to 215 (Arg) of SEQ ID NO:117); a transmembrane domain (residues
534 (Ile) to 550 (Ile) of SEQ ID NO:117); complete intracellular
signaling domain (residues 551 (Lys) to 662 (Cys) of SEQ ID NO:117)
which contains a "Box I" signaling site (residues 568 (Cys) to 574
(Pro) of SEQ ID NO:117), and a "Box II" signaling site (residues
628 (Glu) to 631 (leu) of SEQ ID NO:117). Conserved residues common
to class I cytokine receptors, are at residues 56 (Cys), 187 (Trp),
and 215 (Arg). A comparison of the human and mouse amino acid
sequences reveals that both the human and orthologous polypeptides
contain corresponding structural features described above (and,
see, FIG. 2). The mature sequence for the mouse zcytor17 begins at
Val.sub.46 (as shown in SEQ ID NO:117), which corresponds to
Ala.sub.33 (as shown in SEQ ID NO:5) in the human sequence. There
is about 61% identity between the mouse and human sequences over
the entire amino acid sequence corresponding to SEQ ID NO:5 and SEQ
ID NO: 117. The above percent identity was determined using a FASTA
program with ktup=1, gap opening penalty=12, gap extension
penalty=2, and substitution matrix=BLOSUM62, with other parameters
set as default. The corresponding polynucleotides encoding the
mouse zcytor17 polypeptide regions, domains, motifs, residues and
sequences described above are as shown in SEQ ID NO:116.
[0116] Moreover, a truncated soluble form of the mouse zcytor17
receptor polypeptide appears to be naturally expressed. A
polynucleotide sequence for a truncated soluble form of the mouse
zcytor17 receptor has been identified and is shown in SEQ ID NO:118
and the corresponding amino acid sequence shown in SEQ ID NO:119.
Analysis of the truncated soluble mouse zcytor17 polypeptide
encoded by the DNA sequence of SEQ ID NO:118 revealed an open
reading frame encoding 547 amino acids (SEQ ID NO:119) comprising a
predicted secretory signal peptide of 45 amino acid residues
(residue 1 (Met) to residue 45 (Ala) of SEQ ID NO:119), and a
mature polypeptide of 502 amino acids (residue46 (Val) to residue
547 (Val) of SEQ ID NO:119). Moreover, an additional Met residue,
Met (28) can be used as a starting methionine; comprising a second
predicted secretory signal peptide of 18 amino acid residues
(residue 28 (Met) to residue 45 (Ala) of SEQ ID NO:119), and the
same mature polypeptide of 502 amino acids (residue46 (Val) to
residue 547 (Val) of SEQ ID NO:119. In addition to the WSXWS motif
(SEQ ID NO:3) corresponding to residues 224-228 of SEQ ID NO:119,
the receptor comprises an extracellular domain from residues 46
(Val) to 533 (Trp) of SEQ ID NO:119) that includes a
cytokine-binding domain of approximately 200 amino acid residues
(residues 46 (Val) to 240 (Pro) of SEQ ID NO:119) and a fibronectin
III domain (residues 241 (His) to 533 (Trp) of SEQ ID NO:119); a
CXW motif (residues 66 (Cys) to 68 (Trp) of SEQ ID NO:119); a
domain linker (residues 142 (Thr) to 145 (Pro) of SEQ ID NO:119); a
penultimate strand region (residues 207 (Phe) to 215 (Arg) of SEQ
ID NO:119); and a C-terminal tail region (residues 534 (Leu) to 547
(Val). Conserved residues common to class I cytokine receptors, are
at residues 56 (Cys), 187 (Trp), and 215 (Arg). A comparison of the
human and mouse amino acid sequences, including the truncated
soluble mouse zcytor17, reveals that both the human and orthologous
polypeptides contain corresponding structural features described
above (and, see, FIG. 2). The corresponding polynucleotides
encoding the truncated soluble mouse zcytor17 polypeptide regions,
domains, motifs, residues and sequences described above are as
shown in SEQ ID NO:118.
[0117] Those skilled in the art will recognize that the sequences
disclosed in SEQ ID NO:110, SEQ ID NO:108 and SEQ ID NO:4 represent
alleles of human zcytor17 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 DNA sequence shown in SEQ ID NO:110, SEQ ID
NO:108 or SEQ ID NO:4, 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 SEQ ID NO:111, SEQ ID NO:109, SEQ ID
NO:5 SEQ ID NO:117 or SEQ ID NO:119. cDNAs generated from
alternatively spliced mRNAs, which retain the properties of the
zcytor17 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. For example, the short-form and long-form soluble zcytor17
receptors described above, and in SEQ ID NO:112 and SEQ ID NO:113
or SEQ ID NO:114 and SEQ ID NO:115 can be considered allelic or
splice variants of zcytor17.
[0118] The present invention also provides isolated zcytor17
polypeptides that are substantially similar to the polypeptides of
SEQ ID NO:111, SEQ ID NO:109 or SEQ ID NO:5 and their orthologs,
e.g., SEQ ID NO:117 and SEQ ID NO:119. The term "substantially
similar" is used herein to denote polypeptides having at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or greater
than 99% sequence identity to the sequences shown in SEQ ID NO:111,
SEQ ID NO:109 or SEQ ID NO:5 or their orthologs, e.g., SEQ ID
NO:117 and SEQ ID NO:119. Such polypeptides will more preferably be
at least 90% identical, and most preferably 95% or more identical
to SEQ ID NO:111, SEQ ID NO:109 and SEQ ID NO:5 or its orthologs.)
Percent sequence identity is determined by conventional methods.
See, for example, Altschul et al., Bull Math. Bio. 48: 603-616,
1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915-10919, 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 "blosum 62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 4 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: Total .times. .times. number .times. .times.
of .times. .times. identical .times. .times. matches [ length
.times. .times. of .times. .times. the .times. .times. longer
.times. .times. sequence .times. .times. plus .times. .times. the
number .times. .times. of .times. .times. gaps .times. .times.
introduced .times. .times. into .times. .times. .times. the .times.
.times. longer sequence .times. .times. in .times. .times. order
.times. .times. to .times. .times. align .times. .times. .times.
the .times. .times. two .times. .times. sequences ] .times. 100
##EQU1## TABLE-US-00004 TABLE 4 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
[0119] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0120] 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 variant zcytor17. 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).
[0121] Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:111, SEQ ID NO:109, SEQ ID NO:5, SEQ ID NO:117 and SEQ ID
NO:119) 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. Preferred
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=, and substitution matrix=BLOSUM62, with
other parameters set as default. 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).
[0122] 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 FASTA program parameters set as default.
[0123] The BLOSUM62 table (Table 4) 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 below), 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).
[0124] Variant zcytor17 polypeptides or substantially homologous
zcytor17 polypeptides are characterized as having one or more amino
acid substitutions, deletions or additions. These changes are
preferably of a minor nature, that is conservative amino acid
substitutions (see Table 5) and other substitutions that do not
significantly affect the folding or activity of the polypeptide;
small deletions, typically of one to about 30 amino acids; and
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide of up to
about 20-25 residues, or an affinity tag. The present invention
thus includes polypeptides that comprise a sequence that is at
least 80%, preferably at least 90%, and more preferably 95% or more
identical to the corresponding region of SEQ ID NO:111, SEQ ID
NO:109, SEQ ID NO:5, SEQ ID NO:117 or SEQ ID NO:119 excluding the
tags, extension, linker sequences and the like. Polypeptides
comprising affinity tags can further comprise a proteolytic
cleavage site between the zcytor17 polypeptide and the affinity
tag. Suitable sites include thrombin cleavage sites and factor Xa
cleavage sites. TABLE-US-00005 TABLE 5 Conservative amino acid
substitutions Basic: arginine lysine histidine Acidic: glutamic
acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine
isoleucine valine Aromatic: phenylalanine tryptophan tyrosine
Small: glycine alanine serine threonine methionine
[0125] The present invention provides an isolated an isolated
multimeric cytokine receptor comprising a polypeptide having at
least 90 percent sequence identity with SEQ ID NO:111, SEQ ID
NO:109, or SEQ ID NO:5; and at least a portion of at least one
class I cytokine receptor, wherein the multimeric cytokine receptor
binds to at least a portion of SEQ ID NO:2. The polypeptide having
at least 90 percent sequence identity with SEQ ID NO:111 may
include, for instance, amino acid residue 20 to amino acid residue
227 of SEQ ID NO:111, amino acid residue 20 to amino acid residue
227 of SEQ ID NO:519, amino acid residue 20 to amino acid residue
543 of SEQ ID NO:111, amino acid residue 544 to amino acid residue
732 of SEQ ID NO: III, amino acid residue 544 to amino acid residue
649 of SEQ ID NO:109, amino acid residue 20 to amino acid residue
732 of SEQ ID NO:111, amino acid residue 20 to amino acid residue
649 of SEQ ID NO:109, and combinations thereof. The at least a
portion of at least one class I cytokine receptor includes, for
example, OSMRbeta (SEQ ID NO:7) and/or WSX-1 (SEQ ID NO:9). For
example, the at least a portion of at least one class I cytokine
receptor comprising at least a portion of SEQ ID NO:7 may comprise
amino acid residue 28 to amino acid residue 429 of SEQ ID NO:7,
amino acid residue 35 to amino acid residue 137 of SEQ ID NO:7,
amino acid residue 240 to amino acid residue 342 of SEQ ID NO:7,
amino acid residue 348 to amino acid residue 429 of SEQ ID NO:7,
amino acid residue 28 to amino acid residue 739 of SEQ ID NO:7,
amino acid residue 28 to amino acid residue 761 of SEQ ID NO:7,
amino acid residue 762 to amino acid residue 979 of SEQ ID NO:7,
and/or combinations thereof. The multimeric cytokine receptor may
be a heterodimer, trimer, tetramer, pentamer, or the like. In
addition, the multimeric cytokine receptor may be soluble,
immobilized on a solid support, or membrane-bound. Optionally, the
multimeric cytokine receptor may antagonize an activity of SEQ ID
NO:2, such as inhibit or reduce the proliferation of hematopoietic,
immune, and/or inflammatory cells, or inhibit or reduce the
enhancement of the hematopoietic, immune, and/or inflammatory
process, or inhibit or reduce the differentiation of hematopoietic
cells, for instance lymphoid cells such as monocytic cells,
macrophages and/or T cells. The multimeric cytokine receptor of the
present invention may also comprise an affinity tag selected from
the group of polyhistidine, protein A, glutathione S transferase,
Glu-Glu, substance P, Flag.TM. peptide, streptavidin binding
peptide, and an immunoglobulin F, polypeptide.
[0126] The present invention also provides for a ligand/receptor
complex comprising a polypeptide which includes at least a portion
of SEQ ID NO:2; and a soluble multimeric cytokine receptor
comprising at least a portion of at least one polypeptide selected
from the group of SEQ ID NO:111, SEQ ID NO:109, SEQ ID NO:7, and
SEQ ID NO:9, wherein the polypeptide is attached to the soluble
multimeric cytokine receptor. The soluble multimeric cytokine
receptor may comprise the extracellular domain and/or transmembrane
domain of zcytor17 (SEQ ID NO:111), OSMRbeta (SEQ ID NO:7), and/or
WSX-1 (SEQ ID NO:9). For example, the soluble multimeric cytokine
receptor may comprise amino acid residue 20 to 227 to SEQ ID
NO:111, amino acid residue 20 to 519 to SEQ ID NO:111, amino acid
residue 20 to 543 to SEQ ID NO:111, amino acid residue 28 to 739 to
SEQ ID NO:7, amino acid residue 28 to 429 to SEQ ID NO:7, amino
acid residue 35 to 137 to SEQ ID NO:7, amino acid residue 240 to
342 to SEQ ID NO:7, amino acid residue 348 to 429 to SEQ ID NO:7,
or combinations thereof. The soluble multimeric cytokine receptor
may be a heterodimer, trimer, tetramer, pentamer, or the like. The
multimeric cytokine receptor of the present invention may also
comprise an affinity tag as described herein. The polypeptide of
the ligand/receptor complex may comprise amino acid residues of SEQ
ID NO:2 selected from the group of 38 to 152, 27 to 164, 24 to 164,
1 to 164, 38 to 52, 83 to 98, 104 to 117, 137 to 152, and
combinations thereof.
[0127] The ligand/receptor complex of the present may also comprise
a fusion protein. The fusion protein may comprise at least four
polypeptides, wherein the order of polypeptides from N-terminus to
C-terminus are a first polypeptide comprising amino acid residues
38-52 of SEQ ID NO:2; a first spacer of 6-27 amino acid residues; a
second polypeptide comprising amino acid residues selected from the
group of (a) IL-2 helix B residues of SEQ ID NO:183; (b) IL-4 helix
B residues 65-83 of SEQ ID NO:179; (c) IL-3 helix B residues 73-86
of SEQ ID NO:102; (d) GM-CSF helix B residues 72-81 of SEQ ID
NO:181; and (e) amino acid residues 83-98 of SEQ ID NO:2; a second
spacer of 5-11 amino acid residues; a third polypeptide comprising
amino acid residues selected from the group of (a) IL-2 helix C
residues 102-116 of SEQ ID NO:177; (b) IL-4 helix C residues 94-118
of SEQ ID NO:179; (c) IL-3 helix C residues 91-103 of SEQ ID
NO:102; (d) GM-CSF helix C residues 85-103 of SEQ ID NO:181; and
(e) amino acid residues 104-117 of SEQ ID NO:2; a third spacer of
3-29 amino acid residues; and a fourth polypeptide comprising amino
acid residues selected from the group of (a) IL-2 helix D residues
134-149 of SEQ ID NO:177; (b) IL-3 helix D residues 123-141 of SEQ
ID NO:102; (c) IL-4 helix D residues 133-151 of SEQ ID NO:179; (d)
GM-CSF helix D residues 120-131 of SEQ ID NO:181; and (e) amino
acid residues 137-152 of SEQ ID NO:2; and a multimeric cytokine
receptor comprising at least a portion of at least one polypeptide
selected from the group of SEQ ID NO: III, SEQ ID NO:109, SEQ ID
NO:7, and SEQ ID NO:9; wherein the fusion protein is attached to
the multimeric cytokine receptor.
[0128] Alternatively, the fusion protein may comprise at least four
polypeptides, wherein the order of polypeptides from N-terminus to
C-terminus are a first polypeptide comprising amino acid residues
selected from a group of (a) IL-2 helix A residues 27-48 of SEQ ID
NO:177; (b) IL-4 helix A residues 30-42 of SEQ ID NO:179; (c) IL-3
helix A residues 35-45 of SEQ ID NO:102; (d) GM-CSF helix A
residues 30-44 of SEQ ID NO:181; and (e) amino acids residues 38-52
of SEQ ID NO:2; a first spacer of 6-27 amino acid residues; a
second polypeptide comprising amino acid residues selected from the
group of (a) IL-2 helix B residues of SEQ ID NO:183; (b); IL-4
helix B residues 65-83 of SEQ ID NO:179; (c) IL-3 helix B residues
73-86 of SEQ ID NO:102; (d) GM-CSF helix B residues 72-81 of SEQ ID
NO:181; and (e) amino acid residues 83-98 of SEQ ID NO:2; a second
spacer of 5-11 amino acid residues; a third polypeptide comprising
amino acid residues selected from the group of (a) IL-2 helix C
residues 102-116 of SEQ ID NO:177; (b) IL-4 helix C residues 94-118
of SEQ ID NO:179; (c) IL-3 helix C residues 91-103 of SEQ ID
NO:102; (d) GM-CSF helix C residues 85-103 of SEQ ID NO:181; and
(e) amino acid residues 104-117 of SEQ ID NO:2; a third spacer of
3-29 amino acid residues; and a fourth polypeptide comprising amino
acid residues from 137-152 of SEQ ID NO:2; and a multimeric
cytokine receptor comprising at least a portion of at least one
polypeptide selected from the group of SEQ ID NO:111, SEQ ID
NO:109, SEQ ID NO:7, and SEQ ID NO:9; wherein the fusion protein is
attached to the multimeric cytokine receptor. A multimeric cytokine
receptor may comprise at least one of the following polypeptides of
SEQ ID NO: 111, SEQ ID NO:109, SEQ ID NO:7, SEQ ID NO:9, or the
extracellular domains thereof. The ligand/receptor complex can be
soluble and may additionally include an affinity tag as described
herein.
[0129] The present invention also provides an isolated and purified
polynucleotide that encodes a polypeptide comprising an amino acid
sequence having at least 90 percent sequence identity with SEQ ID
NO:1 .mu.l, SEQ ID NO:109, or SEQ ID NO:5, wherein the polypeptide
and at least a portion of at least one class I cytokine receptor
form a multimeric cytokine receptor, and wherein the multimeric
cytokine receptor binds to at least a portion of SEQ ID NO:2. The
present invention also provides an isolated and purified
polynucleotide that encodes a polypeptide comprising at least a
portion of at least one of SEQ ID NO:111, SEQ ID NO:109, or SEQ ID
NO:5, wherein the polypeptide and at least a portion of a class I
cytokine receptor form a multimeric cytokine receptor, and wherein
the multimeric cytokine receptor binds to at least a portion of SEQ
ID NO:2. The polynucleotide may encode a polypeptide that is
included in a soluble multimeric cytokine receptor and that may
also include an affinity tag as described herein. The polypeptide
may comprise, for example, amino acid residue 20 to 227 to SEQ ID
NO:111, amino acid residue 20 to 519 to SEQ ID NO: 111, amino acid
residue 20 to 543 to SEQ ID NO:111, and/or combinations thereof. In
addition, the at least a portion of at least one class I cytokine
receptor may comprise, for example, amino acid residue 28 to 739 to
SEQ ID NO:7, amino acid residue 28 to 429 to SEQ ID NO:7, amino
acid residue 35 to 137 to SEQ ID NO:7, amino acid residue 240 to
342 to SEQ ID NO:7, amino acid residue 348 to 429 to SEQ ID NO:7,
and/or combinations thereof. The soluble multimeric cytokine
receptor may be a heterodimer, trimer, tetramer, pentamer, or the
like. The at least a portion of SEQ ID NO:2 may include, for
instance amino acid residues of SEQ ID NO:2 selected from the group
of 38 to 152, 27 to 164, 24 to 164, 1 to 164, 38 to 52, 83 to 98,
104 to 117, 137 to 152, and combinations thereof. Optionally, the
multimeric cytokine receptor may antagonize an activity of SEQ ID
NO:2 as described herein.
[0130] The present invention further provides a variety of other
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a zcytor17 polypeptide
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. Immunoglobulin-zcytor17 polypeptide fusions can be
expressed in genetically engineered cells to produce a variety of
multimeric zcytor17 analogs. Auxiliary domains can be fused to
zcytor17 polypeptides to target them to specific cells, tissues, or
macromolecules (e.g., collagen). A zcytor17 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. For
example, one or more domains from zcytor17 soluble receptor can be
joined to a soluble cytokine receptor, such as OSMRbeta and/or
WSX-1, which may enhance their biological properties or efficiency
of production. Additionally, the soluble multimeric cytokine
receptor may further include an affinity tag. An affinity tag can
be, for example, a tag selected from the group of polyhistidine,
protein A, glutathione S transferase, Glu-Glu, substance P,
Flag.TM. peptide, streptavidin binding peptide, and an
immunoglobulin F.sub.c polypeptide.
[0131] 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 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 259:806-9, 1993; and Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-9, 1993). 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. 271:19991-8, 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. 33:7470-7476, 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. 2:395-403, 1993).
[0132] 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 zcytor17 amino acid residues.
[0133] 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: 1081-5, 1989; Bass
et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991). 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 (e.g. ligand binding and signal
transduction) as disclosed below to identify amino acid residues
that are critical to the activity of the molecule. See also, Hilton
et al., J. Biol. Chem. 271:4699-4708, 1996. Sites of
ligand-receptor, protein-protein or other biological interaction
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 255:306-312, 1992; Smith
et al., J. Mol. Biol 224:899-904, 1992; Wlodaver et al., FEBS Lett.
309:59-64, 1992. The identities of essential amino acids can also
be inferred from analysis of homologies with related receptors.
[0134] Determination of amino acid residues that are within regions
or domains that are critical to maintaining structural integrity
can be determined. Within these regions one can determine specific
residues that will be more or less tolerant of change and maintain
the overall tertiary structure of the molecule. Methods for
analyzing sequence structure include, but are not limited to,
alignment of multiple sequences with high amino acid or nucleotide
identity and computer analysis using available software (e.g., the
Insight II viewer and homology modeling tools; MSI, San Diego,
Calif.), secondary structure propensities, binary patterns,
complementary packing and buried polar interactions (Barton,
Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al.,
Current Opin. Struct. Biol. 6:3-10, 1996). In general, when
designing modifications to molecules or identifying specific
fragments determination of structure will be accompanied by
evaluating activity of modified molecules.
[0135] Amino acid sequence changes are made in zcytor17
polypeptides so as to minimize disruption of higher order structure
essential to biological activity. For example, when the zcytor17
polypeptide comprises one or more helices, changes in amino acid
residues will be made so as not to disrupt the helix geometry and
other components of the molecule where changes in conformation
abate some critical function, for example, binding of the molecule
to its binding partners. The effects of amino acid sequence changes
can be predicted by, for example, computer modeling as disclosed
above or determined by analysis of crystal structure (see, e.g.,
Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Other
techniques that are well known in the art compare folding of a
variant protein to a standard molecule (e.g., the native protein).
For example, comparison of the cysteine pattern in a variant and
standard molecules can be made. Mass spectrometry and chemical
modification using reduction and alkylation provide methods for
determining cysteine residues which are associated with disulfide
bonds or are free of such associations (Bean et al., Anal. Biochem.
201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and
Patterson et al., Anal. Chem. 66:3727-3732, 1994). It is generally
believed that if a modified molecule does not have the same
disulfide bonding pattern as the standard molecule folding would be
affected. Another well known and accepted method for measuring
folding is circular dichrosism (CD). Measuring and comparing the CD
spectra generated by a modified molecule and standard molecule is
routine (Johnson, Proteins 7:205-214, 1990). Crystallography is
another well known method for analyzing folding and structure.
Nuclear magnetic resonance (NMR), digestive peptide mapping and
epitope mapping are also known methods for analyzing folding and
structural similarities between proteins and polypeptides (Schaanan
et al., Science 257:961-964, 1992).
[0136] A Hopp/Woods hydrophilicity profile of the zcytor17 protein
sequence as shown in SEQ ID NO:2, SEQ ID NO:46, SEQ ID NO:54, SEQ
ID NO:57 and SEQ ID NO:93 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).
See, FIG. 1. 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 zcytor17, hydrophilic regions include
amino acid residues 43 through 48 of SEQ ID NO:2 and SEQ ID NO:46
(residues 56 through 61 of SEQ ID NO:54), amino acid residues 157
through 162 of SEQ ID NO:2 and SEQ ID NO:46 (residues 170 through
175 of SEQ ID NO:54), amino acid residues 158 through 163 of SEQ ID
NO:2 and SEQ ID NO:46 (residues 171 through 176 of SEQ ID NO:54),
amino acid residues 221 through 226 of SEQ ID NO:2 and SEQ ID NO:46
(residues 234 through 239 of SEQ ID NO:54), and amino acid residues
426 through 431 of SEQ ID NO:2 and SEQ ID NO:46 (residues 439
through 444 of SEQ ID NO:54).
[0137] Those skilled in the art will recognize that hydrophilicity
or hydrophobicity will be taken into account when designing
modifications in the amino acid sequence of a zcytor17 polypeptide,
so as not to disrupt the overall structural and biological profile.
Of particular interest for replacement are hydrophobic residues
selected from the group consisting of Val, Leu and Ile or the group
consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example,
residues tolerant of substitution could include such residues as
shown in SEQ ID NO:2, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:57 and
SEQ ID NO:93. However, Cysteine residues would be relatively
intolerant of substitution.
[0138] The identities of essential amino acids can also be inferred
from analysis of sequence similarity between class I cytokine
receptor family members with zcytor17. Using methods such as
"FASTA" analysis described previously, regions of high similarity
are identified within a family of proteins and used to analyze
amino acid sequence for conserved regions. An alternative approach
to identifying a variant zcytor17 polynucleotide on the basis of
structure is to determine whether a nucleic acid molecule encoding
a potential variant zcytor17 polynucleotide can hybridize to a
nucleic acid molecule having the nucleotide sequence of SEQ ID
NO:1, SEQ ID NO:45 or SEQ ID NO:53, as discussed above.
[0139] Other methods of identifying essential amino acids in the
polypeptides of the present invention are 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. Natl. 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 as
disclosed below 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).
[0140] The present invention also includes a multimeric cytokine
receptor which includes functional fragments of zcytor17
polypeptides and nucleic acid molecules encoding such functional
fragments. A "functional" zcytor17 or fragment thereof defined
herein is characterized by its ability to mediate proliferative or
differentiating activity, by its ability to induce or inhibit
specialized cell functions, or by its ability to bind specifically
to an anti-zcytor17 antibody or zcytor17 ligand (either soluble or
immobilized). Moreover, functional fragments also include the
signal peptide, intracellular signaling domain, and the like. As
previously described herein, zcytor17 is characterized by a class I
cytokine receptor structure. Thus, the present invention further
provides fusion proteins encompassing: (a) polypeptide molecules
comprising an extracellular domain, cytokine-binding domain, or
intracellular domain described herein; and (b) functional fragments
comprising one or more of these domains. The other polypeptide
portion of the fusion protein may comprise at least a portion of
one or more of another class I cytokine receptor, for example,
gp130, LIF, IL-112, WSX-1, IL-2 receptor .beta.-subunit and the
.beta.-common receptor (i.e., IL3, IL-5, and GM-CSF receptor
.beta.-subunits), or by a non-native and/or an unrelated secretory
signal peptide that facilitates secretion of the fusion
protein.
[0141] Routine deletion analyses of nucleic acid molecules can be
performed to obtain functional fragments of a nucleic acid molecule
that encodes a zcytor17 polypeptide. As an illustration, DNA
molecules having the nucleotide sequence of SEQ ID NO:1, SEQ ID
NO:45 or SEQ ID NO:53 or fragments thereof, can be digested with
Bal31 nuclease to obtain a series of nested deletions. These DNA
fragments are then inserted into expression vectors in proper
reading frame, and the expressed polypeptides are isolated and
tested for zcytor17 activity, or for the ability to bind
anti-zcytor17 antibodies or zcytor17 ligand. One alternative to
exonuclease digestion is to use oligonucleotide-directed
mutagenesis to introduce deletions or stop codons to specify
production of a desired zcytor17 fragment. Alternatively,
particular fragments of a zcytor17 polynucleotide can be
synthesized using the polymerase chain reaction.
[0142] Standard methods for identifying functional domains are
well-known to those of skill in the art. For example, 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
(ijhoff 1987); Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation 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).
[0143] 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 241:53-57, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 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. 30:10832-10837, 1991; Ladner
et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO
92/062045) and region-directed mutagenesis (Derbyshire et al., Gene
46:145, 1986; Ner et al., DNA 7:127, 1988).
[0144] Variants of the disclosed zcytor17 DNA and polypeptide
sequences can be generated through DNA shuffling as disclosed by
Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci.
USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly,
variant DNAs 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 DNAs, such as
allelic variants or DNAs 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.
[0145] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized zcytor17 receptor polypeptides in host cells.
Preferred assays in this regard include cell proliferation assays
and biosensor-based ligand-binding assays, which are described
below. Mutagenized DNA molecules that encode active receptors or
portions thereof (e.g., ligand-binding fragments, signaling
domains, and the like) 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.
[0146] The present invention also provides a novel multimeric
cytokine receptor in which a segment comprising at least a portion
of one or more of the domains of zcytor17, for instance, secretory,
extracellular, transmembrane, and intracellular, is fused to
another polypeptide, for example, an extracellular domain of a
class I cytokine receptor, such as OSMRbeta and/or WSX-1. Fusion is
preferably done by splicing at the DNA level to allow expression of
chimeric molecules in recombinant production systems. The resultant
molecules are then assayed for such properties as improved
solubility, improved stability, prolonged clearance half-life,
improved expression and secretion levels, and pharmacodynamics.
Such a multimeric cytokine receptor may further comprise additional
amino acid residues (e.g., a polypeptide linker) between the
component proteins or polypeptides. A domain linker may comprise a
sequence of amino acids from about 3 to about 20 amino acids long,
from about 5 to 15 about amino acids long, from about 8 to about 12
amino acids long, and about 10 amino acids long. One function of a
linker is to separate the active protein regions to promote their
independent bioactivity and permit each region to assume its
bioactive conformation independent of interference from its
neighboring structure.
[0147] Using the methods discussed herein, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptide
fragments or variants of SEQ ID NO:111, SEQ ID NO:109, SEQ ID NO:5,
SEQ ID NO:117 and SEQ ID NO:119 that retain the signal transduction
or ligand binding activity. For example, one can make a zcytor17
"soluble receptor" by preparing a variety of polypeptides that are
substantially homologous to the cytokine-binding domain (residues
20 (Ala) to 227 (Pro) of SEQ ID NO:111 and SEQ ID NO:109; residues
33 (Ala) to 240 (Pro) of SEQ ID NO:5), the extracellular domain
(residues 20 (Ala) to 519 (Glu) of SEQ ID NO:111 and SEQ ID NO:109;
residues 33 (Ala) to 532 (Glu) of SEQ ID NO:5), or allelic variants
or species orthologs thereof (e.g., see SEQ ID NO:117 and SEQ ID
NO:119 and functional fragments thereof as described herein)) and
retain ligand-binding activity of the wild-type zcytor17 protein.
Moreover, variant zcytor17 soluble receptors such as those shown in
SEQ ID NO:113 and SEQ ID NO:115 can be isolated. Such polypeptides
may include additional amino acids from, for example, part or all
of the transmembrane and intracellular domains. Such polypeptides
may also include additional polypeptide segments as generally
disclosed herein such as labels, affinity tags, and the like.
[0148] For any zcytor17 polypeptide, including variants, soluble
receptors, and fusion polypeptides or 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.
[0149] The zcytor17 multimeric cytokine receptors of the present
invention, including full-length polypeptides, biologically active
fragments, and fusion polypeptides, can be produced in genetically
engineered host cells according to conventional techniques.
Suitable host cells are those cell types that can be transformed or
transfected with exogenous DNA and grown in culture, and include
bacteria, fungal cells, and cultured higher eukaryotic cells.
Eukaryotic cells, particularly cultured cells of multicellular
organisms, are preferred. Techniques for manipulating cloned DNA
molecules and introducing exogenous DNA into a variety of host
cells are disclosed by Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, and Ausubel et al., eds., Current
Protocols in Molecular Biology, John Wiley and Sons, Inc., NY,
1987.
[0150] The present invention also provides an expression vector
comprising an isolated and purified DNA molecule including the
following operably linked elements: a first transcription promoter,
a first DNA segment encoding a polypeptide having at least 90
percent sequence identity with SEQ ID NO:11, and a first
transcription terminator; and a second transcription promoter, a
second DNA segment encoding at least a portion of a class I
cytokine receptor, and a second transcription terminator; wherein
the polypeptide and the class I cytokine receptor form a multimeric
cytokine receptor; and wherein the multimeric cytokine receptor
binds to at least a portion of SEQ ID NO:2. The DNA molecule may
further comprise a secretory signal sequence operably linked to the
first and second DNA segments. The multimeric cytokine receptor may
be soluble and/or may further comprise an affinity tag as described
herein. In addition, the multimeric cytokine receptor may
antagonize an activity of SEQ ID NO:2 as described herein. The at
least at portion of a class I cytokine receptor may comprise
portions of SEQ ID NO:7 and/or SEQ ID NO:9, such as, for instance,
amino acid residue 28 to amino acid residue 429 of SEQ ID NO:7,
amino acid residue 35 to amino acid residue 137 of SEQ ID NO:7,
amino acid residue 240 to amino acid residue 342 of SEQ ID NO:7,
amino acid residue 348 to amino acid residue 429 of SEQ ID NO:7,
amino acid residue 28 to amino acid residue 739 of SEQ ID NO:7,
amino acid residue 28 to amino acid residue 761 of SEQ ID NO:7,
amino acid residue 762 to amino acid residue 979 of SEQ ID NO:7, or
combinations thereof. The present invention also provides a
cultured cell containing the above-described expression vector.
[0151] In general, a DNA sequence, for example, encoding a zcytor17
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.
[0152] To direct, for example, a zcytor17 polypeptide into the
secretory pathway of a host cell, a secretory signal sequence (also
known as a leader sequence, prepro sequence or pre sequence) is
provided in the expression vector. The secretory signal sequence
may be that of zcytor17, or may be derived from another secreted
protein (e.g., t-PA) or synthesized de novo. The secretory signal
sequence is operably linked to the zcytor17 DNA sequence, i.e., 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 DNA sequence encoding the polypeptide
of interest, although certain secretory signal sequences may be
positioned elsewhere in the DNA 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).
[0153] Alternatively, the secretory signal sequence contained in
the polypeptides of the present invention is used to direct other
polypeptides into the secretory pathway. The present invention
provides for such fusion polypeptides. A signal fusion polypeptide
can be made wherein a secretory signal sequence derived from amino
acid 1 (Met) to amino acid 19 (Ala) of SEQ ID NO:111 and SEQ ID
NO:109, or wherein a secretory signal sequence derived from amino
acid 1 (Met) to amino acid 32 (Ala) of SEQ ID NO:5, or amino acid 1
(Met) to amino acid 45 (Ala) of SEQ ID NO: 117 or SEQ ID NO:119),
or amino acid 28 (Met) to residue 45 (Ala) of SEQ ID NO:117 or SEQ
ID NO:119), 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 fusions may be used in vivo or
in vitro to direct peptides through the secretory pathway.
[0154] The present invention also provides a cultured cell
comprising a first expression vector comprising a DNA molecule
containing the following operably linked elements: a transcription
promoter, a DNA segment encoding a polypeptide having at least 90
percent sequence identity with SEQ ID NO:111, and a transcription
terminator; and a second expression vector comprising a
transcription promoter, a DNA segment encoding at least a portion
of a class I cytokine receptor, and a transcription terminator;
wherein the polypeptide and the class I cytokine receptor form a
multimeric cytokine receptor. The first and second expression
vectors may further comprise a secretory signal sequence operably
linked to the first and second DNA segments. The multimeric
cytokine receptor may be soluble, may be a heterodimer, and/or may
further comprise an affinity tag as described herein. In addition,
the multimeric cytokine receptor may antagonize an activity of SEQ
ID NO:2 as described herein. The at least at portion of a class I
cytokine receptor may comprise portions of SEQ ID NO:7 and/or SEQ
ID NO:9, such as, for instance, amino acid residue 28 to amino acid
residue 429 of SEQ ID NO:7, amino acid residue 35 to amino acid
residue 137 of SEQ ID NO:7, amino acid residue 240 to amino acid
residue 342 of SEQ ID NO:7, amino acid residue 348 to amino acid
residue 429 of SEQ ID NO:7, amino acid residue 28 to amino acid
residue 739 of SEQ ID NO:7, amino acid residue 28 to amino acid
residue 761 of SEQ ID NO:7, amino acid residue 762 to amino acid
residue 979 of SEQ ID NO:7, or combinations thereof.
[0155] Cultured mammalian cells are suitable hosts within the
present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et
al., ibid.), and liposome-mediated transfection (Hawley-Nelson et
al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and
viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989;
Wang and Finer, Nature Med. 2:714-716, 1996). The production of
recombinant polypeptides in cultured mammalian cells is disclosed,
for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et
al., U.S. Pat. No. 4,784,950; Palmiter et al, U.S. Pat. No.
4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured
mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC
No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL
10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virot.
36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No.
CCL 61) cell lines. Additional suitable cell lines are known in the
art and available from public depositories such as the American
Type Culture Collection, Rockville, Md. In general, strong
transcription promoters are preferred, such as promoters from SV-40
or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other
suitable promoters include those from metallothionein genes (U.S.
Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late
promoter.
[0156] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. 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 preferred 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.
Alternative 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.
[0157] Other higher eukaryotic cells can also be used as hosts,
including plant cells, insect cells and avian cells. The use of
Agrobacterium rhizogenes as a vector for expressing genes in plant
cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore)
11:47-58, 1987. Transformation of insect cells and production of
foreign polypeptides therein is disclosed by Guarino et al., U.S.
Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells
can be infected with recombinant baculovirus, commonly derived from
Autographa californica nuclear polyhedrosis virus (AcNPV). See,
King, L. A. and Possee, R. D., The Baculovirus Expression System: A
Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. et
al., Baculovirus Expression Vectors: A Laboratory Manual, New York,
Oxford University Press., 1994; and, Richardson, C. D., Ed.,
Baculovirus Expression Protocols. Methods in Molecular Biology,
Totowa, N.J., Humana Press, 1995. A second method of making
recombinant zcytor17 baculovirus utilizes a transposon-based system
described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79,
1993). This system, which utilizes transfer vectors, is sold in the
Bac-to-Bac.TM. kit (Life Technologies, Rockville, Md.). This system
utilizes a transfer vector, pFastBac1.TM. (Life Technologies)
containing a Tn7 transposon to move the DNA encoding the zcytor17
polypeptide into a baculovirus genome maintained in E. coli as a
large plasmid called a "bacmid." See, Hill-Perkins, M. S. and
Possee, R. D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J
Gen Virol 75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport,
B., J Biol Chem 270:1543-9, 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 zcytor17 polypeptide, for
example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc.
Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known in the
art, a transfer vector containing zcytor17 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 isolated, using
common techniques, and used to transfect Spodoptera frugiperda
cells, e.g., Sf9 cells. Recombinant virus that expresses zcytor17
is subsequently produced. Recombinant viral stocks are made by
methods commonly used in the art.
[0158] The recombinant virus is used to infect host cells,
typically a cell line derived from the fall armyworm, Spodoptera
frugiperda. See, in general, Glick and Pasternak, Molecular
Biotechnology: Principles and Applications of Recombinant DNA, ASM
Press, Washington, D.C., 1994. Another suitable cell line is the
High FiveO.TM. cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
are used to grow and 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.
Procedures used are generally described in available laboratory
manuals (King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et
al., ibid.; Richardson, C. D., ibid.). Subsequent purification of
the zcytor17 polypeptide from the supernatant can be achieved using
methods described herein.
[0159] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. 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 preferred 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.
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.
Transformation systems for other yeasts, including Hansenula poly
orpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanotica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol
132:3459-3465, 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.
[0160] The use of Pichia methanolica as host for the production of
recombinant proteins is disclosed in WIPO Publications 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, it is preferred that the promoter and terminator in
the plasmid 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 preferred 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), 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, it is preferred to use
host cells in which both methanol utilization genes (AUG1 and AUG2)
are deleted. For production of secreted proteins, host cells
deficient in vacuolar protease genes (PEP4 and PRB1) are preferred.
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. It is preferred to transform P. methanolica
cells 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.
[0161] Prokaryotic host cells, including strains of the bacteria
Escherichia coli, Bacillus and other genera are also useful host
cells within the present invention. Techniques for transforming
these hosts and expressing foreign DNA sequences cloned therein are
well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a zcytor17 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.
[0162] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell. P. methanotica cells
are cultured in a medium comprising adequate sources of carbon,
nitrogen and trace nutrients at a temperature of about 25.degree.
C. to 35.degree. C. Liquid cultures are provided with sufficient
aeration by conventional means, such as shaking of small flasks or
sparging of fermentors. A preferred culture medium for P.
methanotica is YEPD (2% D-glucose, 2% Bacto.TM. Peptone (Difco
Laboratories, Detroit, Mich.), 1% Bacto.TM. yeast extract (Difco
Laboratories), 0.004% adenine and 0.006% L-leucine).
[0163] Within one aspect of the present invention, a zcytor17
multimeric cytokine receptor (including transmembrane and
intracellular domains) is produced by a cultured cell, and the cell
is used to screen for ligands for the receptor, including the
natural ligand (SEQ ID NO:2), 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.
[0164] Mammalian cells suitable for use in expressing the novel
receptors of the present invention and transducing a
receptor-mediated signal include cells that express a 0-subunit,
such as gp130, and cells that co-express gp130 and LIF receptor
(Gearing et al., EMBO J. 10:2839-2848, 1991; Gearing et al., U.S.
Pat. No. 5,284,755). In this regard it is generally preferred to
employ a cell that is responsive to other cytokines that bind to
receptors in the same subfamily, such as IL-6 or LIF, because such
cells will contain the requisite signal transduction pathway(s).
Preferred cells of this type include BaF3 cells (Palacios and
Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol Cell
Biol 6: 4133-4135, 1986), the human TF-1 cell line (ATCC number
CRL-2003) and the DA-1 cell line (Branch et al., Blood 69:1782,
1987; Broudy et al., Blood 75:1622-1626, 1990). In the alternative,
suitable host cells can be engineered to produce a .beta.-subunit
or other cellular component needed for the desired cellular
response. For example, the murine cell line BaF3 (Palacios and
Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell.
Biol. 6: 4133-4135, 1986), a baby hamster kidney (BHK) cell line,
or the CTLL-2 cell line (ATCC TIB-214) can be transfected to
express the mouse gp130 subunit, or mouse gp130 and LIF receptor,
in addition to zcytor17. It is generally preferred to use a host
cell and receptor(s) from the same species, however this approach
allows cell lines to be engineered to express multiple receptor
subunits from any species, thereby overcoming potential limitations
arising from species specificity. In the alternative, species
homologs of the human 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 IL-3, can thus be engineered to become
dependent upon a zcytor17 ligand or anti-zcytor17 antibody.
[0165] Cells expressing functional zcytor17 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 the 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 reduction or metabolic breakdown of Alymar Bluem
(AccuMed, Chicago, Ill.) or
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, STAT
or SRE (see, for example, 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. 269:19094-29101, 1994; Schenborn
and Goiffin, Promega Notes 41:11, 1993). Luciferase 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-
or tissue-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 cell 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, retransfection,
subculturing, and re-assay of positive cells to isolate a clonal
cell line expressing the ligand. Media samples conditioned by
kidney, liver, spleen, thymus, other lymphoid tissues, or T-cells
are preferred sources of ligand for use in screening
procedures.
[0166] The present invention also provides a method of producing a
multimeric cytokine receptor. The method includes culturing a cell
as described herein, and isolating the multimeric cytokine receptor
produced by the cell.
[0167] The present invention also provides a method for detecting a
multiple cytokine receptor ligand in a test sample. The method
includes contacting the test sample with a multimeric cytokine
receptor comprising a polypeptide comprising amino acid residue 20
to amino acid residue 227 of SEQ ID NO:111 or SEQ ID NO:109 (amino
acid residue 33 to amino acid residue 240 of SEQ ID NO:5), and at
least a portion of at least one class I cytokine receptor; and
detecting the binding of the multimer cytokine receptor to the
ligand in the test sample. The at least a portion of at least one
class I cytokine receptor can include, for example, a portion of
SEQ ID NO:9 and/or a portion of SEQ ID NO:7, such as, for instance,
amino acid residue 28 to amino acid residue 429 of SEQ ID NO:7,
amino acid residue 35 to amino acid residue 137 of SEQ ID NO:7,
amino acid residue 240 to amino acid residue 342 of SEQ ID NO:7,
amino acid residue 348 to amino acid residue 429 of SEQ ID NO:7,
amino acid residue 28 to amino acid residue 739 of SEQ ID NO:7,
and/or combinations thereof.
[0168] A natural ligand for a zcytor17 multimeric cytokine receptor
of the present invention can also be identified by mutagenizing a
cytokine-dependent cell line expressing zcytor17 and culturing it
under conditions that select for autocrine growth. See WIPO
publication WO 95/21930. Within a typical procedure, cells
expressing zcytor17 are mutagenized, such as with EMS. The cells
are then allowed to recover in the presence of the required
cytokine, then transferred to a culture medium lacking the
cytokine. Surviving cells are screened for the production of a
ligand for zcytor17 multimeric cytokine receptor, such as by adding
soluble receptor polypeptide comprising the zcytor17
cytokine-binding domain and at least a portion of a class I
cytokine receptor, such as the cytokine-binding domain of OSMRbeta
(SEQ ID NO:7) and/or WSX-1 (SEQ ID NO:9), as described herein to
the culture medium to compete against the ligand or by assaying
conditioned media on wild-type cells compared to transfected cells
expressing the zcytor17 multimeric cytokine receptor. Preferred
cell lines for use within this method include cells that are
transfected to express gp130 or gp130 in combination with LIF
receptor. Preferred such host cell lines include transfected CTLL-2
cells (Gillis and Smith, Nature 268:154-156, 1977) and transfected
BaF3 cells.
[0169] Moreover, a secretion trap method employing zcytor17 soluble
multimeric cytokine receptor can be used to isolate a zcytor17
ligand, such as SEQ ID NO:2 (Aldrich, et al, Cell 87: 1161-1169,
1996). A cDNA expression library prepared from a known or suspected
ligand source is transfected into COS-7 cells. The cDNA library
vector generally has an SV40 origin for amplification in COS-7
cells, and a CMV promoter for high expression. The transfected
COS-7 cells are grown in a monolayer and then fixed and
permeabilized. Tagged or biotin-labeled zcytor17 soluble multimeric
cytokine receptor, described herein, is then placed in contact with
the cell layer and allowed to bind cells in the monolayer that
express an anti-complementary molecule, i.e., a zcytor17 ligand. A
cell expressing a ligand will thus be bound with receptor
molecules. An anti-tag antibody (anti-Ig for Ig fusions, M2 or
anti-FLAG for FLAG-tagged fusions, streptavidin, anti-Glu-Glu tag,
and the like) which is conjugated with horseradish peroxidase (HRP)
is used to visualize these cells to which the tagged or
biotin-labeled zcytor17 soluble multimeric cytokine receptor has
bound. The HRP catalyzes deposition of a tyramide reagent, for
example, tyramide-FITC. A commercially-available kit can be used
for this detection (for example, Renaissance TSA-Direct.TM. Kit;
NEN Life Science Products, Boston, Mass.). Cells which express
zcytor17 multimeric cytokine receptor ligand will be identified
under fluorescence microscopy as green cells and picked for
subsequent cloning of the ligand using procedures for plasmid
rescue as outlined in Aldrich, et al, supra., followed by
subsequent rounds of secretion trap assay, or conventional
screening of cDNA library pools, until single clones are
identified.
[0170] As a multimeric receptor complex, the activity of zcytor17
polypeptide can be measured 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.TM. 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, H. M. et al.,
Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol
228:84-108, 1997; Arimilli, S. et al., J. Immunol Meth. 212:49-59,
1998; Van Liefde, I. Et al., Eur. J. Pharmacol. 346:87-95, 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 the
zcytor17 polypeptide. Preferably, the microphysiometer is used to
measure responses of a zcytor17-expressing eukaryotic cell,
compared to a control eukaryotic cell that does not express
zcytor17 polypeptide. Zcytor17-expressing eukaryotic cells comprise
cells into which zcytor17 has been transfected or infected via
adenovirus vector, and the like, as described herein, creating a
cell that is responsive to zcytor17-modulating stimuli, or are
cells naturally expressing zcytor17, such as zcytor17-expressing
cells derived from lymphoid, spleen, thymus tissue or PBLs.
Differences, measured by an increase or decrease in extracellular
acidification, in the response of cells expressing zcytor17,
relative to a control, are a direct measurement of
zcytor17-modulated cellular responses. Moreover, such
zcytor17-modulated responses can be assayed under a variety of
stimuli. Also, using the microphysiometer, there is provided a
method of identifying agonists and antagonists of zcytor17
multimeric cytokine receptor, comprising providing cells expressing
a zcytor17 multimeric cytokine receptor, culturing a first portion
of the cells in the absence of a test compound, culturing a second
portion of the cells in the presence of a test compound, and
detecting an increase or a decrease in a cellular response of the
second portion of the cells as compared to the first portion of the
cells. Antagonists and agonists, including the natural ligand for
zcytor17 multimeric cytokine receptor, can be rapidly identified
using this method.
[0171] A zcytor17 multimeric cytokine receptor can be expressed as
a fusion with an immunoglobulin heavy chain constant region,
typically an F.sub.c fragment, which contains two constant region
domains and lacks the variable region. Methods for preparing such
fusions are disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584.
Such fusions are typically secreted as multimeric molecules wherein
the F.sub.c portions are disulfide bonded to each other and two
non-Ig polypeptides are arrayed in closed proximity to each other.
Fusions of this type can be used for example, for dimerization,
increasing stability and in vivo half-life, to affinity purify
ligand, as in vitro assay tool or antagonist. For use in assays,
the chimeras are bound to a support via the F.sub.c region and used
in an ELISA format.
[0172] Additional assays provided by the present invention include
the use of hybrid receptor polypeptides. These hybrid polypeptides
fall into two general classes. Within the first class, the
intracellular domain of zcytor17, comprising approximately residues
544 (Lys) to 732 (Val) of SEQ ID NO:111, residues 544 (Lys) to 649
(Ile) of SEQ ID NO:109, or residues 557 (Lys) to 662 (Ile) of SEQ
ID NO:5, or residues 551 (Lys) to 662 (Cys) of SEQ ID NO:117 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, for instance, 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 and assayed for a
response. This system provides a means for analyzing signal
transduction mediated by zcytor17 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
zcytor17. A second class of hybrid receptor polypeptides comprise
the extracellular (ligand-binding) domain (approximately residues
20 (Ala) to 519 (Glu) of SEQ ID NO:111 and SEQ ID NO:109;
approximately residues 33 (Ala) to 532 (Glu) of SEQ ID NO:5) or
cytokine-binding domain of zcytor17 (approximately residues 20
(Ala) to 227 (Pro) of SEQ ID NO:111 and SEQ ID NO:109; or
approximately residues 33 (Ala) to 240 (Pro) of SEQ ID NO:5;
approximately residues 46 (Val) to 533 (Glu) of SEQ ID NO:117; or
approximately residues 46 (Val) to 533 (Trp) of SEQ ID NO:119) with
a cytoplasmic domain of a second receptor, preferably a cytokine
receptor, and a transmembrane domain. The transmembrane domain may
be derived from either receptor. Hybrid 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 use of a broad spectrum of
cell types within receptor-based assay systems.
[0173] The expression of WSX-1 is strongest in thymus, spleen, PBL,
and lymph node, as well as increased expression observed for
activated T-cells. The tissue distribution for OSMRbeta is
described as very broad. The tissue distribution of these three
receptors suggests that a target for zcytor17lig is hematopoietic
lineage cells, in particular T-cells, monocytes/macrophages and
lymphoid progenitor cells and lymphoid cells. Other known
four-helical-bundle cytokines that act on lymphoid cells include
IL-2, IL-4, IL-7, and IL-15. For a review of four-helical-bundle
cytokines, see, Nicola et al., Advances in Protein Chemistry
52:1-65, 1999 and Kelso, A., Immunol Cell Biol 76:300-317,
1998.
[0174] Conditioned media (CM) from CD3+ selected,
PMA/Tonomycin-stimulated human peripheral blood cells supported the
growth of BaF3 cells that expressed the zcytor17 receptor, OSMRbeta
and WSX-1 receptor and were otherwise dependent on IL-3.
Conditioned medias from cells that were not: 1)
PMA/Tonomycin-stimulated; or were not: 2) CD3 selected (with or
without PMA/Tonomycin stimulation) did not support the growth of
Baf3 cells expressing zcytor17, OSMRbeta and WSX-1
(BaF3/zcytor17/WSX-1/OSMRbeta) receptor-expressing cells. Control
experiments demonstrated that this proliferative activity was not
attributable to other known growth factors, and that the ability of
such conditioned media to stimulate proliferation of
zcytor17/WSX-1/OSMRbeta receptor-expressing cells could be
neutralized by a soluble form of the zcytor17 receptor.
[0175] Conditioned-media from CD3+ selected cells activated with
PMA/Tonomycin also supported growth of BaF3 cells that expressed
the zcytor17 receptor and OSMRbeta receptor (zcytor17/OSMRbeta),
while BaF3 cells expressing only zcytor17 receptor and WSX-1
receptor (zcytor17/WSX-1), or containing only the OSMRbeta
receptor, were not stimulated by this conditioned-media.
[0176] Proliferation of zcytor17/WSX-1/OSMRbeta receptor-expressing
BaF3 cells exposed to CM from CD3+ selected,
PMA/Ionomycin-stimulated human peripheral blood cells were
identified by visual inspection of the cultures and/or by
proliferation assay. Many suitable proliferation assays are known
in the art, and include assays for reduction of a dye such as
AlamarBlue.TM. (AccuMed International, Inc. Westlake, Ohio),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(Mosman, J. Immunol Meth. 65: 55-63, 1983); 3-(4,5 dimethyl
thiazol-2yl)-5-3-carboxymethoxyphenyl-2H-tetrazolium;
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tet-
razolium hydroxide; and cyanoditolyl-tetrazolium chloride (which
are commercially available from Polysciences, Inc., Warrington,
Pa.); mitogenesis assays, such as measurement of incorporation of
.sup.3H-thymidine; dye exclusion assays using, for example,
naphthalene black or trypan blue; dye uptake using diacetyl
fluorescein; and chromium release. See, in general, Freshney,
Culture of Animal Cells: A Manual of Basic Technique, 3rd ed.,
Wiley-Liss, 1994, which is incorporated herein by reference.
[0177] A cDNA library was prepared from CD3+ selected, PMA- and
Tonomycin-stimulated primary human peripheral blood cells. The CD3+
selected, PMA- and Tonomycin-stimulated human peripheral blood
cells cDNA library was divided into pools containing multiple cDNA
molecules and was transfected into a host cell line, for example,
BHK 570 cells (ATCC accession no. 10314). The transfected host
cells were cultured in a medium that did not contain exogenous
growth factors (e.g., 5% FBS) and conditioned medium was collected.
The conditioned media were assayed for the ability to stimulate
proliferation of BaF3 cells transfected with the zcytor17, WSX-1,
and OSMRbeta receptors. cDNA pools producing conditioned medium
that stimulated BaF3/zcytor17/WSX-1/OSMRbeta receptor cells were
identified. This pooled plasmid cDNA was electroporated into E.
coli. cDNA was isolated from single colonies and transfected
individually into BHK 570 cells. Positive clones were identified by
a positive result in the BaF3/zcytor17/WSX-1/OSMRbeta receptor
proliferation assay, and the activity was confirmed by
neutralization of proliferation using the soluble zcytor17
receptor.
[0178] In view of the tissue distribution observed for zcytor17
receptor agonists (including the natural
zcytor17lig/substrate/cofactor/etc.) and/or antagonists have
enormous potential in both in vitro and in vivo applications.
Compounds identified as zcytor17lig agonists are useful for
expansion, proliferation, activation, differentiation, and/or
induction or inhibition of specialized cell functions of cells
involved in homeostasis of hematopoiesis and immune function. For
example, zcytor17lig and agonist compounds are useful as components
of defined cell culture media, and may be used alone or in
combination with other cytokines and hormones to replace serum that
is commonly used in cell culture. Agonists are thus useful in
specifically promoting the growth and/or development of T-cells,
B-cells, monocytes/macrophages, NK cells, cytotoxic lymphocytes,
and other cells of the lymphoid and myeloid lineages in
culture.
[0179] Antagonists are also useful as research reagents for
characterizing sites of ligand-receptor interaction. Antagonists
are useful to inhibit expansion, proliferation, activation, and/or
differentiation of cells involved in regulating hematopoiesis.
Inhibitors of zcytor17lig activity (zcytor17lig antagonists)
include anti-zcytor17lig antibodies and soluble multimeric cytokine
receptors, as well as other peptidic and non-peptidic agents
(including ribozymes).
[0180] A zcytor17lig-binding protein, such as a multimeric cytokine
receptor of the present invention, can also be used for
purification of ligand. The multimeric cytokine receptor is
immobilized on a solid support, such as beads of agarose,
cross-linked agarose, glass, cellulosic resins, silica-based
resins, polystyrene, cross-linked polyacrylamide, or like materials
that are stable under the conditions of use. Methods for linking
polypeptides to solid supports are known in the art, and include
amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide
activation, epoxide activation, sulfhydryl activation, and
hydrazide activation. The resulting medium will generally be
configured in the form of a column, and fluids containing ligand
are passed through the column one or more times to allow ligand to
bind to the receptor polypeptide. The ligand is then eluted using
changes in salt concentration, chaotropic agents (guanidine HCR),
or pH to disrupt ligand-receptor binding.
[0181] An assay system that uses a ligand-binding receptor (or an
antibody, one member of a complement/anti-complement pair) or a
binding fragment thereof, and a commercially available biosensor
instrument (BIAcore, Pharmacia Biosensor, Piscataway, N.J.) may be
advantageously employed. Such receptor, antibody, member of a
complement/anti-complement pair or fragment is immobilized onto the
surface of a receptor chip. Use of this instrument is disclosed by
Karlsson, J. Immunol. Methods 145:229-40 (1991) and Cunningham and
Wells, J. Mol. Biol 234:554-63 (1993). A receptor, antibody, member
or fragment is covalently attached, using amine or sulfhydryl
chemistry, to dextran fibers that are attached to gold film within
the flow cell. A test sample is passed through the cell. If a
ligand, epitope, or opposite member of the
complement/anti-complement pair is present in the sample, it will
bind to the immobilized receptor, antibody or member, respectively,
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. Alternatively, ligand/receptor binding
can be analyzed using SELDI.TM. technology (Ciphergen, Inc., Palo
Alto, Calif.).
[0182] Ligand-binding receptor polypeptides can also be used within
other assay systems known in the art. Such systems include
Scatchard analysis for determination of binding affinity (see
Scatchard, Ann. NY Acad. Sci. 51: 660-72 (1949)) and calorimetric
assays (Cunningham et al., Science 253:545-48 (1991); and
Cunningham et al., Science 245:821-25 (1991)).
[0183] The present invention also provides an antibody that
specifically binds to a polypeptide or at least at portion of a
multimeric cytokine receptor as described herein.
[0184] Zcytor17 multimeric cytokine receptors can also be used to
prepare antibodies that bind to epitopes, peptides or polypeptides
thereof. The multimeric cytokine receptor or a fragment thereof
serves as an antigen (immunogen) to inoculate an animal and elicit
an immune response. One of skill in the art would recognize that
antigenic, epitope-bearing polypeptides may contain a sequence of
at least 6, preferably at least 9, and more preferably at least 15
to about 30 contiguous amino acid residues of a polypeptide(s) of
the multimeric cytokine receptor, such as zcytor17 (SEQ ID NO:111),
OSMRbeta (SEQ ID NO:7), and/or WSX-1 (SEQ ID NO:9). Polypeptides
comprising a larger portion of a multimeric cytokine receptor,
i.e., from 30 to 100 residues up to the entire length of the amino
acid sequence are included. Antigens or immunogenic epitopes can
also include attached tags, adjuvants, carriers and vehicles, as
described herein.
[0185] Antibodies from an immune response generated by inoculation
of an animal with these antigens can be isolated and purified as
described herein. Methods for preparing and isolating polyclonal
and monoclonal antibodies are well known in the art. See, for
example, Current Protocols in Immunology, Cooligan, et al. (eds.),
National Institutes of Health, John Wiley and Sons, Inc., 1995;
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R.,
Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications,
CRC Press, Inc., Boca Raton, Fla., 1982.
[0186] As would be evident to one of ordinary skill in the art,
polyclonal antibodies can be generated from inoculating a variety
of warm-blooded animals such as horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, and rats with a multimeric cytokine
receptor or a fragment thereof. The immunogenicity of a multimeric
cytokine receptor may be increased through the use of an adjuvant,
such as alum (aluminum hydroxide) or Freund's complete or
incomplete adjuvant. Multimeric cytokine receptors useful for
immunization also include fusion polypeptides, such as fusions of
zcytor17, OSMRbeta, and/or WSX-1, 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.
[0187] As used herein, the term "antibodies" includes polyclonal
antibodies, affinity-purified polyclonal antibodies, monoclonal
antibodies, and antigen-binding fragments, such as F(ab').sub.2 and
Fab proteolytic fragments. Genetically engineered intact antibodies
or fragments, such as chimeric antibodies, Fv fragments, single
chain antibodies and the like, as well as synthetic antigen-binding
peptides and polypeptides, are also included. Non-human antibodies
may be humanized by grafting non-human CDRs onto human framework
and constant regions, or by incorporating the entire non-human
variable domains (optionally "cloaking" them with a human-like
surface by replacement of exposed residues, wherein the result is a
"veneered" antibody). In some instances, humanized antibodies may
retain non-human residues within the human variable region
framework domains to enhance proper binding characteristics.
Through humanizing antibodies, biological half-life may be
increased, and the potential for adverse immune reactions upon
administration to humans is reduced. Moreover, human antibodies can
be produced in transgenic, non-human animals that have been
engineered to contain human immunoglobulin genes as disclosed in
WIPO Publication WO 98/24893. It is preferred that the endogenous
immunoglobulin genes in these animals be inactivated or eliminated,
such as by homologous recombination.
[0188] Antibodies are considered to be specifically binding if: 1)
they exhibit a threshold level of binding activity, and 2) they do
not significantly cross-react with related polypeptide molecules. A
threshold level of binding is determined if anti-multimeric
cytokine receptor antibodies herein bind to a multimeric cytokine
receptor, peptide or epitope with an affinity at least 10-fold
greater than the binding affinity to control (non-multimeric
cytokine receptor) protein. It is preferred that the antibodies
exhibit 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, G., Ann. NY Acad. Sci. 51: 660-672
(1949)).
[0189] Whether anti-multimeric cytokine receptor antibodies do not
significantly cross-react with related polypeptide molecules is
shown, for example, by the antibody detecting zcytor17 multimeric
cytokine receptor but not known related polypeptides using a
standard Western blot analysis (Ausubel et al., ibid.). Examples of
known related polypeptides are those disclosed in the prior art,
such as known orthologs, and paralogs, and similar known members of
a protein family. Screening can also be done using non-human
multimeric cytokine receptor, and multimeric cytokine receptor
mutant polypeptides. Moreover, antibodies can be "screened against"
known related polypeptides, to isolate a population that
specifically binds to the multimeric cytokine receptor. For
example, antibodies raised to multimeric cytokine receptor are
adsorbed to related polypeptides adhered to insoluble matrix;
antibodies specific to multimeric cytokine receptor will flow
through the matrix under the proper buffer conditions. Screening
allows isolation of polyclonal and monoclonal antibodies
non-crossreactive to known closely related polypeptides
(Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold
Spring Harbor Laboratory Press, 1988; Current Protocols in
Immunology, Cooligan, et al. (eds.), National Institutes of Health,
John Wiley and Sons, Inc., 1995). Screening and isolation of
specific antibodies is well known in the art. See, Fundamental
Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in
Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and
Practice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjamin
et al., Ann. Rev. Immunol. 2: 67-101, 1984. Specifically binding
anti-multimeric cytokine receptor antibodies can be detected by a
number of methods in the art, and disclosed below.
[0190] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which bind to multimeric cytokine
receptor proteins or polypeptides. Exemplary assays are described
in detail in Antibodies: A Laboratory Manual, Harlow and Lane
(Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative
examples of such assays include: concurrent immunoelectrophoresis,
radioimmunoassay, radioimmuno-precipitation, enzyme-linked
immunosorbent assay (ELISA), dot blot or Western blot assay,
inhibition or competition assay, and sandwich assay. In addition,
antibodies can be screened for binding to wild-type versus mutant
multimeric cytokine receptor protein or polypeptide.
[0191] Within another aspect the present invention provides an
antibody produced by the method as disclosed above, wherein the
antibody binds to at least a portion of a multimer cytokine
receptor comprising at least a portion of SEQ ID NO:111, SEQ ID
NO:109, or SEQ ID NO:5. In one embodiment, the antibody disclosed
above specifically binds to a polypeptide shown in SEQ ID NO:111,
SEQ ID NO:109, or SEQ ID NO:5. In another embodiment, the antibody
can be a monoclonal antibody or a polyclonal antibody.
[0192] Antibodies to multimeric cytokine receptor may be used for
tagging cells that express multimeric cytokine receptor; for
isolating multimeric cytokine receptor by affinity purification;
for diagnostic assays for determining circulating levels of
multimeric cytokine receptor; for detecting or quantitating soluble
multimeric cytokine receptor as a marker of underlying pathology or
disease; in analytical methods employing FACS; for screening
expression libraries; for generating anti-idiotypic antibodies; and
as neutralizing antibodies or as antagonists to block multimeric
cytokine receptor activity in vitro and in vivo. Suitable direct
tags or labels include radionuclides, enzymes, substrates,
cofactors, 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 multimeric cytokine receptor or fragments thereof may
be used in vitro to detect denatured multimeric cytokine receptor
or fragments thereof in assays, for example, Western Blots or other
assays known in the art.
[0193] Suitable detectable molecules may be directly or indirectly
attached to the multimeric cytokine receptor or antibody, and
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,
saporin, 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). Multimeric cytokine receptors 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 polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/anticomplementary
pair.
[0194] A soluble multimeric cytokine receptor can also act as
zcytor17lig "antagonists" to block zcytor17lig binding and signal
transduction in vitro and in vivo. These anti-zcytor17lig binding
proteins would be useful for inhibiting zcytor17lig activity or
protein-binding.
[0195] 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 polypeptide has multiple functional domains
(i.e., an activation domain or a receptor 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 domain only fusion protein
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.
[0196] Moreover, inflammation is a protective response by an
organism to fend off an invading agent. Inflammation is a cascading
event that involves many cellular and humoral mediators. On one
hand, suppression of inflammatory responses can leave a host
immunocompromised; however, if left unchecked, inflammation can
lead to serious complications including chronic inflammatory
diseases (e.g., rheumatoid arthritis, multiple sclerosis,
inflammatory bowel disease and the like), septic shock and multiple
organ failure. Importantly, these diverse disease states share
common inflammatory mediators. The collective diseases that are
characterized by inflammation have a large impact on human
morbidity and mortality. Therefore it is clear that
anti-inflammatory antibodies and binding polypeptides, such as
anti-zcytor17lig antibodies and binding polypeptides described
herein, could have crucial therapeutic potential for a vast number
of human and animal diseases, from asthma and allergy to
autoimmunity and septic shock. As such, use of anti-inflammatory
anti zcytor17lig antibodies and binding polypeptides described
herein can be used therapeutically as zcytor17lig antagonists
described herein, particularly in diseases such as arthritis,
endotoxemia, inflammatory bowel disease, psoriasis, related disease
and the like.
[0197] Arthritis
[0198] Arthritis, including osteoarthritis, rheumatoid arthritis,
arthritic joints as a result of injury, and the like, are common
inflammatory conditions which would benefit from the therapeutic
use of anti-inflammatory antibodies and binding polypeptides, such
as anti-zcytor17lig antibodies and binding polypeptides of the
present invention. For example, rheumatoid arthritis (RA) is a
systemic disease that affects the entire body and is one of the
most common forms of arthritis. It is characterized by the
inflammation of the membrane lining the joint, which causes pain,
stiffness, warmth, redness and swelling. Inflammatory cells release
enzymes that may digest bone and cartilage. As a result of
rheumatoid arthritis, the inflamed joint lining, the synovium, can
invade and damage bone and cartilage leading to joint deterioration
and severe pain amongst other physiologic effects. The involved
joint can lose its shape and alignment, resulting in pain and loss
of movement.
[0199] Rheumatoid arthritis (RA) is an immune-mediated disease
particularly characterized by inflammation and subsequent tissue
damage leading to severe disability and increased mortality. A
variety of cytokines are produced locally in the rheumatoid joints.
Numerous studies have demonstrated that IL-1 and TNF-alpha, two
prototypic pro-inflammatory cytokines, play an important role in
the mechanisms involved in synovial inflammation and in progressive
joint destruction. Indeed, the administration of TNF-alpha and IL-1
inhibitors in patients with RA has led to a dramatic improvement of
clinical and biological signs of inflammation and a reduction of
radiological signs of bone erosion and cartilage destruction.
However, despite these encouraging results, a significant
percentage of patients do not respond to these agents, suggesting
that other mediators are also involved in the pathophysiology of
arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002).
One of those mediators could be zcytor17lig, and as such a molecule
that binds or inhibits zcytor17lig, such as anti zcytor17lig
antibodies or binding partners, could serve as a valuable
therapeutic to reduce inflammation in rheumatoid arthritis, and
other arthritic diseases.
[0200] There are several animal models for rheumatoid arthritis
known in the art. For example, in the collagen-induced arthritis
(CIA) model, mice develop chronic inflammatory arthritis that
closely resembles human rheumatoid arthritis. Since CIA shares
similar immunological and pathological features with RA, this makes
it an ideal model for screening potential human anti-inflammatory
compounds. The CIA model is a well-known model in mice that depends
on both an immune response, and an inflammatory response, in order
to occur. The immune response comprises the interaction of B-cells
and CD4+ T-cells in response to collagen, which is given as
antigen, and leads to the production of anti-collagen antibodies.
The inflammatory phase is the result of tissue responses from
mediators of inflammation, as a consequence of some of these
antibodies cross-reacting to the mouse's native collagen and
activating the complement cascade. An advantage in using the CIA
model is that the basic mechanisms of pathogenesis are known. The
relevant T-cell and B-cell epitopes on type II collagen have been
identified, and various immunological (e.g., delayed-type
hypersensitivity and anti-collagen antibody) and inflammatory
(e.g., cytokines, chemokines, and matrix-degrading enzymes)
parameters relating to immune-mediated arthritis have been
determined, and can thus be used to assess test compound efficacy
in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20, 1999;
Williams et al., Immunol. 89:9784-788, 1992; Myers et al., Life
Sci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959,
1995).
[0201] The administration of soluble zcytor17 comprising
polypeptides (including heterodimeric and multimeric receptors
described herein), such as zcytor17-Fc4 or other zcytor17 soluble
and fusion proteins to these CIA model mice was used to evaluate
the use of zcytor17 to ameliorate symptoms and alter the course of
disease. As a molecule that modulates immune and inflammatory
response, zcytor17lig, may induce production of SAA, which is
implicated in the pathogenesis of rheumatoid arthritis, zcytor17lig
antagonists may reduce SAA activity in vitro and in vivo, the
systemic or local administration of zcytor17lig antagonists such as
anti-zcytor17lig antibodies or binding partners, zcytor17
comprising polypeptides (including heterodimeric and multimeric
receptors described herein), such as zcytor17-Fc4 or other zcytor17
soluble and fusion proteins can potentially suppress the
inflammatory response in RA. Other potential therapeutics include
zcytor17 polypeptides, soluble heterodimeric and multimeric
receptor polypeptides, or anti zcytor17lig antibodies or binding
partners of the present invention, and the like.
[0202] Endotoxemia
[0203] Endotoxemia is a severe condition commonly resulting from
infectious agents such as bacteria and other infectious disease
agents, sepsis, toxic shock syndrome, or in immunocompromised
patients subjected to opportunistic infections, and the like.
Therapeutically useful of anti-inflammatory antibodies and binding
polypeptides, such as anti-zcytor17lig antibodies and binding
polypeptides of the present invention, could aid in preventing and
treating endotoxemia in humans and animals. Other potential
therapeutics include zcytor17 polypeptides, soluble heterodimeric
and multimeric receptor polypeptides, or anti zcytor17lig
antibodies or binding partners of the present invention, and the
like, could serve as a valuable therapeutic to reduce inflammation
and pathological effects in endotoxemia.
[0204] Lipopolysaccharide (LPS) induced endotoxemia engages many of
the proinflammatory mediators that produce pathological effects in
the infectious diseases and LPS induced endotoxemia in rodents is a
widely used and acceptable model for studying the pharmacological
effects of potential pro-inflammatory or immunomodulating agents.
LPS, produced in gram-negative bacteria, is a major causative agent
in the pathogenesis of septic shock (Glausner et al., Lancet
338:732, 1991). A shock-like state can indeed be induced
experimentally by a single injection of LPS into animals. Molecules
produced by cells responding to LPS can target pathogens directly
or indirectly. Although these biological responses protect the host
against invading pathogens, they may also cause harm. Thus, massive
stimulation of innate immunity, occurring as a result of severe
Gram-negative bacterial infection, leads to excess production of
cytokines and other molecules, and the development of a fatal
syndrome, septic shock syndrome, which is characterized by fever,
hypotension, disseminated intravascular coagulation, and multiple
organ failure (Dumitru et al. Cell 103:1071-1083, 2000).
[0205] These toxic effects of LPS are mostly related to macrophage
activation leading to the release of multiple inflammatory
mediators. Among these mediators, TNF appears to play a crucial
role, as indicated by the prevention of LPS toxicity by the
administration of neutralizing anti-TNF antibodies (Beutler et al.,
Science 229:869, 1985). It is well established that lug injection
of E. coli LPS into a C57B1/6 mouse will result in significant
increases in circulating IL-6, TNF-alpha, IL-1, and acute phase
proteins (for example, SAA) approximately 2 hours post injection.
The toxicity of LPS appears to be mediated by these cytokines as
passive immunization against these mediators can result in
decreased mortality (Beutler et al., Science 229:869, 1985). The
potential immunointervention strategies for the prevention and/or
treatment of septic shock include anti-TNF mAb, IL-1 receptor
antagonist, LIF, IL-10, and G-CSF. Since LPS induces the production
of pro-inflammatory factors possibly contributing to the pathology
of endotoxemia, the neutralization of zcytor17lig activity, SAA or
other pro-inflammatory factors by antagonizing zcytor17lig
polypeptide can be used to reduce the symptoms of endotoxemia, such
as seen in endotoxic shock. Other potential therapeutics include
zcytor17 polypeptides, soluble heterodimeric and multimeric
receptor polypeptides, or anti-zcytor17lig antibodies or binding
partners of the present invention, and the like.
[0206] Inflammatory Bowel Disease. IBD
[0207] In the United States approximately 500,000 people suffer
from Inflammatory Bowel Disease (IBD) which can affect either colon
and rectum (Ulcerative colitis) or both, small and large intestine
(Crohn's Disease). The pathogenesis of these diseases is unclear,
but they involve chronic inflammation of the affected tissues.
Potential therapeutics include zcytor17 polypeptides, soluble
heterodimeric and multimeric receptor polypeptides, or
anti-zcytor17lig antibodies or binding partners of the present
invention, and the like, could serve as a valuable therapeutic to
reduce inflammation and pathological effects in IBD and related
diseases.
[0208] Ulcerative colitis (UC) is an inflammatory disease of the
large intestine, commonly called the colon, characterized by
inflammation and ulceration of the mucosa or innermost lining of
the colon. This inflammation causes the colon to empty frequently,
resulting in diarrhea. Symptoms include loosening of the stool and
associated abdominal cramping, fever and weight loss. Although the
exact cause of UC is unknown, recent research suggests that the
body's natural defenses are operating against proteins in the body
which the body thinks are foreign (an "autoimmune reaction").
Perhaps because they resemble bacterial proteins in the gut, these
proteins may either instigate or stimulate the inflammatory process
that begins to destroy the lining of the colon. As the lining of
the colon is destroyed, ulcers form releasing mucus, pus and blood.
The disease usually begins in the rectal area and may eventually
extend through the entire large bowel. Repeated episodes of
inflammation lead to thickening of the wall of the intestine and
rectum with scar tissue. Death of colon tissue or sepsis may occur
with severe disease. The symptoms of ulcerative colitis vary in
severity and their onset may be gradual or sudden. Attacks may be
provoked by many factors, including respiratory infections or
stress.
[0209] Although there is currently no cure for UC available,
treatments are focused on suppressing the abnormal inflammatory
process in the colon lining. Treatments including corticosteroids
immunosuppressives (eg. azathioprine, mercaptopurine, and
methotrexate) and aminosalicytates are available to treat the
disease. However, the long-term use of immunosuppressives such as
corticosteroids and azathioprine can result in serious side effects
including thinning of bones, cataracts, infection, and liver and
bone marrow effects. In the patients in whom current therapies are
not successful, surgery is an option. The surgery involves the
removal of the entire colon and the rectum.
[0210] There are several animal models that can partially mimic
chronic ulcerative colitis. The most widely used model is the
2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis
model, which induces chronic inflammation and ulceration in the
colon. When TNBS is introduced into the colon of susceptible mice
via intra-rectal instillation, it induces T-cell mediated immune
response in the colonic mucosa, in this case leading to a massive
mucosal inflammation characterized by the dense infiltration of
T-cells and macrophages throughout the entire wall of the large
bowel. Moreover, this histopathologic picture is accompanies by the
clinical picture of progressive weight loss (wasting), bloody
diarrhea, rectal prolapse, and large bowel wall thickening (Neurath
et al. Intern. Rev. Immunol 19:51-62, 2000).
[0211] Another colitis model uses dextran sulfate sodium (DSS),
which induces an acute colitis manifested by bloody diarrhea,
weight loss, shortening of the colon and mucosal ulceration with
neutrophil infiltration. DSS-induced colitis is characterized
histologically by infiltration of inflammatory cells into the
lamina propria, with lymphoid hyperplasia, focal crypt damage, and
epithelial ulceration. These changes are thought to develop due to
a toxic effect of DSS on the epithelium and by phagocytosis of
lamina propria cells and production of TNF-alpha and IFN-gamma.
Despite its common use, several issues regarding the mechanisms of
DSS about the relevance to the human disease remain unresolved. DSS
is regarded as a T cell-independent model because it is observed in
T cell-deficient animals such as SCID mice.
[0212] The administration of anti-zcytor17lig antibodies or binding
partners, soluble zcytor17 comprising polypeptides (including
heterodimeric and multimeric receptors), such as zcytor17-Fc4 or
other zcytor17 soluble and fusion proteins to these TNBS or DSS
models can be used to evaluate the use of zcytor17lig antagonists
to ameliorate symptoms and alter the course of gastrointestinal
disease. Zcytor17lig may play a role in the inflammatory response
in colitis, and the neutralization of zcytor17lig activity by
administrating zcytor17lig antagonists is a potential therapeutic
approach for IBD. Other potential therapeutics include zcytor17
polypeptides, soluble heterodimeric and multimeric receptor
polypeptides, or anti-zcytor17lig antibodies or binding partners of
the present invention, and the like.
[0213] Psoriasis
[0214] Psoriasis is a chronic skin condition that affects more than
seven million Americans. Psoriasis occurs when new skin cells grow
abnormally, resulting in inflamed, swollen, and scaly patches of
skin where the old skin has not shed quickly enough. Plaque
psoriasis, the most common form, is characterized by inflamed
patches of skin ("lesions") topped with silvery white scales.
Psoriasis may be limited to a few plaques or involve moderate to
extensive areas of skin, appearing most commonly on the scalp,
knees, elbows and trunk. Although it is highly visible, psoriasis
is not a contagious disease. The pathogenesis of the diseases
involves chronic inflammation of the affected tissues. Zcytor17
polypeptides, soluble heterodimeric and multimeric receptor
polypeptides, or anti-zcytor17lig antibodies or binding partners of
the present invention, and the like, could serve as a valuable
therapeutic to reduce inflammation and pathological effects in
psoriasis, other inflammatory skin diseases, skin and mucosal
allergies, and related diseases.
[0215] Psoriasis is a T-cell mediated inflammatory disorder of the
skin that can cause considerable discomfort. It is a disease for
which there is no cure and affects people of all ages. Psoriasis
affects approximately two percent of the populations of European
and North America. Although individuals with mild psoriasis can
often control their disease with topical agents, more than one
million patients worldwide require ultraviolet or systemic
immunosuppressive therapy. Unfortunately, the inconvenience and
risks of ultraviolet radiation and the toxicities of many therapies
limit their long-term use. Moreover, patients usually have
recurrence of psoriasis, and in some cases rebound, shortly after
stopping immunosuppressive therapy.
[0216] Differentiation is a progressive and dynamic process,
beginning with pluripotent stem cells and ending with terminally
differentiated cells. Pluripotent stem cells that can regenerate
without commitment to a lineage express a set of differentiation
markers that are lost when commitment to a cell lineage is made.
Progenitor cells express a set of differentiation markers that may
or may not continue to be expressed as the cells progress down the
cell lineage pathway toward maturation. Differentiation markers
that are expressed exclusively by mature cells are usually
functional properties such as cell products, enzymes to produce
cell products, and receptors. The stage of a cell population's
differentiation is monitored by identification of markers present
in the cell population.
[0217] There is evidence to suggest that factors that stimulate
specific cell types down a pathway towards terminal differentiation
or dedifferentiation affect the entire cell population originating
from a common precursor or stem cell.
[0218] A multimeric cytokine receptor of the present invention can
be useful for stimulating proliferation, activation,
differentiation and/or induction or inhibition of specialized cell
function of cells of the involved homeostasis of the hematopoiesis
and immune function. In particular, multimeric cytokine receptors
as described herein are useful for stimulating proliferation,
activation, differentiation, induction or inhibition of specialized
cell functions of cells of the hematopoietic lineages, including,
but not limited to, T cells, B cells, monocytes/macrophages, NK
cells, neutrophils, endothelial cells, fibroblasts, eosinophils,
chondrocytes, mast cells, langerhan cells, monocytes, and
macrophages, as well as epithelial cells. Epithelial cells include,
for example, ameloblasts, chief cells, chromatophores,
enterochramaffin cells, enterochromaffin-like cells, goblet cells,
granulosa cells, keratinocytes, dendritic cells, labyrinth
supporting cells, melanocytes, merkel cells, paneth cells, parietal
cells, sertoli cells, and the like.
[0219] The present invention also provides a method for reducing
hematopoietic cells and hematopoietic cell progenitors of a mammal.
The method includes culturing bone marrow or peripheral blood cells
with a composition comprising an effective amount of a soluble
multimeric cytokine receptor to produce a decrease in the number of
lymphoid cells in the bone marrow or peripheral blood cells as
compared to bone marrow or peripheral blood cells cultured in the
absence of the multimeric cytokine receptor. The hematopoietic
cells and hematopoietic cell progenitors can be lymphoid cells,
such as monocytic cells, macrophages, or T cells.
[0220] The present invention also provides a method of inhibiting
an immune response in a mammal exposed to an antigen or pathogen.
The method includes (a) determining directly or indirectly the
level of antigen or pathogen present in the mammal; (b)
administering a composition comprising a soluble multimeric
cytokine receptor in an acceptable pharmaceutical vehicle; (c)
determining directly or indirectly the level of antigen or pathogen
in the mammal; and (d) comparing the level of the antigen or
pathogen in step (a) to the antigen or pathogen level in step (c),
wherein a change in the level is indicative of inhibiting an immune
response. The method may further include (e) re-administering a
composition comprising a multimeric cytokine receptor in an
acceptable pharmaceutical vehicle; (f) determining directly or
indirectly the level of antigen or pathogen in the mammal; and (g)
comparing the number of the antigen or pathogen level in step (a)
to the antigen level in step (f), wherein a change in the level is
indicative of inhibiting an immune response.
[0221] Alternatively, the method can include (a) determining a
level of an antigen- or pathogen-specific antibody; (b)
administering a composition comprising a soluble multimeric
cytokine receptor in an acceptable pharmaceutical vehicle; (c)
determining a post administration level of antigen- or
pathogen-specific antibody; (d) comparing the level of antibody in
step (a) to the level of antibody in step (c), wherein a decrease
in antibody level is indicative of inhibiting an immune
response.
[0222] Zcytor17lig was isolated from tissue known to have important
immunological function and which contain cells that play a role in
the immune system. Zcytor17lig is expressed in CD3+ selected,
activated peripheral blood cells, and it has been shown that
zcytor17lig expression increases after T cell activation. Moreover,
results of experiments described in the Examples section herein
suggest that a multimeric or heterodimeric cytokine receptor of the
present invention can have an effect on the growth/expansion of
monocytes/macrophages, T-cells, B-cells, NK cells and/or
differentiated state of monocytes/macrophages, T-cells, B-cells, NK
cells or these cells' progenitors. Factors that both stimulate
proliferation of hematopoietic progenitors and activate mature
cells are generally known, however, proliferation and activation
can also require additional growth factors. For example, it has
been shown that IL-7 and Steel Factor (c-kit ligand) were required
for colony formation of NK progenitors. IL-15 plus IL-2 in
combination with IL-7 and Steel Factor was more effective
(Mr{umlaut over (.smallcircle.)}zek et al., Blood 87:2632-2640,
1996). However, unidentified cytokines may be necessary for
proliferation of specific subsets of NK cells and/or NK progenitors
(Robertson et. al., Blood 76:2451-2438, 1990). Similarly,
zcytor17lig may act alone or in concert or synergy with other
cytokines to enhance growth, proliferation expansion and
modification of differentiation of monocytes/macrophages, T-cells,
B-cells or NK cells.
[0223] Assays measuring differentiation include, for example,
measuring cell markers associated with stage-specific expression of
a tissue, enzymatic activity, functional activity or morphological
changes (Watt, FASEB, 5:281-284 (1991); Francis, Differentiation
57:63-75 (1994); and Raes, Adv. Anim. Cell Biol. Technol
Bioprocesses, 161-171 (1989)). Alternatively, zcytor17lig
polypeptide itself can serve as an additional cell-surface or
secreted marker associated with stage-specific expression of a
tissue. As such, direct measurement of zcytor17lig polypeptide, or
its loss of expression in a tissue as it differentiates, can serve
as a marker for differentiation of tissues.
[0224] Similarly, direct measurement of zcytor17lig polypeptide, or
its loss of expression in a tissue can be determined in a tissue or
in cells as they undergo tumor progression. Increases in
invasiveness and motility of cells, or the gain or loss of
expression of zcytor17lig in a pre-cancerous or cancerous
condition, in comparison to normal tissue, can serve as a
diagnostic for transformation, invasion and metastasis in tumor
progression. As such, knowledge of a tumor's stage of progression
or metastasis will aid the physician in choosing the most proper
therapy, or aggressiveness of treatment, for a given individual
cancer patient. Methods of measuring gain and loss of expression
(of either mRNA or protein) are well known in the art and described
herein and can be applied to zcytor17lig expression. For example,
appearance or disappearance of polypeptides that regulate cell
motility can be used to aid diagnosis and prognosis of prostate
cancer (Banyard, J. and Zetter, B. R., Cancer and Metast. Rev.
17:449-458, 1999). As an effector of cell motility, zcytor17lig
gain or loss of expression may serve as a diagnostic for lymphoid,
B-cell, epithelial, hematopoietic and other cancers.
[0225] Moreover, the activity and effect of zcytor17lig on tumor
progression and metastasis can be measured in vivo. Several
syngeneic mouse models have been developed to study the influence
of polypeptides, compounds or other treatments on tumor
progression. In these models, tumor cells passaged in culture are
implanted into mice of the same strain as the tumor donor. The
cells will develop into tumors having similar characteristics in
the recipient mice, and metastasis will also occur in some of the
models. Appropriate tumor models for our studies include the Lewis
lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No.
CRL-6323), amongst others. These are both commonly used tumor
lines, syngeneic to the C57BL6/J mouse, that are readily cultured
and manipulated in vitro. Tumors resulting from implantation of
either of these cell lines are capable of metastasis to the lung in
C57BL6/J mice. The Lewis lung carcinoma model has recently been
used in mice to identify an inhibitor of angiogenesis (O'Reilly M
S, et al. Cell 79: 315-328, 1994). C57BL6/J mice are treated with
an experimental agent either through daily injection of recombinant
protein, agonist or antagonist or a one time injection of
recombinant adenovirus. Three days following this treatment,
10.sup.5 to 10.sup.6 cells are implanted under the dorsal skin.
Alternatively, the cells themselves may be infected with
recombinant adenovirus, such as one expressing zcytor17lig, before
implantation so that the protein is synthesized at the tumor site
or intracellularly, rather than systemically. The mice normally
develop visible tumors within 5 days. The tumors are allowed to
grow for a period of up to 3 weeks, during which time they may
reach a size of 1500-1800 mm.sup.3 in the control treated group.
Tumor size and body weight are carefully monitored throughout the
experiment. At the time of sacrifice, the tumor is removed and
weighed along with the lungs and the liver. The lung weight has
been shown to correlate well with metastatic tumor burden. As an
additional measure, lung surface metastases are counted. The
resected tumor, lungs and liver are prepared for histopathological
examination, immunohistochemistry, and in situ hybridization, using
methods known in the art and described herein. The influence of the
expressed polypeptide in question, e.g., zcytor17lig, on the
ability of the tumor to recruit vasculature and undergo metastasis
can thus be assessed. In addition, aside from using adenovirus, the
implanted cells can be transiently transfected with zcytor17lig.
Use of stable zcytor17lig transfectants as well as use of
induceable promoters to activate zcytor17lig expression in vivo are
known in the art and can be used in this system to assess
zcytor17lig induction of metastasis. Moreover, purified zcytor17lig
or zcytor17lig conditioned media can be directly injected in to
this mouse model, and hence be used in this system. For general
reference see, O'Reilly M S, et al. Cell 79:315-328, 1994; and
Rusciano D, et al. Murine Models of Liver Metastasis. Invasion
Metastasis 14:349-361, 1995.
[0226] A soluble multimeric cytokine receptor of the present
invention or antibodies thereto may be useful in treating
tumorgenesis, and therefore would be useful in the treatment of
cancer. Zcytor17lig is expressed in activated T-cells, monocytes
and macrophages, and is linked to a region of the human chromosome
wherein translocations are common in leukemias. Moreover, the
zcytor17lig is shown to act through a cytokine receptor, zcytor17
multimeric cytokine receptor, which is also expressed in activated
T-cells, monocytes and macrophages. Over stimulation of activated
T-cells, monocytes and macrophages by zcytor17lig could result in a
human disease state such as an immune cell cancer. As such,
identifying zcytor17lig expression, polypeptides (e.g., by
anti-zcytor17lig antibodies, zcytor17 soluble multimeric cytokine
receptors (e.g., zcytor17 receptor, heterodimers (e.g.,
zcytor17/OSMRbeta, zcytor17/WSX-1), multimers (e.g.,
zcytor17/OSMRbeta/WSX-1)), or other zcytor17lig binding partners)
can serve as a diagnostic, and can serve as antagonists of
zcytor17lig proliferative activity. The ligand could be
administered in combination with other agents already in use
including both conventional chemotherapeutic agents as well as
immune modulators such as interferon alpha. Alpha/beta interferons
have been shown to be effective in treating some leukemias and
animal disease models, and the growth inhibitory effects of
interferon-alpha and zcytor17lig may be additive.
[0227] NK cells are thought to play a major role in elimination of
metastatic tumor cells and patients with both metastases and solid
tumors have decreased levels of NK cell activity (Whiteside et.
al., Curr. Top. Microbiol. Immunol 230:221-244, 1998). An agent
that stimulates NK cells would be useful in the elimination of
tumors.
[0228] The present invention provides a method of reducing
proliferation of a neoplastic monocytes/macrophages comprising
administering to a mammal with a monocyte/macrophage neoplasm an
amount of a composition including a soluble multimeric cytokine
receptor or antibody thereto sufficient to reduce proliferation of
the neoplastic monocytes/macrophages.
[0229] The present invention provides a method for inhibiting
activation or differentiation of monocytes/macrophages. Monocytes
are incompletely differentiated cells that migrate to various
tissues where they mature and become macrophages. Macrophages play
a central role in the immune response by presenting antigen to
lymphocytes and play a supportive role as accessory cells to
lymphocytes by secreting numerous cytokines. Macrophages can
internalize extracellular molecules and upon activation have an
increased ability to kill intracellular microorganisms and tumor
cells. Activated macrophages are also involved in stimulating acute
or local inflammation.
[0230] In another aspect, the present invention provides a method
of reducing proliferation of a neoplastic B or T-cells comprising
administering to a mammal with a B or T cell neoplasm an amount of
a composition including a soluble multimeric cytokine receptor
sufficient to reducing proliferation of the neoplastic
monocytes/macrophages. Furthermore, the zcytor17lig antagonist can
be a ligand/toxin fusion protein.
[0231] A zcytor17 multimeric cytokine receptor-saporin fusion toxin
may be employed against a similar set of leukemias and lymphomas,
extending the range of leukemias that can be treated with a
cytokine antagonsist. For example, such leukemias can be those that
over-express zcytor17 receptors (e.g., zcytor17 receptor,
heterodimers (e.g., zcytor17/OSMRbeta, zcytor17/WSX-1), multimers
(e.g., zcytor17/OSMRbeta/WSX)). Fusion toxin mediated activation of
the zcytor17 receptor, zcytor17 receptor heterodimers or multimers
(e.g., zcytor17/OSMRbeta, zcytor17/WSX-1 or zcytor17/WSX-1/OSMR)
provides two independent means to inhibit the growth of the target
cells, the first being identical to the effects seen by the ligand
alone, and the second due to delivery of the toxin through receptor
internalization. The lymphoid and monocyte restricted expression
pattern of the zcytor17 receptor suggests that the ligand-saporin
conjugate can be tolerated by patients.
[0232] The tissue distribution of receptors for a given cytokine
offers a strong indication of the potential sites of action of that
cytokine. Expression of zcytor17 was seen in monocytes and B-cells,
with a dramatic increase of expression upon activation for CD3+,
CD4+, and CD8+ T-cells. In addition, two monocytic cell lines,
THP-1 (Tsuchiya et al., Int. J. Cancer 26:171-176, 1980) and U937
(Sundstrom et al., Int. J. Cancer 17:565-577, 1976), were also
positive for zcytor17 expression.
[0233] Northern analysis of WSX-1 receptor revealed transcripts in
all tissues examined, with increased levels of expression in human
spleen, thymus, lymph node, bone marrow, and peripheral blood
leukocytes. Also, expression levels of WSX-1 increased upon
activation of T-cells.
[0234] Expression of OSMR is reported to be very broad (Mosley et
al, JBC 271:32635-32643, 1996). This distribution of zcytor17,
WSX-1, and OSMRbeta receptors supports a role for zcytor17lig in
immune responses, especially expansion of T-cells upon activation
or a role in the monocyte/macrophage arm of the immune system.
[0235] Thus, particular embodiments of the present invention are
directed toward use of a soluble multimeric cytokine receptor, for
instance zcytor17/WSX-1/OSMR, and zcytor17/OSMR 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, CD4+ and CD8+ cells),
suppression of immune response to a pathogen or antigen. Moreover
the presence of zcytor17 expression in activated immune cells such
as activated CD4+ and CD19+cells showed that zcytor17 receptor 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. As such, antibodies and binding partners of the present
invention that are agonistic or antagonistic to zcytor17 receptor
function, such as a soluble zcytor17 multimeric cytokine receptor,
can be used to modify immune response and inflammation.
[0236] The zcytor17lig structure and tissue expression suggests a
role in early hematopoietic or thymocyte development and immune
response regulation or inflammation. These processes involve
stimulation of cell proliferation and differentiation in response
to the binding of one or more cytokines to their cognate receptors.
In view of the tissue distribution observed for this zcytor17lig,
agonists (including the natural receptor(s)) and antagonists have
enormous potential in both in vitro and in vivo applications.
Compounds identified as zcytor17lig agonists are useful for
stimulating proliferation and development of target cells in vitro
and in vivo. For example, agonist compounds, zcytor17lig, or
anti-zcytor17lig antibodies, are useful as components of defined
cell culture media, and may be used alone or in combination with
other cytokines and hormones to replace serum that is commonly used
in cell culture. Agonists are thus useful in specifically promoting
the growth and/or development or activation of monocytes, T-cells,
B-cells, and other cells of the lymphoid and myeloid lineages, and
hematopoietic cells in culture.
[0237] The molecules of the present invention have particular use
in the monocyte/macrophage arm of the immune system. Methods are
known that can assess such activity. For example, interferon gamma
(IFN.gamma.) is a potent activator of mononuclear phagocytes. For
example, an increase in expression of zcytor17 upon activation of
THP-1 cells (ATCC No. TIB-202) with interferon gamma could suggest
that this receptor is involved in monocyte activation. Monocytes
are incompletely differentiated cells that migrate to various
tissues where they mature and become macrophages. Macrophages play
a central role in the immune response by presenting antigen to
lymphocytes and play a supportive role as accessory cells to
lymphocytes by secreting numerous cytokines. Macrophages can
internalize extracellular molecules and upon activation have an
increased ability to kill intracellular microorganisms and tumor
cells. Activated macrophages are also involved in stimulating acute
or local inflammation. Moreover, monocyte-macrophage function has
been shown to be abnormal in a variety of diseased states. For
example see, Johnston, R B, New Eng. J. Med. 318:747-752, 1998.
[0238] One of skill in the art would recognize that agonists of
zcytor17 multimeric cytokine receptor, such as zcytor17lig, are
useful. For example, depressed migration of monocytes has been
reported in populations with a predisposition to infection, such as
newborn infants, patients receiving corticosteroid or other
immunosuppressive therapy, and patients with diabetes mellitus,
burns, or AIDS. Agonists for zcytor17 multimeric cytokine receptor,
such as zcytor17lig, could result in an increase in the ability of
monocytes to migrate and possibly prevent infection in these
populations. There is also a profound defect of phagocytic killing
by mononuclear phagocytes from patients with chronic granulomatous
disease. This results in the formation of subcutaneous abscesses,
as well as abscesses in the liver, lungs, spleen, and lymph nodes.
An agonist of zcytor17 multimeric cytokine receptor, such as
zcytor17lig, could correct or improve this phagocytic defect. In
addition, defective monocyte cytotoxicity has been reported in
patients with cancer and Wiskott-Aldrich syndrome (eczema,
thrombocytopenia, and recurrent infections). Activation of
monocytes by agonists of zcytor17 multimeric cytokine receptor,
such as zcytor17lig, could aid in treatment of these conditions.
The monocyte-macrophage system is prominently involved in several
lipid-storage diseases (sphingolipidoses) such as Gaucher's
disease. Resistance to infection can be impaired because of a
defect in macrophage function, which could be treated by agonists
to zcytor17 multimeric cytokine receptor such as zcytorI7
.mu.g.
[0239] Moreover, one of skill in the art would recognize that
antagonists of a zcytor17 multimeric cytokine receptor are useful.
For example, in atherosclerotic lesions, one of the first
abnormalities is localization of monocyte/macrophages to
endothelial cells. These lesions could be prevented by use of
antagonists to zcytor17lig. Zcytor17 soluble multimeric cytokine
receptors, such as, for instance, heterodimers and trimers, can
also be used as antagonists to the zcytor17lig. Moreover,
monoblastic leukemia is associated with a variety of clinical
abnormalities that reflect the release of the biologic products of
the macrophage, examples include high levels of lysozyme in the
serum and urine and high fevers. Moreover, such leukemias exhibit
an abnormal increase of monocytic cells. These effects could
possibly be prevented by antagonists to zcytor17lig, such as
described herein.
[0240] Using methods known in the art, and disclosed herein, one of
skill could readily assess the activity of a zcytor17 multimeric
cytokine receptor in the disease states disclosed herein,
inflammation, cancer, or infection as well as other disease states
involving monocytic cells. In addition, as zcytor17lig is expressed
in a T-cell, macrophage and monocyte-specific manner, and these
diseases involve abnormalities in monocytic cells, such as cell
proliferation, function, localization, and activation, the
polynucleotides, polypeptides, and antibodies of the present
invention can be used to as diagnostics to detect such monocytic
cell abnormalities, and indicate the presence of disease. Such
methods involve taking a biological sample from a patient, such as
blood, saliva, or biopsy, and comparing it to a normal control
sample. Histological, cytological, flow cytometric, biochemical and
other methods can be used to determine the relative levels or
localization of zcytor17lig, or cells expressing zcytor17lig, i.e.,
monocytes, in the patient sample compared to the normal control. A
change in the level (increase or decrease) of zcytor17lig
expression, or a change in number or localization of monocytes
(e.g., increase or infiltration of monocytic cells in tissues where
they are not normally present) compared to a control would be
indicative of disease. Such diagnostic methods can also include
using radiometric, fluorescent, and colorimetric tags attached to
polynucleotides, polypeptides or antibodies of the present
invention. Such methods are well known in the art and disclosed
herein.
[0241] Amino acid sequences having zcytor17lig activity can be used
to modulate the immune system by binding soluble zcytor17
multimeric cytokine receptor, and thus, preventing the binding of
zcytor17lig with endogenous zcytor17lig receptor. Zcytor17lig
antagonists, such as a zcytor17 multimeric cytokine receptor, can
also be used to modulate the immune system by inhibiting the
binding of Zcytor17 ligand with the endogenous zcytor17lig
receptor. Accordingly, the present invention includes the use of a
multimeric cytokine receptor that can be also used to treat a
subject which produces an excess of either zcytor17lig or Zcytor17
comprising receptor(s). Suitable subjects include mammals, such as
humans or veterinary animals.
[0242] Zcytor17lig has been shown to be expressed in activated
mononuclear cells, and may be involved in regulating inflammation.
As such, polypeptides of the present invention can be assayed and
used for their ability to modify inflammation, or can be used as a
marker for inflammation. Methods to determine proinflammatory and
antiinflammatory qualities of zcytor17lig are known in the art and
discussed herein. Moreover, it may be involved in up-regulating the
production of acute phase reactants, such as serum amyloid A (SAA),
.alpha.1-antichymotrypsin, and haptoglobin, and that expression of
zcytor17 receptor ligand may be increased upon injection of
lipopolysaccharide (LPS) in vivo that are 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 a 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, where a ligand such as zcytor17lig that
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 the ligand. Such antagonists are provided by the present
invention. For example, a method of reducing inflammation comprises
administering to a mammal with or at risk of developing
inflammation an amount of a composition of a soluble multimeric
cytokine receptor that is 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 multimeric 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.
[0243] Like zcytor17lig, analysis of the tissue distribution of the
mRNA corresponding it's zcytor17 receptor cDNA showed that mRNA
level was highest in monocytes and prostate cells, and is elevated
in activated monocytes, and activated CD4+, activated CD8+, and
activated CD3+ cells. Hence, zcytor17 receptor is also implicated
in inducing inflammatory and immune response. Thus, particular
embodiments of the present invention are directed toward use of
zcytor17lig-antibodies, and zcytor17lig, as well as soluble
zcytor17 receptor 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, CD4+ and CD8+ cells), suppression of immune response to a
pathogen or antigen. Moreover the presence of zcytor17 receptor and
zcytor17lig expression in activated immune cells such as activated
CD3+, monocytes, CD4+ and CD19+ cells showed that zcytor17 receptor
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. As such, zcytor17lig and zcytor17lig-antibodies of the
present invention that are agonistic or antagonistic to zcytor17
receptor function, can be used to modify immune response and
inflammation.
[0244] Moreover, zcytor17lig polypeptides that bind zcytor17
multimeric cytokine receptors and antibodies thereto are useful
to:
[0245] Antagonize or block signaling via a zcytor17 multimeric
cytokine receptor 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.
[0246] Antagonize or block signaling via the zcytor17 multimeric
cytokine receptor 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 zcytor17 receptor (Hughes C et al., J. Immunol 153:
3319-3325 (1994)). Asthma, allergy and other atopic disease may be
treated with an MAb against, for example, soluble zcytor17
multimeric cytokine receptors or zcytor17/CRF2-4 heterodimers, to
inhibit the immune response or to deplete offending cells. Blocking
or inhibiting signaling via zcytor17 multimeric cytokine receptor,
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. Zcytor17 multimeric cytokine receptor 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 zcytor17 receptor 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 TI diabetes (IDDM), renal tumors and other diseases.
[0247] Agonize or initiate signaling via the zcytor17 multimeric
cytokine receptor in the treatment of autoimmune diseases such as
IDDM, MS, SLE, myasthenia gravis, rheumatoid arthritis, and IBD.
Zcytor17lig 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, zcytor17lig may be used to
signal, deplete and deviate immune cells involved in asthma,
allergy and atopoic disease. Signaling via zcytor17 multimeric
cytokine receptor may also benefit diseases of the pancreas,
kidney, pituitary and neuronal cells. IDDM, NIDDM, pancreatitis,
and pancreatic carcinoma may benefit. Zcytor17 multimeric cytokine
receptor 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 T-cell specific leukemias, lymphomas,
plasma cell dyscrasia (e.g., multiple myeloma), and carcinoma may
be treated with monoclonal antibodies (e.g., neutralizing antibody)
to zcytor17-comprising soluble receptors of the present
invention.
[0248] Soluble zcytor17 multimeric cytokine receptors as described
herein can be used to neutralize/block zcytor17 receptor ligand
activity in the treatment of autoimmune disease, atopic disease,
NIDDM, pancreatitis and kidney dysfunction as described above. A
soluble form of zcytor17 multimeric cytokine receptor may be used
to promote an antibody response mediated by T cells and/or to
promote the production of IL-4 or other cytokines by lymphocytes or
other immune cells.
[0249] A soluble zcytor17 multimeric cytokine receptor may be
useful as antagonists of zcytor17lig. Such antagonistic effects can
be achieved by direct neutralization or binding of its natural
ligand. In addition to antagonistic uses, the soluble receptors can
bind zcytor17lig and act as carrier or vehicle proteins, in order
to transport zcytor17lig to different tissues, organs, and cells
within the body. As such, the soluble receptors 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, monocytes, or tumor. For example, in
acute infection or some cancers, benefit may result from induction
of inflammation and local acute phase response proteins. Thus, the
soluble receptors described herein or antibodies thereto can be
used to specifically direct the action of a pro-inflammatory
zcytor17lig ligand. See, Cosman, D. Cytokine 5: 95-106 (1993); and
Femandez-Botran, R. Exp. Opin. Invest. Drugs 9:497-513 (2000).
[0250] Moreover, the soluble zcytor17 multimeric cytokine receptors
can be used to stabilize the zcytor17lig, 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,
Zcytor17 may be combined with a cognate ligand such as its ligand
to comprise a ligand/soluble receptor complex. Such complexes may
be used to stimulate responses from cells presenting a companion
receptor subunit. The cell specificity of zcytor17 multimeric
cytokine receptor/zcytor17lig 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. Zcytor17
multimeric cytokine receptor/ligand 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); and
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.
[0251] Zcytor17lig may also be used within diagnostic systems for
the detection of circulating levels of ligand, and in the detection
of acute phase inflammatory response. Within a related embodiment,
antibodies or other agents that specifically bind to zcytor17lig
can be used to detect circulating zcytor17lig polypeptides;
conversely, zcytor17lig itself can be used to detect circulating or
locally-acting receptor polypeptides. Elevated or depressed levels
of ligand or receptor polypeptides may be indicative of
pathological conditions, including inflammation or cancer.
Moreover, detection of acute phase proteins or molecules such as
zcytor17lig 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.
[0252] Polynucleotides encoding a zcytor17 multimeric cytokine
receptor are useful within gene therapy applications where it is
desired to increase or inhibit zcytor17lig activity. If a mammal
has a mutated or absent zcytor17 gene, the zcytor17 gene of the
present invention can be introduced into the cells of the mammal.
In one embodiment, a gene encoding a zcytor17 multimeric cytokine
receptor is introduced in vivo in a viral vector. Such vectors
include an attenuated or defective DNA virus, such as, but not
limited to, herpes simplex virus (HSV), papillomavirus, Epstein
Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the
like. Defective viruses, which entirely or almost entirely lack
viral genes, are preferred. A defective virus is not infective
after introduction into a cell. Use of defective viral vectors
allows for administration to cells in a specific, localized area,
without concern that the vector can infect other cells. Examples of
particular vectors include, but are not limited to, a defective
herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell.
Neurosci. 2:320-30 (1991)); an attenuated adenovirus vector, such
as the vector described by Stratford-Perricaudet et al., J. Clin.
Invest. 90:626-30 (1992); and a defective adeno-associated virus
vector (Samulski et al., J. Virol. 61:3096-101 (1987); and Samulski
et al., J. Virol. 63:3822-8 (1989)).
[0253] A zcytor17 gene of the present invention can be introduced
in a retroviral vector, e.g., as described in Anderson et al., U.S.
Pat. No. 5,399,346; Mann et al. Cell 33:153 (1983); Temin et al.,
U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;
Markowitz et al., J. Virol. 62:1120 (1988); Temin et al., U.S. Pat.
No. 5,124,263; International Patent Publication No. WO 95/07358,
published Mar. 16, 1995 by Dougherty et al.; and Kuo et al., Blood
82:845 (1993). Alternatively, the vector can be introduced by
lipofection in vivo using liposomes. Synthetic cationic lipids can
be used to prepare liposomes for in vivo transfection of a gene
encoding a marker (Felgner et al., Proc. Natl. Acad. Sci. USA
84:7413-7 (1987); Mackey et al., Proc. Natl. Acad. Sci. USA
85:8027-31 (1988)). The use of lipofection to introduce exogenous
genes into specific organs in vivo has certain practical
advantages. Molecular targeting of liposomes to specific cells
represents one area of benefit. More particularly, directing
transfection to particular cells represents one area of benefit.
For instance, directing transfection to particular cell types would
be particularly advantageous in a tissue with cellular
heterogeneity, such as the immune system, pancreas, liver, kidney,
and brain. Lipids may be chemically coupled to other molecules for
the purpose of targeting. Targeted peptides (e.g., hormones or
neurotransmitters), proteins such as antibodies, or non-peptide
molecules can be coupled to liposomes chemically.
[0254] It is possible to remove the target cells from the body; to
introduce the vector as a naked DNA plasmid; and then to re-implant
the transformed cells into the body. Naked DNA vectors for gene
therapy can be introduced into the desired host cells by methods
known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun or use of a DNA vector
transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7 (1992);
and Wu et al., J. Biol. Chem. 263:14621-4 (1988).
[0255] Antisense methodology can be used to inhibit zcytor17
multimeric cytokine receptor gene transcription, such as to inhibit
cell proliferation in vivo. Polynucleotides that are complementary
to a segment of a zcytor17-encoding polynucleotide (e.g., a
polynucleotide as set forth in SEQ ID NO:110. SEQ ID NO:108, or SEQ
ID NO:4) are designed to bind to zcytor17lig-encoding mRNA and to
inhibit translation of such mRNA. Such antisense polynucleotides
are used to inhibit expression of zcytor17lig polypeptide-encoding
genes in cell culture or in a subject.
[0256] Mice engineered to express the zcytor17lig gene, referred to
as "transgenic mice," and mice that exhibit a complete absence of
zcytor17lig 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
zcytor17lig, 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 zcytor17lig polypeptide, polypeptide fragment or a
mutant thereof may alter normal cellular processes, resulting in a
phenotype that identifies a tissue in which zcytor17lig expression
is functionally relevant and may indicate a therapeutic target for
the zcytor17lig, its agonists or antagonists. For example, a
preferred transgenic mouse to engineer is one that over-expresses
the zcytor17lig (amino acid residues 23-164 of SEQ ID NO:2; or
24-163 of SEQ ID NO:11). Moreover, such over-expression may result
in a phenotype that shows similarity with human diseases.
Similarly, knockout zcytor17lig mice can be used to determine where
zcytor17lig is absolutely required in vivo. The phenotype of
knockout mice is predictive of the in vivo effects of that a
zcytor17lig antagonist, such as a soluble zcytor17 multimeric
cytokine receptor, may have. The human or mouse zcytor17lig cDNA
described herein can be used to generate knockout mice. These mice
may be employed to study the zcytor17lig gene and the protein
encoded thereby in an in vivo system, and can be used as in vivo
models for corresponding human diseases. Moreover, transgenic mice
expression of zcytor17lig antisense polynucleotides or ribozymes
directed against zcytor17lig, described herein, can be used
analogously to transgenic mice described above. Studies may be
carried out by administration of purified zcytor17lig protein, as
well.
[0257] The present invention also provides a composition which
includes an effective amount of a soluble multimeric cytokine
receptor comprising a polypeptide comprising amino acid residue 20
to amino acid residue 543 of SEQ ID NO:111 and at least a portion
of at least one class I cytokine receptor; and a pharmaceutically
acceptable vehicle. The polypeptide may be comprised of various
fragment or portions of the extracellular domain of SEQ ID NO:111,
SEQ ID NO:109, and/or SEQ ID NO:5, such as for instance, amino acid
residue 20 to amino acid residue 227 of SEQ ID NO:111 and amino
acid residue 20 to amino acid residue 519 of SEQ ID NO:111. The at
least a portion of at least one class I cytokine receptor can
include, for example, a portion of SEQ ID NO:9 and/or a portion of
SEQ ID NO:7, such as, for instance, amino acid residue 28 to amino
acid residue 429 of SEQ ID NO:7, amino acid residue 35 to amino
acid residue 137 of SEQ ID NO:7, amino acid residue 240 to amino
acid residue 342 of SEQ ID NO:7, amino acid residue 348 to amino
acid residue 429 of SEQ ID NO:7, amino acid residue 28 to amino
acid residue 739 of SEQ ID NO:7, and/or combinations thereof. The
multimeric cytokine receptor may further include an affinity tag as
described herein.
[0258] The present invention also provides an immune cell
inhibiting composition which includes an effective amount of a
soluble multimeric cytokine receptor comprising a polypeptide
comprising amino acid residue 20 to amino acid residue 227 of SEQ
ID NO:111 and at least a portion of at least one class I cytokine
receptor; and a pharmaceutically acceptable vehicle, wherein the
soluble multimeric cytokine receptor inhibits the proliferation of
immune cells.
[0259] The present invention also provides an inflammatory cell
inhibiting composition which includes an effective amount of a
soluble multimeric cytokine receptor comprising a polypeptide
comprising amino acid residue 20 to amino acid residue 227 of SEQ
ID NO:111 and at least a portion of at least one class I cytokine
receptor; and a pharmaceutically acceptable vehicle, wherein the
soluble multimeric cytokine receptor inhibits the proliferation of
inflammatory cells.
[0260] Experimental evidence suggests a role for zcytor17lig in the
progression of diseases that involve the skin or epithelium of
internal surfaces, such as, for instance, large intestine, small
intestine, pancrease, lung, prostate, uterus, and the like. First,
as disclosed herein, zcytor17 receptors, including both OSM
receptor beta and zcytor17, are expressed in several cell types
located in epithelial surfaces including cell lines derived from
lung epithelium, lung fibroblast, prostate, colon, breast, liver
epithelium, bone and skin epithelium, bone fibroblast, and the
like. Moreover, as disclosed herein, examples from each of these
cell types also responded to zcytor17lig activation of a STAT
reporter construct. In addition, several cell lines responded to
zcytor17lig stimulation by producing increased levels of IL-6,
IL-8, MCP-1 (a chemotactic factor) as described herein. In whole,
these data suggest a role for zcytor17lig in diseases that involve
the epithelium such as, for instance, atopic dermatitis;
dermatitis; psoriasis; psoriatic arthritis; eczema; gingivitis;
peridontal disease; inflammatory bowel diseases (IBD) (e.g.,
ulcerative colitis, Crohn's disease); reproductive disorders, such
as, for instance, cervical dysplasia, cervical cancer; other skin
diseases like cancers: sarcomas; carcinomas; melanoma, etc. i.e,
not just inflammatory diseases, since immune system is involved in
activating/curing cancers; diseases involving barrier dysfunction,
such as, for instance, graft-versus-host disease (GVHD) and
irritable bowel syndrome (IBS); and diseases that involve lung
epithelium, such as asthma, emphysema, and the like. In addition,
the release of cytokines IL-6, IL-8, and MCP-1 by cells exposed to
zcytor17lig suggests that zcytor17lig is involved in inflammation.
Therefore, regulation of zcytor17lig can be useful in the treatment
of autoimmune, inflammatory, or cancerous diseases associated with
the tissues that express receptor. These diseases include, for
example, prostatitis, hepatitis, osteoarthritis, and the like.
Zcytor17lig may positively or negatively directly or indirectly
regulate these diseases. Therefore, the administration of
zcytor17lig can be used to treat diseases as described herein
directly or with molecules that inhibit zcytor17lig activity
including, for example, both monoclonal antibodies to zcytor17lig
or monoclonal antibodies to zcytor17, or monoclonal antibodies that
recognize the zcytor17 and OSM receptor beta complex.
[0261] Data also suggests that zcytor17lig may be involved in the
regulation of TH2 T cell mediated diseases. First, zcytor17lig is
made by the TH2 subset of activated T cells. TH2 cells express more
zcytor17lig as compared to THI cells. In addition, at least two
lung epithelial cell lines (SK-LU-1, A549) were stimulated to
increase IL13 receptor alpha-2 mRNA in response to zcyto17 ligand
stimulation as described herein. There is an association of IL-13
receptor alpha2 chain and tumorigenicity of human breast and
pancreatic tumors. This suggests that zcytor17lig may play a role
in regulating tumorigenicity of these types of cancers, as well as
other cancers. Therefore, the administration of a zcytor17lig
antagonist or direct use of zcytor17lig may be useful in treatment
of these types of cancers, benign or malignant and at various
grades (grades I-IV) and stages (e.g., TNM or AJC staging methods)
of tumor development, in mammals, preferably humans.
[0262] It is well-known in the art that IL13 is involved in the
generation of activated TH2 cells and in TH2 mediated diseases,
such as asthma, atopic dermatitis, and the like. Zcytor17lig or
zcytor17lig antagonists may be useful in the treatment of diseases
that involved TH2 T cells. This would include diseases such as, for
instance, atopic dermatitis, asthma, as well as other diseases that
are exacerbated by activated TH2 cells. The involvement of
zcytor17lig in diseases, such as, for instance, atopic dermatitis,
is also supported by the phenotype of the transgenic mice that
overexpress zcytor17lig and develop symptoms of atopic dermatitis
as described herein.
[0263] Despite the preferential expression of zcytor17lig by TH2
cells, there is still some expression of zcytor17lig in TH1 cells
and in CD8+ T cells. Therefore, zcytor17lig or its antagonists may
be useful in treating diseases that involve immune modulation of
activated T cells including, for example, viral infection, cancers,
graft rejection, and the like.
[0264] Zcytor17lig may also be involved in the development of
cancer. There is expression of the zcytor17 and OSM receptor beta
receptors in human bone fibroblast osteosarcomas, human skin
fibroblast melanoma, colon epithelial carcinoma, adenocarcinoma,
breast epithelial adenocarcinoma, prostate epithelial adenosarcoma,
and lung epithelial adenocarcinoma and carcinoma. Therefore, it may
be useful to treat tumors of epithelial origin with either
zcytor17lig, fragments thereof, or zcytor17lig antagonists which
include, but are not limited to, carcinoma, adenocarcinoma, and
melanoma. Notwithstanding, zcytor17lig or a zcytor17lig antagonist
may be used to treat a cancer, or reduce one or more symptoms of a
cancer, from a cancer including but not limited to, squamous cell
or epidermoid carcinoma, basal cell carcinoma, adenocarcinoma,
papillary carcinoma, cystadenocarcinoma, bronchogenic carcinoma,
bronchial adenoma, melanoma, renal cell carcinoma, hepatocellular
carcinoma, transitional cell carcinoma, choriocarcinoma, seminoma,
embryonal carcinoma, malignant mixed tumor of salivary gland
origin, Wilms' tumor, immature teratoma, teratocarcinoma, and other
tumors comprising at least some cells of epithelial origin.
[0265] Generally, the dosage of administered zcytor17lig
polypeptide (or 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 zcytor17lig 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. One skilled in the art can readily determine
such dosages, and adjustments thereto, using methods known in the
art.
[0266] Administration of a zcytor17 multimeric receptor agonist or
antagonist to a subject can be topical, inhalant, 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.
[0267] 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 Zcytor17 multimeric receptor agonist or antagonist 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 Detiv. 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 Zcytor17 multimeric receptor
binding activity (Potts et al, Pharm. Biotechnol 10:213
(1997)).
[0268] A pharmaceutical composition comprising a protein,
polypeptide, or peptide having Zcytor17 multimeric receptor 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 vehicle. A composition is said to be in a
"pharmaceutically acceptable vehicle" if its administration can be
tolerated by a recipient patient. Sterile phosphate-buffered saline
is one example of a pharmaceutically acceptable vehicle. Other
suitable vehicles are well-known to those in the art. See, for
example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th
Edition (Mack Publishing Company 1995).
[0269] For purposes of therapy, molecules having Zcytor17
multimeric receptor binding activity and a pharmaceutically
acceptable vehicle are administered to a patient in a
therapeutically effective amount. A combination of a protein,
polypeptide, or peptide having Zcytor17 multimeric receptor binding
activity and a pharmaceutically acceptable vehicle is said to be
administered in a "therapeutically effective amount" or "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 at least a portion of the
inflammatory response.
[0270] A pharmaceutical composition comprising Zcytor17lig (or
Zcytor17lig analog or fusion protein) can be furnished in liquid
form, in an aerosol, or in solid form. Liquid forms, are
illustrated by injectable solutions, aerosols, droplets,
topological 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
at., Pharm. Biotechnot. 10:239 (1997); Ranade, "Implants in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 95-123 (CRC Press 1995); Bremer et at., "Protein Delivery
with Infusion Pumps," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);
Yewey et at., "Delivery of Proteins from a Controlled Release
Injectable Implant," in Protein Delivery: Physical Systems, Sanders
and Hendren (eds.), pages 93-117 (Plenum Press 1997)). Other solid
forms include creams, pastes, other topological applications, and
the like.
[0271] 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 at.,
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.
[0272] 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 at.,
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.
[0273] 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 at., 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 at., Biochim. Biophys. Acta
1068:133 (1991); Allen et at., Biochim. Biophys. Acta 1150:9
(1993)).
[0274] 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 at., Japanese Patent
04-244,018; Kato et at., 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)).
[0275] 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. BuIL20:259 (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.
[0276] 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)).
[0277] Polypeptides having Zcytor17 multimeric receptor 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 at.,
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 at., Biochim. Biophys. Acta 1150:9 (1993)).
[0278] 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 vehicles for intravenous
administration of therapeutic proteins (see, for example, Gref et
al, Pharm. Biotechnol. 10:167 (1997)).
[0279] The present invention also contemplates chemically modified
polypeptides having binding Zcytor17 multimeric receptor activity
such as zcytor17 multimeric receptor heterodimeric or multimeric
soluble receptors, and Zcytor17 multimeric receptor antagonists,
for example anti-zcytor17 multimeric receptor antibodies or binding
polypeptides, which a polypeptide is linked with a polymer, as
discussed above.
[0280] 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).
[0281] As an illustration, pharmaceutical compositions may be
supplied as a kit comprising a container that comprises a
polypeptide with a Zcytor17 multimeric receptor extracellular
domain, e.g., zcytor17 multimeric receptor heterodimeric or
multimeric soluble receptors, or a Zcytor17 multimeric receptor
antagonist (e.g., a neutralizing antibody or antibody fragment that
binds a Zcytor17 multimeric receptor 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 Zcytor17 multimeric receptor
composition is contraindicated in patients with known
hypersensitivity to Zcytor17 multimeric receptor.
[0282] The complete disclosure of all patents, patent applications,
and publications, and electronically available material (e.g.,
GenBank amino acid and nucleotide sequence submissions) cited
herein are incorporated by reference. The foregoing detailed
description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described, for variations obvious to one skilled in the art
will be included within the invention defined by the claims.
EXAMPLES
Example 1
Construction of MPL-zcytor17 Polypeptide Chimera: MPL Extracellular
and TM Domain Fused to the zcytor17 Intracellular Signaling
Domain
[0283] The 5' extracellular domain of the murine MPL receptor was
isolated from a plasmid containing the murine MPL receptor
(PHZ1/MPL plasmid) by digestion with EcoRI and BamHI generating a
1164 bp fragment. The digestion was run on a 1% agarose gel and the
fragment was isolated using the Qiaquick gel extraction kit
(Qiagen) as per manufacturer's instructions. The rest of the MPL
extracellular domain and transmembrane domain were generated using
PCR with primers ZC6,673 (SEQ ID NO:13) and ZC29,082 (SEQ ID
NO:14). The reaction conditions were as follows: 15 cycles at
94.degree. C. for 1 min., 55.degree. C. for 1 min., 72.degree. C.
for 2 min.; followed by 72.degree. C. for 7 min.; then a 4.degree.
C. soak. The PCR product was run on a 1% agarose gel and the
approximately 400 bp MPL receptor fragment was isolated using
Qiaquick.TM. gel extraction kit (Qiagen) as per manufacturer's
instructions.
[0284] The intracellular domain of human zcytor17 was isolated from
a plasmid containing zcytor17 receptor cDNA (#23/pCAP) using PCR
with primers ZC29,083 (SEQ ID NO:15) and ZC29,145 (SEQ ID NO:16).
The polynucleotide sequence that corresponds to the zcytor17
receptor coding sequence is shown in SEQ ID NO:5. The reaction
conditions were as per above. The PCR product was run on a 1%
agarose gel and the approximately 320 bp zcytor17 fragment isolated
using Qiaquick gel extraction kit as per manufacturer's
instructions.
[0285] Each of the isolated PCR fragments described above were
mixed at a 1:1 volumetric ratio and used in a PCR reaction using
ZC6673 (SEQ ID NO:13) and ZC29145 (SEQ ID NO:16) to create all but
the 5' MPL portion of the MPL-zcytor17 chimera. The reaction
conditions were as follows: 15 cycles at 94.degree. C. for 1 min.,
55.degree. C. for 1 min., 72.degree. C. for 2 min.; followed by
72.degree. C. for 7 min.; then a 4.degree. C. soak. The entire PCR
product was run on a 1% agarose gel and the approximately 700 bp
MPL-zcytor17 chimera fragment isolated using Qiaquick gel
extraction kit (Qiagen) as per manufacturer's instructions. The
MPL-zcytor17 chimera fragment was digested with BamHI (BRL) and
XbaI (Boerhinger Mannheim) as per manufacturer's instructions. The
entire digest was run on a 1% agarose gel and the cleaved
MPL-zcytor17 chimera isolated using Qiaquick.TM. gel extraction kit
(Qiagen) as per manufacturer's instructions. The resultant cleaved
MPL-zvytorl7 chimera plus 5' MPL EcoRI/BamHI fragment described
above were inserted into an expression vector to generate the full
MPL-zcytor17 chimeric receptor as described below.
[0286] Recipient expression vector pZP-7 was digested with EcoRI
(BRL) and XbaI (BRL) as per manufacturer's instructions, and gel
purified as described above. This vector fragment was combined with
the EcoRI and XbaI cleaved MPL-zcytor17 PCR chimera isolated above
and the EcoRI and BamHI 5' MPL fragment isolated above in a
ligation reaction. The ligation was run using T4 Ligase (Epicentre
Technologies), at room temperature for 1 hour as per manufacturer's
instructions. A sample of the ligation was electroporated into
DH10B ElectroMAX.TM. electrocompetent E. coli cells (25 .mu.F,
200.OMEGA., 1.8V). Transformants were plated on LB+Ampicillin
plates and single colonies screened by miniprep (Qiagen) and
digestion with EcoRT to check for the MPL-zcytor17 chimera. EcoRT
digestion of correct clones yield about a 2 kb fragment.
Confirmation of the MPL-zcytor17 chimera sequence was made by
sequence analyses. The insert was approximately 3.1 kb, and was
full-length.
Example 2
MPL-zcytor17 Chimera Based Proliferation in BAF3 Assay Using Alamar
Blue
A. Construction of BaF3 Cells Expressing MPL-zcytor17 Chimera
[0287] 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, 1-2 ng/ml murine IL-3 (mTL-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, pZP-7/MPL-zcytor17 plasmid DNA was
prepared and purified using a Qiagen Maxi Prep kit (Qiagen) as per
manufacturer's instructions. BaF3 cells for electroporation were
washed twice in RPMI media and then resuspended in 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 pZP-7/MPL-zcytor17 plasmid DNA and
transferred to separate disposable electroporation chambers (GIBCO
BRL). At room temperature cells were given 5.times.0.1 msec shocks
at 800 volts followed by 5.times.2 ms shocks at 600 volts delivered
by an electroporation apparatus (Cyto-Pulse). Alternatively, cells
were electroporated with two serial pulses (800 .mu.FAD/300 V;
followed by 1180 .mu.FAD/300 V) delivered by a Cell-Porator
(GibcoBRL) electroporation apparatus. 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). Then Geneticin.TM.
(Gibco) selection (1 mg/ml G418) was added to the cells in a T-162
flask to isolate the G418-resistant pool. Pools of the transfected
BaF3 cells, hereinafter called BaF3/MPL-zcytor17 cells, were
assayed for signaling capability as described below.
B. Testing the Signaling Capability of the BaF3/MPL-zcytor17 Cells
Using an Alamar Blue Proliferation Assay
[0288] BaF3/MPL-zcytor17 cells were spun down and washed in the
complete media, described 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.
[0289] Proliferation of the BaF3/MPL-zcytor17 cells was assessed
using murine thrombopoietin (mTPO) diluted with mIL-3 free media to
200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml,
3.1 ng/ml, 1.5 ng/ml concentrations. One hundred microliters of the
diluted mTPO was added to the BaF3/MPL-zcytor17 cells. The total
assay volume was 200 .mu.l. Negative controls were run in parallel
using mIL-3 free media only, without the addition of mTPO. 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. Alamar Blue gives a fluorometric readout based on
the metabolic activity of 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 (Emission), or a Wallac Victor
2 plate reader (PerkinElmer Life Sciences, Boston, Mass.).
[0290] Results confirmed the signaling capability of the
intracellular portion of the zcytor17 receptor, as the
thrombopoietin induced proliferation at approximately 9-13 fold
over background at mTPO concentrations of 50 ng/ml and greater.
Example 3
Construction of Expression Vector Expressing Full-Length zcytor17.
pZp7pX/zcytor17
A. Cloning of Full Length zcytor17 cDNA for Expression:
[0291] To obtain a full-length zcytor17 cDNA, 5' and 3' PCR
products were isolated and joined using an internal PstI site. The
PCR primers were designed using the nucleotide sequence SEQ ID NO:4
and include BamHI and Xho I restriction sites for cloning
purposes.
[0292] A 5' PCR product was generated using a WI-38 cDNA library as
a template and oligonucleotides ZC29,359 (SEQ ID NO:18) and
ZC27,899 (SEQ ID NO:19) as primers. WI-38 is an in-house cDNA
library generated from a human embryonic lung cell line (ATCC
CRL-2221). This 5' PCR reaction was run as follows: 30 cycles at
94.degree. C. for 1 minute, 65.degree. C. for 1 minute, 72.degree.
C. for 2 minutes, then 72.degree. C. for 7 minutes; 10.degree. C.
soak. The PCR reaction used approximately 3 .mu.g of plasmid
prepared from the cDNA library, 20 pmoles of each oligonucleotide,
and five units of PWO DNA polymerase (Roche). About 90% of the 5'
PCR product was ethanol precipitated, digested with BamHI and PstI
and gel purified on a 1.0% agarose gel. The approximately 600 bp
band was excised and used for ligation to the cloning vector pUC18
digested with BamHI and PstI. The resulting transformants were
sequenced to confirm the zcytor17 cDNA sequence. For one of these
transformants, plasmid DNA was prepared and digested with BamHI and
PstI. The resulting approximately 600 bp band was gel purified and
used for a ligation below to form a full-length cDNA.
[0293] A 3' PCR product was generated using a human testes in-house
cDNA library as a template and oligonucleotides ZC27,895 (SEQ ID
NO:20) and ZC29,122 (SEQ ID NO:21) as primers. This 3' PCR reaction
was run as follows: 30 cycles at 94.degree. C. for 45 seconds,
65.degree. C. for 45 seconds, 72.degree. C. for 2 minutes, then
72.degree. C. for 7 minutes; 10.degree. C. soak. The entire 3' PCR
reaction was gel purified on a 1.0% agarose gel and the major 1500
bp band excised. This band was cloned into the PCR Blunt II TOPO
vector using the Zeroblunt TOPO kit (Invitrogen). The resulting
transformants were sequenced to confirm the zcytor17 cDNA sequence.
For one of these transformants, plasmid DNA was prepared and
digested with PstI and XhoI. The resulting approximately 1500 bp
band was gel purified. A three-part ligation was performed with the
5' BamHI to Pst I fragment above, the 3' PstI to XhoI fragment, and
the expression vector pZp7pX digested with BamHI and XhoI. This
generated a pZp7pX plasmid containing a full-length cDNA for
zcytor17 (SEQ ID NO:4), designated pZp7p/zcytor17. The full length
zcytor17 cDNA in pZp7p/zcytor17 has a silent mutation that changes
the T to G at position 1888 of SEQ ID NO:4 (encoding a Gly residue
at residue 464 of SEQ ID NO:5). As this mutation is silent, the
zcytor17 cDNA in pZp7p/zcytor17 encodes the polypeptide as shown in
SEQ ID NO:5. Plasmid pZp7pX is a mammalian expression vector
containing an expression cassette having the CMV promoter, intron
A, multiple restriction sites for insertion of coding sequences,
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, enhancer and origin of
replication, a puromycin resistance gene and the SV40
terminator.
B. Construction of Expression Vector Expressing Full-Length
WSX-1
[0294] The entire WSX-1 receptor (SEQ ID NO:9) was isolated from a
plasmid containing the WSX-1 receptor cDNA (SEQ ID NO:8) (U.S. Pat.
No. 5,925,735). hWSX-1/pBluescript SK(+) plasmid DNA (Stratagene,
La Jolla, Calif.) was digested with EcoRI and XhoI to generate a
1075 bp fragment, and also digested with XhoI and XbaI to generate
a 900 bp fragment. Both digests were run on a 1% agarose gel and
the cleaved WSX-1 fragments isolated.
[0295] Recipient expression vector pZp7Z was digested with EcoRI
and XbaI and gel purified as described above. This vector fragment
was combined with the two cleaved zcytor17 fragments isolated above
in a ligation reaction using T4 Ligase (BRL). The ligation was
incubated at room temperature overnight. A sample of the ligation
was electroporated in to DH10B electroMAX.TM. electrocompetent E.
coli cells (25 .mu.F, 200.OMEGA., 2.3V). Six colonies were grown in
culture and miniprepped DNA was prepared and digested to confirm
the correct WSX-1 full-length insert of 2.0 kb. The resulting
plasmid is pZPZ7Z/WSX-1.
Example 4
Zcytor17 Based Proliferation in BAF3 Assay Using Alamar Blue
A. Construction of BaF3 Cells Expressing zcytor17 Receptor, WSX-1
Receptor and OSMR
[0296] BaF3 cells expressing the full-length zcytor17 receptor were
constructed as per Example 2A above, using 30 .mu.g of the zcytor17
expression vector, described in Example 3A. One exception is that
in place of Geneticin selection, 2 .mu.g/ml of Puromycin (ClonTech)
was added to the transfected cells in a T-162 flask to isolate the
puromycin-resistant pool. The BaF3 cells expressing the zcytor17
receptor mRNA were designated as BaF3/zcytor17. To obtain clones,
Baf3/zcytor17 cells were counted in a hemocytometer and plated at 1
cell/well, 0.5 cell/well, 0.1 cell/well, and 0.01 cell/well in
96-well dishes. Fifteen clones were scaled up to T75 flasks, and
five clones were assayed for zcytor17 expression. Total RNA was
isolated from cell pellets using a S.N.A.P..TM. total RNA Isolation
Kit (InVitrogen). First-strand cDNA was synthesized using the
proSTAR.TM. First Strand RT-PCR kit, and then PCR with zcytor17
specific primers ZC29,180 (SEQ ID NO:22) and ZC29,122 (SEQ ID
NO:23) was performed to screen the clones for expression of
zcytor17. One clone, BaF3/zcytor17#15 was chosen to expand and
transfect with the WSX-1 expression vector.
[0297] BaF3 cells expressing zcytor17 and full-length WSX-1 were
constructed as per Example 2A above, using 30 .mu.g of the WSX-1
expression vector WSX-1/pZp7Z (Example 3B) to electroporate the
BaF3/zcytor17#15 cells. One exception is that in place of Geneticin
selection, 200 .mu.g/ml Zeocin (InVitrogen) was added to the
transfected cells in a T-162 flask to isolate the zeocin-resistant
pool. The BaF3 cells expressing zcytor17 and WSX-1 were designated
BaF3/zcytor17/hWSX-1. To obtain clones, pools of
BaB3/zcytor17/hWSX-1 cells were plated at limiting dilution in
96-well plates. The resulting clones were expanded and total RNA
was isolated using a S.N.A.P..TM. total RNA Isolation Kit
(InVitrogen). First-strand cDNA was synthesized using the
proSTAR.TM. First Strand RT-PCR kit, and then PCR with WSX-1
specific primers ZC9791 (SEQ ID NO:24) and ZC9793 (SEQ ID NO:25)
was used to screen the clones for expression of WSX-1. One clone,
BaF3/zcytor17/hWSX-1#5 was chosen to expand further and transfect
with the OSMRbeta expression vector.
[0298] BaF3 cells expressing zcytor17, WSX-1 and full-length
OSMRbeta were constructed as per Example 2A above, using 30 .mu.g
of the OSMRbeta expression vector OSMR/pZp7NX described in Example
29 to electroporate the BaF3/zcytor17/hWSX-1#5 cells. The BaF3
cells expressing zcytor17, WSX-1, and OSMRbeta mRNA were designated
BaF3/zcytor17/WSX-1/OSMR. To obtain clones, pools of
BaF3/zcytor17/WSX-1/OSMRbeta cells were plated at limiting dilution
in 96-well plates. Individual clones were expanded and total RNA
was isolated using a S.N.A.P..TM. total RNA Isolation Kit
(InVitrogen). First-strand cDNA was synthesized using the
proSTAR.TM. First Strand RT-PCR kit, and then PCR with OSMRbeta
specific primers ZC40109 (SEQ ID NO:26) and ZC40112 (SEQ ID NO:27)
was used to screen the clones for expression of zcytor17, WSX-1,
and OSMR. One clone, BaF3/zcytor17/WSX-1/OSMR#5 was selected and
these cells were used to screen for zcytor17lig as described below
in Examples 5 and 6.
B. Construction of BaF3 Cells Expressing zcytor17 Receptor and
OSMR
[0299] BaF3 cells expressing the full-length zcytor17 receptor were
constructed as per Example 2A above, using 30 .mu.g of the zcytor17
expression vector, described in Example 3A. One exception is that
in place of Geneticin selection, 2 .mu.g/ml of Puromycin (ClonTech)
was added to the transfected cells in a T-162 flask to isolate the
puromycin-resistant pool. The BaF3 cells expressing the zcytor17
receptor mRNA were designated as BaF3/zcytor17. To obtain clones,
pools of Baf3/zcytor17 cells were plated at limiting dilution in
96-well plates. These clones were expanded in culture and total RNA
was isolated using a S.N.A.P..TM. total RNA Isolation Kit
(InVitrogen). First-strand cDNA was synthesized using the
proSTAR.TM. First Strand RT-PCR kit, and then PCR was used to
screen the clones for expression of zcytor17. One clone,
BaF3/zcytor17 #15 was chosen to expand and transfect with the
OSMRbeta expression vector.
[0300] BaF3 cells expressing zcytor17 and full-length OSMRbeta were
constructed as per Example 2A above, using 30 .mu.g of the OSMRbeta
expression vector OSMR/pZp7NX (example 29) to electroporate the
BaF3/zcytor17#15 cells. The BaF3 cells expressing zcytor17 and
OSMRbeta mRNA were designated BaF3/zcytor17/OSMR. These cells were
used to screen for zcytor17lig as described below in Example 5.
Example 5
Screening for zcytor17lig Using BaF3/Zcytor17/WSX-1/OSMRbeta Cells
Using an Alamar Blue Proliferation Assay
A. Activation of CCRF-CEM and CCRF-HSB2 Cells to Test for Presence
of zcytor17lig
[0301] CCRF-CEM and CCRF-HSB2 cells were obtained from ATCC and
stimulated in culture to produce conditioned media to test for the
presence of zcytor17lig activity as described below. The suspension
cells were seeded at 2.times.10.sup.5 cells/ml or 5.times.10.sup.5
cells/ml in RPMI-1640 media supplemented with 10% FBS, 2 mM
L-glutamine (GibcoBRL), 1.times.PSN (GibcoBRL), and activated with
10 ng/ml Phorbol-12-myristate-13-acetate (PMA) (Calbiochem, San
Diego, Calif.) and 0.5 .mu.g/ml Ionomycin.TM. (Calbiochem) for 24
or 48 hrs. The supernatant from the stimulated cells was used to
assay proliferation of the BaF3/zcytor17/WSX-1/OSMRbeta cells or
BaF3/zcytor17/OSMRbeta cells as described below.
B. Screening for zcytor17lig Using BaF3/Zcytor17/WSX-1/OSMRbeta
cells or BaF3/zcytor17/OSMRbeta Cells Using an Alamar Blue
Proliferation Assay
[0302] BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta
cells were spun down and washed in 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.
[0303] Proliferation of the BaF3/zcytor17/WSX-1/OSMRbeta cells or
BaF3/zcytor17/OSMRbeta cells was assessed using conditioned media
from activated CCRFCEM and CCRF-HSB2 cells (see Example 5A).
Conditioned media was diluted with mIL-3 free media to 50%, 25%,
12.5%, 6.25%, 3.125%, 1.5%, 0.75%, and 0.375% concentrations. 100
.mu.l of the diluted conditioned media was added to the
BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells.
The total assay volume is 200 .mu.l. The assay plates were
incubated at 37.degree. C., 5% CO.sub.2 for 3-5 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. Plates were read on the Fmax.TM. plate reader (Molecular
devices) as described above (Example 2).
[0304] Results confirmed the proliferative response of the
BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells
to a factor present in the activated CCRF-CEM and CCRF-HSB2
conditioned media. The response, as measured, was approximately
10-fold over background at the 25% concentration. The untransfected
BaF3 cells did not proliferate in response to this factor, nor did
BaF3 cells transfected with zcytor17 and WSX-1 (BaF3/zcytor17/WXS-1
cells), showing that this factor was specific for Zcytor17/OSMRbeta
or zcytor17/OSMRbeta/WSX-1 receptors. Moreover soluble zcytor17
receptor diminished this proliferative activity of zcytor17lig in
the BaF3/zcytor17/WSX-1/OSMRbeta cells (see, Example 11). Similar
results are expected in BaF3/zcytor17/OSMRbeta cells.
C. Human Primary Source Used to Isolate zcytor17lig
[0305] One hundred milliliters blood draws were taken from each of
six donors. The blood was drawn using 10.times.10 ml vacutainer
tubes containing heparin. Blood was pooled from six donors (600
ml), diluted 1:1 in PBS, and separated using a Ficoll-Paque.RTM.
PLUS (Pharmacia Biotech). The isolated primary human cell yield
after separation on the ficoll gradient was 1.2.times.10.sup.9
cells.
[0306] Cells were suspended in 9.6 ml MACS buffer (PBS, 0.5% EDTA,
2 mM EDTA). 1.6 ml of cell suspension was removed and 0.4 ml CD3
microbeads (Miltenyi Biotec, Auburn, Calif.) added. The mixture was
incubated for 15 min. at 4.degree. C. These cells labeled with CD3
beads were washed with 30 ml MACS buffer, and then resuspended in 2
ml MACS buffer.
[0307] A VS+ column (Miltenyi) was prepared according to the
manufacturer's instructions. The VS+ column was then placed in a
VarioMACS.TM. magnetic field (Miltenyi). The column was
equilibrated with 5 ml MACS buffer. The isolated primary human
cells were then applied to the column. The CD3 negative cells were
allowed to pass through. The column was rinsed with 9 ml (3.times.3
ml) MACS buffer. The column was then removed from the magnet and
placed over a 15 ml falcon tube. CD3+ cells were eluted by adding 5
ml MACS buffer to the column and bound cells flushed out using the
plunger provided by the manufacturer. The incubation of the cells
with the CD3 magnetic beads, washes, and VS+ column steps
(incubation through elution) above were repeated five more times.
The resulting CD3+ fractions from the six column separations were
pooled. The yield of CD3+ selected human cells were
3.times.10.sup.8 total cells.
[0308] A sample of the pooled CD3+ selected human cells was removed
for staining and sorting on a fluorescent antibody cell sorter
(FACS) to assess their purity. The human CD3+ selected cells were
91% CD3+ cells.
[0309] The human CD3+ selected cells were activated by incubating
in RPMI+5% FBS+PMA 10 ng/ml and Tonomycin 0.5 .mu.g/ml (Calbiochem)
for 13 hours 37.degree. C. The supernatant from these activated
CD3+ selected human cells was tested for zcytor17lig activity as
described below. Moreover, the activated CD3+ selected human cells
were used to prepare a cDNA library, as described in Example 6,
below.
D. Testing Supernatant from Activated CD3+ Selected Human Cells for
zcytor17lig Using BaF3/Zcytor17/WSX-1/OSMRbeta Cells and an Alamar
Blue Proliferation Assay
[0310] BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta
cells were spun down and washed in 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.
[0311] Proliferation of the BaF3/zcytor17/WSX-1/OSMRbeta cells or
BaF3/zcytor17/OSMRbeta cells were assessed using conditioned media
from activated CD3+ selected human cells (see Example 5C) diluted
with mIL-3 free media to 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75%,
0.375% and 0.187% concentrations. 100 .mu.l of the diluted
conditioned media was added to the BaF3/zcytor17/WSX-1/OSMRbeta
cells or BaF3/zcytor17/OSMRbeta cells. The total assay volume was
200 .mu.l. The assay plates were incubated and assayed as described
in Example 5B.
[0312] Results confirmed the proliferative response of the
BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells
to a factor present in the activated CD3+ selected human Cell
conditioned media. The response, as measured, was approximately
15-fold over background at the 25% concentration. The untransfected
BaF3 cells did not proliferate in response to this factor, nor did
BaF3 cells transfected with zcytor17 and WSX-1 (BaF3/zcytor17/WXS-1
cells), showing that this factor is specific for Zcytor17/OSMRbeta
or zcytor17/OSMRbeta/WSX-1 receptors.
Example 6
Cloning of Human zcytor17lig from a Human CD3+ Selected Cell
Library
[0313] Screening of a primary human activated CD3+ selected cell
cDNA library revealed an isolated cDNA that is a novel member of
the four-helix bundle cytokine family. This cDNA encoded the
zcytor17lig. The cDNA was identified by screening for activity of
the zcytor17lig using the zcytor17/WSX-1/OSM receptors.
A. The Vector for CD3+ Selected Library Construction
[0314] The vector for CD3+ selected library construction was
pZP7NX. The pZP7NX vector was constructed as follows: The coding
region for the DHFR selective marker in vector pZP7 was removed by
DNA digestion with NcoI and PstI restriction enzymes (Boehringer
Mannheim). The digested DNA was run on 1% agarose gel, cut out and
gel purified using the Qiagen Gel Extraction Kit (Qiagen) as per
manufacturer's instructions. A DNA fragment representing the coding
region of Zeocin selective marker was amplified by PCR method with
primers ZC13,946 (SEQ ID NO:28) and ZC13,945 (SEQ ID NO:29), and
pZeoSV2(+) as a template. There are additional PstI and BclI
restriction sites in primer ZC13,946 (SEQ ID NO:28), and additional
NcoI and SfuI sites in primer ZC13,945 (SEQ ID NO:29). The PCR
fragment was cut with PstI and NcoI restriction enzymes and cloned
into pZP7 vector prepared by cleaving with the same two enzymes and
subsequent gel purification. This vector was named pZP7Z. Then the
Zeocin coding region was removed by DNA digestion of vector pZP7Z
with BclI and SfuI restriction enzymes. The digested DNA was run on
1% agarose gel, cut out and gel purified, and then ligated with a
DNA fragment of Neomycin coding region cut from pZem228 vector
(deposited at the American Type Culture Collection (ATCC),
Manassas, Va.; ATCC Deposit No. 69446) with the same restriction
enzymes (BclI and SfuI).
[0315] This new vector was named pZP7N, in which the coding region
for DHFR selective marker was replaced by the coding region for a
Neomycin selective marker from vector pZem228. A stuffer fragment
including an XhoI site was added to pZP7N to create a vector
suitable for high efficiency directional cloning of cDNA; this new
vector was called pZP7NX. To prepare the vector for cDNA, 20 .mu.g
of pZP7NX was digested with 20 units of EcoRI (Life Technologies
Gaithersberg, Md.) and 20 units of XhoI (Boehringer Mannheim
Indianapolis, IN) for 5 hours at 37.degree. C., then 68.degree. C.
for 15 minutes. The digest was then run on a 0.8% low melt agarose
1.times.TAE gel to separate the stuffer from the vector. The vector
band was excised and digested with "beta-Agarase" (New England
Biolabs, Beverly, Mass.) following the manufacturer's
recommendations. After ethanol precipitation the digested vector
was resuspended in water to 45 ng/ml in preparation for ligation of
CD3+ selected cDNA library described below.
B. Preparation of Primary Human Activated CD3+ Selected Cell cDNA
Library
[0316] Approximately 1.5.times.10.sup.8 primary human CD3+ selected
cells stimulated in ionomycin/PMA were isolated by centrifugation
after culturing at 37.degree. C. for 13 hours (Example 5C). Total
RNA was isolated from the cell pellet using the "RNeasy Midi" kit
from Qiagen, Inc. (Valencia, Calif.). mRNA was isolated from 225
micrograms of total RNA using the "MPG mRNA purification kit" from
CPG Inc. (Lincoln Park, N.J.). 3.4 micrograms of mRNA was isolated
and converted to double stranded cDNA using the following
procedure.
[0317] First strand cDNA from stimulated human CD3+ selected cells
was synthesized as follows. Nine .mu.l Oligo d(T)-selected poly(A)
CD3+ RNA at a concentration of 0.34 .mu.g/.mu.l and 1.0 .mu.l of 1
.mu.g/.mu.l first strand primer ZC18,698 (SEQ ID NO:30) containing
an XhoI restriction site were mixed and heated at 65.degree. C. for
4 minutes and cooled by chilling on ice. First strand cDNA
synthesis was initiated by the addition of 9 .mu.l of first strand
buffer (5.times. SUPERSCRIPTED buffer; (Life Technologies), 4 .mu.l
of 100 mM dithiothreitol and 2 .mu.l of a deoxynucleotide
triphosphate solution containing 10 mM each of DATP, dGTP, dTTP and
5-methyl-dCTP (Pharmacia Biotech Inc.) to the RNA-primer mixture.
The reaction mixture was incubated at 45.degree. C. for 4 minutes
followed by the addition of 8 .mu.l of 200 U/.mu.l
SuperscriptII.RTM., RNase H-reverse transcriptase (Life
technologies). The reaction was incubated at 45.degree. C. for 45
minutes followed by an incubation ramp of 1.degree. C. every 2
minutes to 50.degree. C. where the reaction was held for 10
minutes. To denature any secondary structure and allow for
additional extension of the cDNA the reaction was then heated to
70.degree. C. for 2 minutes then dropped to 55.degree. C. for 4
minutes after which 2 .mu.l of SuperscriptII.RTM. RT was added and
incubated an additional 15 minutes followed by a ramp up to
70.degree. C. 1 minute/1.degree. C. Unincorporated nucleotides were
removed from the cDNA by twice precipitating in the presence of 2
.mu.g of glycogen carrier, 2.0 M ammonium acetate and 2.5 volume
ethanol, followed by a 100 .mu.l wash with 70% ethanol. The cDNA
was resuspended in 98 .mu.l water for use in second strand
synthesis.
[0318] Second strand synthesis was performed on the first strand
cDNA under conditions that promoted first strand priming of second
strand synthesis resulting in DNA hairpin formation. The second
strand reaction contained 98 .mu.l of the first strand cDNA, 30
.mu.l of 5.times. polymerase I buffer (100 mM Tris: HCl, pH 7.5,
500 mM KCl, 25 mM MgCl.sub.2, 50 mM (H.sub.4).sub.2SO.sub.4), 2
.mu.l of 100 mM dithiothreitol, 6 .mu.l of a solution containing 10
mM of each deoxynucleotide triphosphate, 5 .mu.l of 5 mM b-NAD, 1
.mu.l of 3 U/.mu.l E. coli DNA ligase (New England Biolabs Inc.)
and 4 .mu.l of 10 U/.mu.l E. coli DNA polymerase I (New England
Biolabs Inc.). The reaction was assembled at room temperature and
was incubated at room temperature for 2 minutes followed by the
addition of 4 .mu.l of 3.8 U/.mu.l RNase H (Life Technologies). The
reaction was incubated at 15.degree. C. for two hours followed by a
15 minute incubation at room temperature. 10 .mu.l of 1M TRIS pH7.4
was added to the reaction and extracted twice with
phenol/chloroform and once with chloroform, the organic phases were
then back extracted with 50 .mu.l of TE (10 mM TRIS pH 7.4, 1 mM
EDTA), pooled with the other aqueous and ethanol precipitated in
the presence of 0.3 M sodium acetate. The pellet was washed with
100 .mu.l 70% ethanol air dried and resuspended in 40 .mu.l
water.
[0319] The single-stranded DNA of the hairpin structure was cleaved
using mung bean nuclease. The reaction mixture contained 40 .mu.l
of second strand cDNA, 5 .mu.l of 10.times. mung bean nuclease
buffer (Life technologies), 5 .mu.l of mung bean nuclease
(Pharmacia Biotech Corp.) diluted to IU/.mu.l in 1.times. mung bean
nuclease buffer. The reaction was incubated at 37.degree. C. for 45
minutes. The reaction was terminated by the addition of 10 .mu.l of
1 M Tris: HCl, pH 7.4 followed by sequential phenol/chloroform and
chloroform extractions as described above. Following the
extractions, the cDNA was ethanol precipitated in the presence of
0.3 M sodium acetate. The pellet was washed with 100 .mu.l 70%
ethanol air dried and resuspended in 38 .mu.l water.
[0320] The resuspended cDNA was blunt-ended with T4 DNA polymerase.
The cDNA, which was resuspended in 38 .mu.l of water, was mixed
with 12 .mu.l 5.times.T4 DNA polymerase buffer (250 mM Tris:HCl, pH
8.0, 250 mM KCl, 25 mM MgCl.sub.2), 2 .mu.l 0.1 M dithiothreitol, 6
.mu.l of a solution containing 10 mM of each deoxynucleotide
triphosphate and 2 .mu.l of 1 U/.mu.l T4 DNA polymerase (Boehringer
Mannheim Corp.). After an incubation of 45 minutes at 15.degree.
C., the reaction was terminated by the addition of 30 .mu.l TE
followed by sequential phenol/chloroform and chloroform extractions
and back extracted with 20 .mu.l TE as described above. The DNA was
ethanol precipitated in the presence of 2 .mu.l Pellet Paint.TM.
(Novagen) carrier and 0.3 M sodium acetate and was resuspended 11
.mu.l of water.
[0321] EcoRI adapters were ligated onto the 5' ends of the cDNA
described above to enable cloning into an expression vector. 11
.mu.l of cDNA and 4 .mu.l of 65 pmole/.mu.l of Eco RI
hemiphophorylated adaptor (Pharmacia Biotech Corp) were mixed with
5 .mu.l 5.times. ligase buffer (Life Technologies), 2 .mu.l of 10
mM ATP and 3 .mu.l of 1 U/.mu.l T4 DNA ligase (Life Technologies),
1 .mu.l 10.times. ligation buffer (Promega Corp), 9 .mu.l water.
The extra dilution with 1.times. buffer was to prevent the pellet
paint from precipitating. The reaction was incubated 9 hours in a
water bath temperature ramp from 10.degree. C. to 22.degree. C.
over 9 hours, followed by 45 minutes at 25.degree. C. The reaction
was terminated by incubation at 68.degree. C. for 15 minutes.
[0322] To facilitate the directional cloning of the cDNA into an
expression vector, the cDNA was digested with XhoI, resulting in a
cDNA having a 5' Eco RI cohesive end and a 3' XhoI cohesive end.
The XhoI restriction site at the 3' end of the cDNA had been
previously introduced using the ZC18698 (SEQ ID NO:31) primer.
Restriction enzyme digestion was carried out in a reaction mixture
containing 35 .mu.l of the ligation mix described above, 6 .mu.l of
10.times. H buffer (Boehringer Mannheim Corp.), 1 .mu.l of 2 mg/ml
BSA (Biolabs Corp.), 17 .mu.l water and 1.0 .mu.l of 40 U/.mu.l
XhoI (Boehringer Mannheim). Digestion was carried out at 37.degree.
C. for 1 hour. The reaction was terminated by incubation at
68.degree. C. for 15 minutes followed by ethanol precipitation,
washing drying as described above and resuspension in 30 .mu.l
water.
[0323] The resuspended cDNA was heated to 65.degree. C. for 5
minutes and cooled on ice, 4 .mu.l of 5.times. gel loading dye
(Research Genetics Corp.) was added, the cDNA was loaded onto a
0.8% low melt agarose 1.times.TAE gel (SEA PLAQUE GTG.TM. low melt
agarose; FMC Corp.) and electrophoresed. The contaminating adapters
and cDNA below 0.6 Kb in length were excised from the gel. The
electrodes were reversed, molten agarose was added to fill in the
wells, the buffer was changed and the cDNA was electrophoresed
until concentrated near the lane origin. The area of the gel
containing the concentrated cDNA was excised and placed in a
microfuge tube, and the agarose was melted by heating to 65.degree.
C. for 15 minutes. Following equilibration of the sample to
45.degree. C., 2 .mu.l of 1 U/.mu.l Beta-agarase I (Biolabs, Inc.)
was added, and the mixture was incubated for 90 min. at 45.degree.
C. to digest the agarose. After incubation, 1 tenth volume of 3 M
Na acetate was added to the sample, and the mixture was incubated
on ice for 15 minutes. The sample was centrifuged at 14,000.times.g
for 15 minutes at room temperature to remove undigested agarose,
the cDNA was ethanol precipitated, washed in 70% ethanol, air-dried
and resuspended in 40 .mu.l water.
[0324] To determine the optimum ratio of cDNA to vector several
ligations were assembled and electroporated. Briefly, 2 .mu.l of
5.times.T4 ligase buffer (Life Technologies), 1 .mu.l of 110 mM
ATP, 1 .mu.l pZP7NX digested with EcoRI-XhoI, 1 .mu.l T4 DNA ligase
diluted to 0.25 u/.mu.l (Life Technologies) water to 10 .mu.l and
0.5, 1, 2 or 3 .mu.l of cDNA were mixed in 4 separate ligations,
incubated at 22.degree. C. for 4 hours, 68.degree. C. for 20
minutes, sodium acetate-ethanol precipitated, washed, dried and
resuspended in 10 .mu.l. A single microliter of each ligation was
electroporated into 40 .mu.l DH10b ElectroMax.TM. electrocompetent
bacteria (Life Technologies) using a 0.1 cm cuvette (Biorad) and a
Genepulser, pulse controller.TM. (Biorad) set to 2.5 KV, 251 F, 200
ohms. These cells were immediately resuspended in 1 ml SOC broth
(Manniatis et al. supra.) followed by 500 .mu.L of 50% glycerol-SOC
as a preservative. These "glycerol stocks" were frozen in several
aliquots at -70.degree. C. An aliquot of each was thawed and plated
serially on LB-agar plates supplemented with ampicillin at 100
.mu.g/ml. Colony numbers indicated that the optimum ratio of CD3+
cDNA to pZP7NX vector was 1 .mu.l to 45 ng; such a ligation yielded
4.5 million primary clones.
[0325] For the purpose of screening the library using a BaF3-based
proliferation assay (Example 5) glycerol stocks from above were
diluted into liquid cultures of 100 or 250 clones per pool in deep
well microtiter plates, grown 24 hours at 37.degree. C. with
shaking and plasmid isolated using a Qiagen kit following the
manufacturer's instructions. Such DNA was subsequently transfected
into BHK cells, media conditioned 72 hours, harvested and stored at
-80.degree. C., and subsequently placed on 5K
BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta cells
for 72 hours after which proliferation was assessed using an
"Alamar blue" fluorescence assay (Example 5B and Example 2B).
Example 7
Expression Cloning of Human zcytor17lig
[0326] The glycerol stocks from the activated human CD3+ selected
cell library (Example 6) were added to Super Broth II.TM. (Becton
Dickinson, Cockeysville, Md.)+0.1 mg/ml ampicillin (amp) at a
concentration of 250 cells per 800 microliters. The E. coli were
allowed to equilibrate for 24 hours at room temperature. At the
time of inoculation, 400 microliters was plated on LB+amp plates to
determine the actual titer of the inoculation. After 24 hours the
plates were counted and then the final concentration of the
SuperBrothII.TM.+E. coli was adjusted so that the final
concentration was 250 cells per 1.2 ml. Three times 2 liters were
inoculated for a total of 6 liters. The media were then plated into
96-well deep well blocks (Qiagen). Plating was done on the
8-channel Q-Fill2.TM. dispenser (Genetix, Christchurch, Dorset,
UK). The E. coli were grown overnight at 37.degree. C. shaking at
250 rotations/min. on a New Brunswick Scientific Innova 4900
multi-tier environment shaker. The E. coli were spun out of
solution at 3000 rpm, using a Beckman GS-6KR centrifuge. These E.
coli pellets were frozen at -20.degree. C. or used fresh before
miniprepping the plasmid DNA. Each pellet contains approximately
250 cDNA clones from the human CD3+ selected cell library.
[0327] These pools of 250 cDNA clones were then mini-prepped using
QIAprep.TM. 96 Turbo Miniprep kit (Qiagen). Plasmid DNA was eluted
using 125 .mu.l of TE (10 mM Tris pH 8, 1 mM EDTA). This plasmid
DNA was then used to transfect BHK cells.
BHK Transfection
[0328] BHK cells were plated in 96-well tissue culture plates at a
density of 12,000 cells per well in a volume of 100 .mu.l. per
well. Culture media was DMEM (GibcoBRL), 5% heat-inactivated fetal
bovine serum, 2 mM L-glutamine (GibcoBRL), 1.times.PSN (GibcoBRL),
1 mM NaPyruvate (GibcoBRL).
[0329] The following day, BHK cells were washed once with 100 .mu.l
SFA. SFA is serum-free media which is DMEM/F12 or DMEM (Gibco/BRL),
2 mM GlutaMax.TM. (Gibco/BRL), 1 mM NaPyruvate, 10 .mu.g/ml
transferrin, 5 .mu.g/ml insulin, 10 .mu.g/ml fetuin, 2 .mu.g/ml
selenium, 25 mM HEPES (Gibco/BRL), 100 .mu.M non-essential amino
acids (Gibco/BRL).
[0330] A DNA/Lipofectamine.TM. mix was made as follows: 2.2 .mu.l
Lipofectamine.TM. reagent (Gibco/BRL) was combined with 102.8 .mu.l
SFA at room temperature; approximately 5 .mu.l of the plasmid DNA
(200 ng/.mu.l) was then added to the Lipofectamine.TM./SFA to form
the DNA/Lipofectamine.TM. mixture, which was incubated at room
temperature for 30 minutes. The SFA was removed from the BHK cells
and the cells were incubated with 50 .mu.l of the
DNA/lipofectamine.TM. mix for 5 hours at 37.degree. C. with 5%
CO.sub.2. Fifty .mu.l of the DNA/Lipofectamine.TM. mixture was
added to each of two wells of the BHK cells, so that transfections
were done in duplicate.
[0331] After BHK cells were incubated with DNA/Lipofectamine.TM.
mix for 5 hours, the DNA/Lipofectamine.TM. mix was removed and 100
.mu.l culture media was added. Cells were incubated overnight, the
media was removed and replaced with 100 .mu.l. culture media. After
culturing cells for 48-72 hours, conditioned media was removed,
frozen at -80.degree. C. for a minimum of 20 minutes, thawed, and
then 50 .mu.l was assayed in the Baf3 proliferation assay,
described in Example 5, to identify pools of 250 clones with ligand
activity.
[0332] Twenty 96-well plates were screened in a single assay. This
represented approximately 250 cDNAs/well or 480,000 cDNAs total. Of
these, conditioned media from approximately 60 wells (representing
250 cDNAs per well) tested positive in the proliferation assay. One
of these positive pools was chosen to break down and isolate a
single cDNA that would encode the zcytor17lig. This was pool
62A12.
[0333] For pool 62A12, 1 .mu.l. of DNA was used to transform
ElectroMax.TM. DH10B cells (Gibco/BRL) by electroporation. The
transformants were plated on LB+amp (100 .mu.g/ml) plates to give
single colonies. From the electroporated pool, 672 individual
colonies were selected by toothpick into seven 96-well plates
containing 1.2 ml of SuperBrothII.TM. per well. These plates were
numbered #62.1 through #62.7. These were cultured overnight and the
plasmid DNA miniprepped as above. For all seven plates, plasmid DNA
from the breakdown plates was transfected into BHK cells and
assayed by proliferation as above, except that transfections were
not done in duplicate.
[0334] Two positive clones 62.6C7 and 62.6E9 were identified by
activity from a total of 672 clones. Plasmid DNA miniprepped from
clone 62.6E9 was sequenced and a tentative identification was
obtained, but a mixed sequence was obtained from this positive
clones. To further isolate the zcytor17lig cDNA to a single clone,
1 .mu.l of DNA from pool 62.6E9 was used to electroporate DH10B
cells and the transformants plated on LB+amp (100 .mu.g/ml) plates
to give single colonies. Plasmid DNA miniprepped from several
colonies was sequenced to give the exact DNA sequence. The
polynucleotide sequence of zcytor17lig was full-length (SEQ ID
NO:1) and its corresponding amino acid sequence is shown (SEQ ID
NO:2).
Example 8
Construction of Mammalian Expression Vectors that Express zcytor17
Soluble Receptors: zcytor17CEE, zcytor17CFLG, zcytor17CHIS and
zcytor17-Fc4
A. Construction of zcytor17 Mammalian Expression Vector Containing
zcytor17CEE, zcytor17CFLG and zcytor17CHIS
[0335] An expression vector was prepared for the expression of the
soluble, extracellular domain of the zcytor17 polypeptide,
pZp9zcytor17CEE, where the construct is designed to express a
zcytor17 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:32).
[0336] An approximately 1500 bp PCR product was generated using
ZC29,451 (SEQ ID NO:33) and ZC29,124 (SEQ ID NO:34) as PCR primers
to add EcoRI and BamHI restriction sites. A human HPVS in-house
cDNA library was used as a template and PCR amplification was
performed as follows: 30 cycles at 94.degree. C. for 1 minute,
65.degree. C. for 1 minute, 72.degree. C. for 1.5 minutes, then
72.degree. C. for 7 minutes; 10.degree. C. soak. The PCR reaction
was ethanol precipitated and digested with EcoRI and BamHI
restriction enzymes. The digested PCR product was gel purified on a
1.0% agarose gel and the approximately 1500 bp band excised. This
band was then re-amplified using identical primers with the
following cycling: 30 cycles at 94.degree. C. for 1 minute,
65.degree. C. for 1 minute, 72.degree. C. for 3 minutes, then
72.degree. C. for 7 minutes; 10.degree. C. soak. The PCR reaction
was ethanol precipitated and digested with EcoRI and BamHI
restriction enzymes. The digested PCR product was gel purified on a
1.0% agarose gel and the approximately 1500 bp band excised. The
excised DNA was subcloned into plasmid CEEpZp9 that had been cut
with EcoRI and BamHI, to generate plasmid with a GLU-GLU
C-terminally tagged soluble receptor for zcytor17, zcytor17CEEpZp9.
The extracellular domain in the zcytor17CEE cDNA in zcytor17CEEpZp9
has a silent mutation that changes the T to C at position 1705 of
SEQ ID NO:4 (encoding a Pro residue at residue 403 of SEQ ID NO:5).
As this mutation is silent, the zcytor17 cDNA in zcytor17CEEpZp9
encodes the polypeptide as shown in SEQ ID NO:5. Moreover, because
of the construct used, a Gly-Ser residue pair is inserted
C-terminal to the end of the soluble, extracellular domain of
zcytor17 and prior to the C-terminal Glu-Glu Tag (SEQ ID NO:32). As
such, the tag at the C-terminus of the zcytor17 extracellular
domain, was a Glu-Glu tag as shown in (SEQ ID NO:17). Plasmid
CEEpZp9 is a mammalian expression vector containing an expression
cassette having the mouse metallothionein-1 promoter, multiple
restriction sites for insertion of coding sequences, 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, enhancer and origin of replication, a DHFR
gene and the SV40 terminator. Using standard molecular biological
techniques zcytor17CEEpZp9 was electroporated into DH10B 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 screened by
restriction analysis, or PCR from DNA prepared from individual
colonies. The insert sequence of positive clones was verified by
sequence analysis. A large scale plasmid preparation was done using
a QIAGEN.RTM. Maxi prep kit (Qiagen) according to manufacturer's
instructions.
[0337] The same process is used to prepare the zcytor17 soluble
receptors with a C-terminal his tag, composed of 6 His residues in
a row; and a C-terminal FLAG.RTM. tag (SEQ ID NO:36),
zcytor17CFLAG. To construct these constructs, the aforementioned
vector has either the HIS or the FLAG.RTM. tag in place of the
glu-glu tag (e.g., SEQ ID NO:17; SEQ ID NO:32 or SEQ ID NO:35).
B. Mammalian Expression Construction of Soluble Human zcytor17
Receptor: zcytor17-Fc4
[0338] An expression vector, pEZE-2 hzcytor17/Fc4, was prepared to
express a C-terminally Fc4 tagged soluble version of hzcytor17
(human zcytor17-Fc4) in PF CHO cells. PF CHO cells are an in house
CHO cell line adapted for growth in protein-free medium (ExCell 325
PF medium; JRH Biosciences). The in house CHO cell line was
originally derived from CHO DG44 cells (G. Urlaub, J. Mitchell, E.
Kas, L. A. Chasin, V. L. Funanage, T. T. Myoda and J. L. Hamlin,
"The Effect Of Gamma Rays at the Dihydrofolate Reductase Locus:
Deletions and Inversions," Somatic Cell and Molec. Genet., 12:
555-566 (1986). A fragment of zcytor17 cDNA that includes the
polynucleotide sequence from extracellular domain of the zcytor17
receptor was fused in frame to the Fc4 polynucleotide sequence (SEQ
ID NO:37) to generate a zcytor17-Fc4 fusion (SEQ ID NO:38 and SEQ
ID NO:39). The pEZE-2 vector is a mammalian expression vector that
contains the Fc4 polynucleotide sequence and a cloning site that
allows rapid construction of C-terminal Fc4 fusions using standard
molecular biology techniques.
[0339] A 1566 base pair fragment was generated by PCR, containing
the extracellular domain of human zcytor17 and the first two amino
acids of Fc4 (Glu and Pro) with FseI and BglII sites coded on the
5' and 3' ends, respectively. This PCR fragment was generated using
primers ZC29,157 (SEQ ID NO:40) and ZC29,150 (SEQ ID NO:41) by
amplification from a plasmid containing the extracellular domain of
human zcytor17 (pZp9zcytor17CEE) (Example 8). The PCR reaction
conditions were as follows: 25 cycles of 94.degree. C. for 1
minute, 60.degree. C. for 1 minute, and 72.degree. C. for 2
minutes; 1 cycle at 72.degree. C. for 10 minutes; followed by a
4.degree. C. soak. The fragment was digested with FseI and BglII
restriction endonucleases and subsequently purified by 1% gel
electrophoresis and band purification using QiaQuick gel extraction
kit (Qiagen). The resulting purified DNA was ligated for 5 hours at
room temperature into a pEZE-2 vector previously digested with FseI
and BglII containing Fc4 3' of the FseI and BglII sites.
[0340] Two .mu.l of the ligation mix was electroporated in 37 .mu.l
DH10B electrocompetent E. coli (Gibco) according to the
manufacturer's directions. The transformed cells were diluted in
400 .mu.l of LB media and plated onto LB plates containing 100
.mu.g/ml ampicillin. Clones were analyzed by restriction digests
and positive clones were sent for DNA sequencing to confirm the
sequence of the fusion construct. 1 .mu.l of a positive clone was
transformed into 37 .mu.l of DH10B electrocompetent E. coli and
streaked on a LB/amp plate. A single colony was picked from this
streaked plate to start a 250 ml LB/amp culture that was then grown
overnight at 37.degree. C. with shaking at 250 rpm. This culture
was used to generate 750 .mu.g of purified DNA using a Qiagen
plasmid Maxi kit (Qiagen).
Example 9
Transfection and Expression Of Zcytor17 Soluble Receptor
Polypeptides
[0341] BHK 570 cells (ATCC No. CRL-10314), DG-44 CHO, or other
mammalian cells are plated at about 1.2.times.10.sup.6 cells/well
(6-well plate) in 800 .mu.l of appropriate serum free (SF) media
(e.g., DMEM, Gibco/BRL High Glucose) (Gibco BRL, Gaithersburg,
Md.). The cells are transfected with expression plasmids containing
zcytor17CEE, zcytor17CFLG, zcytor17CHIS or zcytor17-Fc4 (Example
8), using Lipofectin.TM. (Gibco BRL), in serum free (SF) media
according to manufacturer's instruction. Single clones expressing
the soluble receptors are isolated, screened and grown up in cell
culture media, and purified using standard techniques.
A. Mammalian Expression of Soluble Human zcytor17CEE Receptor
[0342] BHK 570 cells (ATCC NO: CRL-10314) were plated in T-75
tissue culture flasks and allowed to grow to approximately 50 to
70% confluence at 37.degree. C., 5% CO.sub.2, in DMEM/FBS media
(DMEM, Gibco/BRL High Glucose, (Gibco BRL, Gaithersburg, Md.), 5%
fetal bovine serum, 1 mM L-glutamine (JRH Biosciences, Lenea, K S),
1 mM sodium pyruvate (Gibco BRL)). The cells were then transfected
with the plasmid containing zcytor17CEE (Example 8) using
Lipofectamine.TM. (Gibco BRL), in serum free (SF) media formulation
(DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1%
L-glutamine and 1% sodium pyruvate). Ten .mu.g of the plasmid DNA
pZp9zcytor17CEE (Example 8) was diluted into a 15 ml tube to a
total final volume of 500 .mu.l with SF media. 50 .mu.l of
Lipofectamine was mixed with 450 .mu.l of SF medium. The
Lipofectamine mix was added to the DNA mix and allowed to incubate
approximately 30 minutes at room temperature. Four ml of SF media
was added to the DNA:Lipofectamine mixture. The cells were rinsed
once with 5 ml of SF media, aspirated, and the DNA:Lipofectamine
mixture was added. The cells were incubated at 37.degree. C. for
five hours, and then 5 ml of DMEM/10% FBS media was added. The
flask was incubated at 37.degree. C. overnight after which time the
cells were split into the selection media (DMEM/FBS media from
above with the addition of 1 .mu.M methotrexate or 10 .mu.M
Methotrexate (Sigma Chemical Co., St. Louis, Mo.) in 150 mm plates
at 1:2, 1:10, and 1:50. Approximately 10 days post-transfection,
one 150 mm plate of 1 .mu.M methotrexate resistant colonies was
trypsinized, the cells were pooled, and one-half of the cells were
replated in 10 .mu.M methotrexate; to further amplify expression of
the zcytor17CEE protein. A conditioned-media sample from this pool
of amplified cells was tested for expression levels using SDS-PAGE
and Western analysis.
B. Mammalian Expression of Soluble Human zcytor17-Fc4 Receptor
[0343] Five replicates of 200 .mu.g of pEZE-2 hzcytor17Fc4 plasmid
DNA (Example 8) were linearized by restriction digestion with FspI,
a restriction enzyme that cuts once within the vector and does not
disturb genes necessary for expression. 200 .mu.g of CHO cell
genomic DNA was added to each replicate as carrier DNA and then the
DNA was precipitated by addition of 0.1 volumes of 3M Sodium
Acetate pH 5.2 and 2.2 volumes ethanol followed by a 15 minute ice
incubation and microcentrifugation at 4.degree. C. The resulting
DNA pellets were washed in 70% ethanol and air dried before being
resuspended in 100 .mu.l protein free (PF) CHO non-selection growth
media (21 g/L PF CHO Ex Cell 325/200 mM L-glutamine (Gibco)/100 mM
sodium pyruvate (Gibco)/1.times. HT Supplement (Gibco). Ten million
PF CHO passage 61 cells were added to the DNA in 600 .mu.l of PF
CHO non-selection growth media and then electroporated in a Gene
Pulser II Electroporation system (BioRad) using 950 .mu.F
capacitance and 300 Kv using a 0.4 cm gap Gene Pulser (BioRad)
electroporation cuvette. All 5 replicates of the electroporated
cells were pooled and directly selected in -HT media (21 g/L PF CHO
Ex Cell 325/200 mM L-glutamine (Gibco)/100 mM sodium pyruvate
(Gibco). Cells were selected for 15 days in -HT media before being
passaged at 4.times.10.sup.5 ml into 50 nm MTX selection. Eight
days later cells were seeded at 3.5.times.10.sup.5 cells/ml into
200 mM MTX selection. After one week, cells were seeded at
4.times.10.sup.5 cells/ml into 1 .mu.M MTX selection. After two
weeks at 1 .mu.M MTX, cells were seeded at 1.times.10.sup.6
cells/ml into 50 ml to generate conditioned medium. The resulting
72 hour conditioned media was analyzed by probing western blots
with an antibody generated against human Ig. The cells produced
hzcytor17/Fc4 protein at approximately 1 mg/L.
C. Larger-Scale Mammalian Expression of Soluble Human zcytor17-Fc4
Receptor
[0344] Two hundred micrograms of pEZE-2 hzcytor17Fc4 plasmid DNA
(Example 8) was linearized by restriction digestion with FspI, a
restriction enzyme that cuts once within the pEZE-2 vector and does
not disturb genes necessary for expression. 200 .mu.g of CHO
genomic DNA (prepared in-house) was added as carrier DNA and then
the DNA was precipitated by addition of 0.1 volumes of 3M Sodium
Acetate pH 5.2 and 2.5 volumes ethanol followed by
microcentrifugation at Room temperature. Five replicate DNA pellets
were made and transformed. The resulting DNA pellet was washed in
70% ethanol and air dried before being resuspended in 100 .mu.l PF
CHO non-selection growth media (21 g/L PF CHO Ex Cell 325 /200 mM
L-glutamine (Gibco)/100 mM sodium pyruvate (Gibco)/1.times. HT
Supplement (Gibco). Ten million PF CHO cells were added to the DNA
in 600 .mu.l of PF CHO non-selection growth media and then
electroporated in a Gene Pulser II Electroporation system (BioRad)
using 950 .mu.F capacitance and 300 volts using a 0.4 cm gap Gene
Pulser (BioRad) electroporation cuvette. The electroporated cells
were pooled and put directly into selection in -HT media (21 g/L PF
CHO Ex Cell 325/200 mM L-glutamine (Gibco)/100 mM sodium pyruvate
(Gibco). Cells were selected for 14 days in --HT media before being
passaged at 4.times.10.sup.5/ml into 50 nm MTX selection. Cells
were amplified to 200 nM MTX and then to 1 uM MTX. The --HT, 50 nM,
and 1 uM pools were seeded at 1.times.10.sup.6 c/ml for 48 hours,
and the resulting conditioned media was analyzed by probing western
blots with an antibody generated against human Ig.
Example 10
Purification of zcytor17 Soluble Receptors from BHK 570 and CHO
Cells
A. Transient Mammalian Expression and Purification of Soluble Human
zcytor17-Fc4 Receptor
[0345] pEZE-2 hzcytor17Fc4 plasmid DNA (Example 1B) was introduced
into 40 maxi plates of BHK cells using Lipofectamine (Gibco BRL) as
described herein and in manufacturer's instructions. Cells were
allowed to recover overnight, then were rinsed and refed with
serum-free medium (SL7V4, made in-house). After 72 hours, the media
was collected and filtered, and cells were refed with serum-free
medium. After 72 hours, the media was again collected and
filtered.
[0346] The serum-free conditioned media (2.times.1.5 L batches)
from transiently transfected BHK cells was pumped over a 1.5 ml
Protein A-agarose column in 20 mM Tris, pH 7.5, 0.5 M NaCl. The
column was washed extensively with this buffer and then the bound
protein was eluted with 1 ml of 0.2 M glycine, pH 2.5, 0.5 M NaCl.
The eluted protein was collected into 0.1 ml of 2 M Tris, pH
8.5.
[0347] Aliquots were collected for SDS-polyacrylamide gel
electrophoresis and the bulk zcytor17-Fc was dialyzed overnight
against PBS. The soluble receptor was sterile filtered and placed
in aliquots at -80.degree. C.
B. Purification of zcytor17-Fc4
[0348] Recombinant carboxyl terminal Fc4 tagged zcytor17 (Example 8
and Example 9) was produced from transfected CHO cells. The CHO
transfection was performed using methods known in the art.
Approximately five-liters of conditioned media were harvested and
sterile filtered using Nalgene 0.2 .mu.m filters.
[0349] Protein was purified from the filtered media by a
combination of Poros 50 protein A affinity chromatography
(PerSeptive Biosystems, 1-5559-01, Framingham, Mass.) and Superdex
200 gel exclusion chromatography column (Amersham Pharmacia
Biotech, Piscataway, N.J.). Culture medium was directly loaded onto
a 10.times.70 mm (5.5-ml bed volume) protein A affinity column at a
flow of about 3-10 ml minute. Following column washing for ten
column volumes of PBS, bound protein was eluted by five column
volumes of 0.1 M glycine, pH 3.0 at 10 ml/minute). Fractions of 2
ml each were collected into tubes containing 100 .mu.p of 2.0 M
Tris, pH 8.0, in order to neutralize the eluted proteins. Samples
from the affinity column were analyzed by SDS-PAGE with coomassie
staining and Western blotting for the presence of zcytor17-Fc4
using human Ig-HRP. Zcytor17-Fc4-containing fractions were pooled
and concentrated to 1-2 ml using Biomax-30 concentrator
(Millipore), and loaded onto a 20.times.580 mm Superdex 200 gel
filtration column. The fractions containing purified zcytor17-Fc4
were pooled, filtered through 0.2 .mu.m filter, aliquoted into 100
.mu.l each, and frozen at -80.degree. C. The concentration of the
final purified protein was determined by BCA assay (Pierce,
Rockford, Ill.).
C. SDS-PAGE and Western Blotting Analysis of zcytor17/Fc4
[0350] Recombinant zcytor17-Fc4 was analyzed by SDS-PAGE (Nupage
4-12%, Invitrogen, Carlsbad, Calif.) with coomassie staining method
and Western blotting using human 1 g-HRP. Either the conditioned
media or purified protein was electrophoresed using an Invitrogen
Novex's Xcell II mini-cell, and transferred to nitrocellulose (0.2
mm; Invitrogen, Carlsbad, Calif.) at room temperature using Novex's
Xcell II blot module with stirring according to directions provided
in the instrument manual. The transfer was run at 500 mA for one
hour in a buffer containing 25 mM Tris base, 200 mM glycine, and
20% methanol. The filters were then blocked with 10% non-fat dry
milk in PBS for 10 minutes at room temperature. The nitrocellulose
was quickly rinsed, then the human Ig-HRP antibody (1:2000) was
added in PBS containing 2.5% non-fat dry milk. The blots were
incubated for two hours at room temperature, or overnight at
4.degree. C., with gentle shaking. Following the incubation, the
blots were washed three times for 10 minutes each in PBS, then
quickly rinsed in H.sub.2O. The blots were developed using
commercially available chemiluminescent substrate reagents
(SuperSignal.RTM. ULTRA reagents 1 and 2 mixed 1:1; reagents
obtained from Pierce, Rockford, Ill.), and the signal was captured
using Lumi-Imager's Lumi Analyst 3.0 software (Boehringer Mannheim
GmbH, Germany) for exposure times ranging from 10 second to 5
minutes or as necessary.
[0351] The purified zcytor17-Fc4 appeared as a single band with
either the coomassie or silver staining at about 220 kDa under
non-reducing conditions, and at about 120 kDa under reducing
conditions, suggesting the dimeric form of zcytor17-Fc4 under
non-reducing conditions as expected.
Example 11
Assay Using zcytor17 Soluble Receptor zcytor17-Fc4 Soluble Receptor
in Competitive Inhibition Assay
[0352] BaF3/zcytor17/WSX-1/OSMRbeta cells or BaF3/zcytor17/OSMRbeta
cells were spun down and washed in 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.
[0353] Both conditioned media from the CCRF-CEM and CCRF-HSB2 cell
activation and the human CD3+ selected cells, described in Example
5, were added in separate experiments at 25%, 12.5%, 6.25%, 3.125%,
1.5%, 0.75%, 0.375%, and 0.187% concentrations, with or without
zcytor17 soluble receptors (Zcytor17-Fc4; See, Example 9 and
Example 10) at 1-10 .mu.g/ml. The total assay volume was 200
.mu.l.
[0354] The assay plates were incubated at 37.degree. C., 5%
CO.sub.2 for 3-5 days at which time Alamar Blue (Accumed) was added
at 20 .mu.l well. Plates were again incubated at 37.degree. C., 5%
CO.sub.2 for 16-24 hours. Plates were read on the Fmax.TM. plate
reader (Molecular Devices) as described in Example 2. Results
demonstrated partial inhibition of cell growth with zcytor17-Fc4
soluble receptor at 10 .mu.g/ml, confirming that the factor in each
sample was specific for the zcytor17 receptor.
[0355] Titration curves, diluting out the soluble receptor, or
soluble receptor heterodimers and trimers comprising zcytor17
receptor (e.g., zcytor17/OSMR, zcytor17/WSX-1, or
zcytor17/OSMR/WSX-1, or other Class I cytokine receptor subunits)
are also run using the above stated assay to determine whether
zcytor17 receptors are able to completely inhibit growth, for
example, at low or physiologic concentrations.
Example 12
Secretion Trap Assay
[0356] A secretion trap assay is used to test the binding of the
zcytor17lig to receptors comprising zcytor17 receptor, such as the
zcytor17 receptor or receptor heterodimers and trimers comprising
zcytor17 receptor (e.g., zcytor17/OSMR, zcytor17/WSX-1, or
zcytor17/OSMR/WSX-1, or other Class I cytokine receptor subunits).
Zcytor17lig plasmid DNA is transfected into COS cells, and used to
assess binding of the zcytor17lig to receptors comprising zcytor17
receptor by secretion trap as described below.
A. COS Cell Transfections
[0357] The COS cell transfection is performed as follows: Mix about
800 ng of zcytor17lig cDNA and 5 .mu.l Lipofectamine.TM. in 92
.mu.l serum free DMEM media (55 mg sodium pyruvate, 146 mg
L-glutamine, 5 mg transferrin, 2.5 mg insulin, 1 g selenium and 5
mg fetuin in 500 ml DMEM), incubate at room temperature for 30
minutes and then add 400 .mu.l serum free DMEM media. Add this 500
.mu.L mixture onto 1.5.times.10.sup.5 COS cells/well plated on
12-well tissue culture plate and incubate for 5 hours at 37.degree.
C. Add 500[L 20% FBS DMEM media (100 ml FBS, 55 mg sodium pyruvate
and 146 mg L-glutamine in 500 ml DMEM) and incubate overnight.
B. Secretion Trap Assay
[0358] The secretion trap is performed as follows: Media is rinsed
off cells with PBS and then fixed for 15 minutes with 1.8%
Formaldehyde in PBS. Cells are then washed with TNT (0.1M Tris-HCL,
0.15M NaCl, and 0.05% Tween-20 in H.sub.2O), and permeated with
0.1% Triton-X in PBS for 15 minutes, and again washed with TNT.
Cells are blocked for 1 hour with TNB (0.1M Tris-HCL, 0.15M NaCl
and 0.5% Blocking Reagent (EN Renaissance TSA-Direct Kit) in
H.sub.2O), and washed again with TNT. If using the biotinylated
receptor protein, the cells are blocked for 15 minute incubations
with Avidin and then Biotin (Vector Labs) washing in-between with
TNT. Depending on which soluble receptor is used, the cells are
incubated for 1 hour in TNB with: (A) 1-3 .mu.g/ml zcytor17 soluble
receptor zcytor17-Fc4 fusion protein (Example 10); (B) 1-3 .mu.g/ml
zcytor17/OSMRbeta soluble receptor protein; (C) 1-3 .mu.g/ml
zcytor17/WSX-1 soluble receptor protein; or (D) 1-3 .mu.g/ml
zcytor17/OSMR/WSX-1 soluble receptor protein. Cells are then washed
with TNT. Depending on which soluble receptor is used (e.g., if
labeled with an Fc4 tag (SEQ ID NO:37), C-terminal FLAG tag (SEQ ID
NO:36), or CEE tag (SEQ ID NO:32; SEQ ID NO:35)), cells are
incubated for another hour with: (A) 1:200 diluted goat-anti-human
Ig-HRP (Fc specific); (B) 1:1000 diluted M2-HRP; (C) 1:1000 diluted
anti-GluGlu antibody-HRP; or (D) 1:300 diluted streptavidin-HRP
(NEN kit) in TNB, for example. Again cells are washed with TNT.
[0359] Positive binding is detected with fluorescein tyramide
reagent diluted 1:50 in dilution buffer (EN kit) and incubated for
4-6 minutes, and washed with TNT. Cells are preserved with
Vectashield Mounting Media (Vector Labs Burlingame, Calif.) diluted
1:5 in TNT. Cells are visualized using a FITC filter on fluorescent
microscope.
Example 13
Chromosomal Assignment and Placement of the Gene Sequence for the
zcytor17lig
[0360] The zcytor17lig gene sequence was mapped to human chromosome
12 using the commercially available version of the "Stanford G3
Radiation Hybrid Mapping Panel" (Research Genetics, Inc.,
Huntsville, Ala.). The "Stanford G3 RH Panel" contains DNA from
each of 83 radiation hybrid clones of the whole human genome, plus
two control DNAs (the RM donor and the A3 recipient). A publicly
available WWW server (e.g., Standford University) allows
chromosomal localization of markers and genes.
[0361] For the mapping of the zcytor17lig gene sequence with the
"Stanford G3 RH Panel", 20 .mu.l reactions were set up in a 96-well
microtiter plate compatible for PCR (Stratagene, La Jolla, Calif.)
and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene).
Each of the 95 PCR reactions consisted of 2 .mu.l 10.times.PCR
reaction buffer (Qiagen, Inc., Valencia, Calif.), 1.6 .mu.l dNTPs
mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 .mu.l sense
primer, ZC41,458 (SEQ ID NO:42), 1 .mu.l antisense primer, ZC41,457
(SEQ ID NO:43), 2 .mu.l "RediLoad" (Research Genetics, Inc.,
Huntsville, Ala.), 0.1 .mu.l Qiagen HotStarTaq DNA Polymerase (5
units/.mu.l), 25 ng of DNA from an individual hybrid clone or
control and distilled water for a total volume of 20 .mu.l. The
reactions were overlaid with an equal amount of mineral oil and
sealed. The PCR cycler conditions were as follows: an initial 1
cycle 15 minute denaturation at 95.degree. C., 35 cycles of a 45
second denaturation at 95.degree. C., 1 minute annealing at
53.degree. C. and 1 minute and 15 seconds extension at 72.degree.
C., followed by a final 1 cycle extension of 7 minutes at
72.degree. C. The reactions were separated by electrophoresis on a
2% agarose gel (EM Science, Gibbstown, N.J.) and visualized by
staining with ethidium bromide.
[0362] The results showed linkage of the zcytor17lig gene sequence
to the chromosome 12 marker SHGC-83339 with a LOD score of >11
and at a distance of 17 cR.sub.--10000 from the marker. This marker
positions zcytor17lig gene in the 12q24.31 chromosomal region.
Example 14
Identification and Cloning of Murine zcytor17lig
A. Identification of Full Length Murine zcytor17lig
[0363] Using the human zcytor17lig peptide sequence (SEQ ID NO:2)
to query an in house DNA database, a murine cDNA, Genbank Accession
No. AK005939, was identified as a potential partial sequence for
the murine zcytor17lig. The AK005939 cDNA sequence was used to
query a database containing murine genomic fragments. A genomic
contig of the murine zcytor17lig was assembled (SEQ ID NO:76).
Prediction of coding potential on this genomic fragment with the
program Genscan revealed a likely cDNA sequence, with the same gene
structure as the human zcytor17lig. A murine cDNA sequence is
represented in SEQ ID NO:10, and corresponding polypeptide sequence
is shown in SEQ ID NO:11.
B. Cloning of Mouse zcytor17lig from a Mouse Testis cDNA Library by
PCR
[0364] Based on the genomic sequence (SEQ ID NO:76), two PCR
primers were designed and used to identify a cDNA source of mouse
zcytor17lig by PCR. These Primers ZC41498 (SEQ ID NO:86) and
ZC41496 (SEQ ID NO:87) were designed to the putative 5' and 3'
untranslated regions of the mouse sequences (SEQ ID NO:76 and SEQ
ID NO:10). Several cDNA sources were screened by PCR, including
Marathon-ready cDNAs (Clontech) and aliquots of locally made cDNA
libraries. Products were visualized on 1% agarose gels. Bands of
the expected size were observed in reactions utilizing a mouse
testis cDNA library template. These PCR reactions were successfully
performed in approximately 50 .mu.l volumes with or without 10%
DMSO, using pfu turbo polymerase (Stratagene) according to the
manufacturer's recommendations; with an additional application of a
wax hot-start employing hot start 50s (Molecular Bioproducts, Inc.
San Diego, Calif.). PCR thermocycling was performed with a single
cycle of 94.degree. C. for 4 min; followed by 40 cycles of
94.degree. C.: 30 seconds, 48.degree. C.: 30 seconds, 72.degree.
C.: 50 seconds; with additional final 72.degree. C. extension for 7
minutes. The two PCR reactions were pooled and purified using low
melt agarose and Gelase agarose digesting enzyme (Epicenter, Inc.
Madison, Wis.) according to the manufacturer's recommendations.
[0365] DNA sequence determination of these PCR products revealed a
murine zcytor17 cDNA sequence (SEQ ID NO:90) which comprised an ORF
identical to SEQ ID NO:10, confirming that SEQ ID NO:10 encoded the
mouse zcytor17lig polypeptide. PCR primers, ZC41583 (SEQ ID NO:88)
and ZC41584 (SEQ ID NO:89), were then used to add FseI and AscI
restriction sites and a partial Kozak sequence to the mcytorl7
.mu.g open reading frame and termination codon (SEQ ID NO:92). A
Robocycler 40 thermocycler (Stratagene) was used to run a
temperature gradient of annealing temperatures and cycling as
follows. Pfu turbo polymerase (Stratagene) was applied as described
above, but only in 10% DMSO. Cycling was performed with a single
cycle of 94.degree. C. for 4 min; followed by 20 cycles of
94.degree. C.: 30 seconds, 65.degree. C. to 51.degree. C. gradient:
30 seconds, 72.degree. C.: 1 minute; and a single 72.degree. C.
extension for 7 minutes. The template for this second thermocycling
reaction was 1 .mu.l of the initial gel-purified mcytor17 .mu.g PCR
product, above. Resulting PCR product from the three lowest
temperature reactions were pooled and gel purified using the Gelase
(Epicenter) method described above. This purified mzcytor17lig was
digested with FseI and AscI and ligated into a pZP7X vector
modified to have FseI and AscI sites in its cloning site. Plasmid
pZP7X is a mammalian expression vector containing an expression
cassette having the mouse metallothionein-1 (MT-1) promoter,
multiple restriction sites for insertion of coding sequences, and a
human growth hormone terminator. The plasmid also has an E. coli
origin of replication, a mammalian selective marker expression unit
having an SV40 promoter, enhancer and origin of replication, a DHFR
gene, and the SV40 terminator. The cloned murine cDNA sequence is
represented in SEQ ID NO:90, and corresponding polypeptide sequence
is shown in SEQ ID NO:91 (which is identical to SEQ ID NO:11).
Example 15
Isolation of Mouse zcytor17lig cDNA Clone from an Activated Mouse
Spleen Library
A. Murine Primary Source Used to Isolate Mouse zcytor17lig
[0366] Mouse spleens from Balb/C mice, are collected and mashed
between frosted-end slides to create a cell suspension. The
isolated primary mouse cell yield is expected to be about
6.4.times.10.sup.8 cells prior to selection described below.
[0367] The spleen cells are suspended in 9.6 ml MACS buffer (PBS,
0.5% EDTA, 2 mM EDTA). 1.6 ml of cell suspension is removed and 0.4
ml CD90 (Thy1.2) microbeads (Miltenyi Biotec) added. The mixture is
incubated for 15 min. at 4.degree. C. These cells labeled with CD90
beads are washed with 30 ml MACS buffer, and then resuspended in 2
ml MACS buffer.
[0368] A VS+ column (Miltenyi) is prepared according to the
manufacturer's instructions. The VS+ column is then placed in a
VarioMACS.TM. magnetic field (Miltenyi). The column is equilibrated
with 5 ml MACS buffer. The isolated primary mouse cells are then
applied to the column. The CD90 negative cells are allowed to pass
through. The column is rinsed with 9 ml (3.times.3 ml) MACS buffer.
The column is then removed from the magnet and placed over a 15 ml
falcon tube. CD90+cells are eluted by adding 5 ml MACS buffer to
the column and bound cells flushed out using the plunger provided
by the manufacturer. The incubation of the cells with the CD90
magnetic beads, washes, and VS+ column steps (incubation through
elution) above are repeated once more. The resulting CD90+
fractions from the 2 column separations are pooled. The yield of
CD90+ selected mouse spleen cells are expected to be about
1.times.10.sup.8 total cells.
[0369] A sample of the pooled CD90+ selected mouse cells is removed
for staining and sorting on a fluorescent antibody cell sorter
(FACS) to assess their purity. A PE-conjugated hamster anti-mouse
CD3.epsilon. antibody (PharMingen) is used for staining and sorting
the CD90+ selected cells. The mouse CD90+ selected cells should be
about 93% CD3+ cells, suggesting the cells are 93% T-cells.
[0370] The murine CD90+ selected cells are activated by incubating
3.times.10.sup.6 cells/ml in RPMI+5% FBS+PMA 10 ng/ml and Tonomycin
0.5 .mu.g/ml (Calbiochem) for overnight at 37.degree. C. The
supernatant from these activated CD90+ selected mouse cells is
tested for zcytor17lig activity as described below. Moreover, the
activated CD90+ selected mouse cells are used to prepare a cDNA
library, as described in Example 16, below.
Example 16
Cloning of Mouse zcytor17lig from a Mouse CD90+ Selected Cell
Library
[0371] Screening of a primary mouse activated CD90+ selected cell
cDNA library can reveal isolated cDNA that is a novel member of the
four-helix bundle cytokine family that would encode the mouse
ortholog of the human zcytor17lig. The cDNA is identified by
hybridization screening.
A. The Vector for CD90+ Selected Library Construction
[0372] The vector, pZP7N is used for CD3+ selected library
construction (See Example 6A)
B. Preparation of Primary Mouse Activated CD90+ Selected Cell cDNA
Library
[0373] Approximately 1.5.times.10.sup.8 primary mouse CD90+
selected cells stimulated in ionomycin/PMA (Example 15) are
isolated by centrifugation. Total RNA is isolated from the cell
pellet, and converted to double stranded cDNA as described in
Example 6B. This DNA is subsequently transfected into BHK cells, as
described in Example 6B, and proliferation is assessed using an
"Alamar blue" fluorescence assay (Example 2B).
[0374] For the purpose of screening the library by secretion trap
cloning, a complex, amplified form of the library is needed to
transfect COS-7 cells. 4.8 million clones are plated on 110 15 cm
LB-agar plates supplemented with 100 .mu.g/ml ampicillin, 10
.mu.g/ml methicillin. After growing the plates overnight at
37.degree. C. the bacteria are harvested by scraping and pelleted.
Plasmid DNA is extracted from the pelleted bacteria using a
Nucleobond-giga.TM. (Clonetech) following the manufacturer's
instructions. This plasmid is then used to transfect COS-7 cells on
slides and screened using the secretion trap technique described
below (Example 17).
C. Screening the Activated Mouse cDNA Library
[0375] Approximately 5.times.10.sup.5 clones are plated on 10
LB/Amp Maxi plates. The colonies are lifted, denatured,
neutralized, and cross-linked using the standard procedure
(Sambrook, J. et al. supra.). Fifty nanograms of the 300 bp 5' RACE
PCR fragment (Example 14) is labeled with .sup.32P using Prime-Itr
RmT random primer labeling kit (Stratagene). The 10 filters are
hybridized with this labeled probe at 65.degree. C. overnight using
ExpressHyb.TM. Hybridization Solution (Clontech). The filters are
then washed sequentially at 60.degree. C. for 1 hour three times
with 0.2.times.SSC (30 mM NaCl, 3 mM sodium citrate, pH 7.0), 0.1%
SDS; and then at 65.degree. C. for 1 hour. The filters are exposed
at -80.degree. C. overnight, and the X-ray film are developed. Agar
plugs containing the positive colonies are pulled, and the clones
plated on 10-cm LB/Amp plates. The colonies are then filter-lifted
and hybridized again following the same procedure described above.
Single DNA clones are isolated and sequenced using standard
methods, to identify the mouse cDNA.
Example 17
Mouse zcytor17lig does not Bind to Human zcytor17 Soluble Receptor
in Secretion Trap Assay
[0376] The DNA for mouse clone mzcytor17lig/pZP7 was transfected
into COS cells, and the binding of zcytor17 comprising soluble
receptors (human zcytor17 soluble receptor zcytor17-Fc4 (Example
10), or soluble receptor heterodimers (zcytor17/WSX-1 or
BaF3/zcytor17/OSMRbeta), to the transfected COS cells were tested
by a secretion trap assay (Example 12). The assay confirmed that
the mouse zcytor17lig does not bind to human zcytor17 soluble
receptor.
[0377] The COS cell transfection was performed as per Example 12
using about 0.7 .mu.g mouse zcytor17lig cDNA (Example 16) in 3
.mu.l.
[0378] The secretion trap was performed as per example 12 using,
for example, 1 .mu.g/ml zcytor17 soluble receptor Fc4 fusion
protein (Example 10) (or zcytor17 comprising soluble receptor
heterodimers as described herein) in TNB, and 1:200 diluted
goat-anti-human Ig-HRP (Fc specific) in TNB for the detectable
antibody. Positive binding of the soluble human zcytor17 receptor
to the prepared fixed cells was not detected with fluorescein
tyramide reagent as per Example 12. Cells were preserved and
visualized according to Example 12.
[0379] Results indicated that the mouse zcytor17lig does not bind
to human zcytor17 soluble receptor (or zcytor17 comprising soluble
receptor heterodimers as described herein).
Example 18
Expression of Mouse zcytor17lig in Mammalian Cells
Mammalian Expression of Mouse zcytor17lig
[0380] BHK 570 cells (ATCC No: CRL-10314) were plated in 10 cm
tissue culture dishes and allowed to grow to approximately 20%
confluence overnight at 37.degree. C., 5% CO.sub.2, in DMEM/FBS
media (DMEM, Gibco/BRL High Glucose media; Gibco BRL, Gaithersburg,
Md.), 5% fetal bovine serum (Hyclone, Logan, Utah), 1 mM
L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodium pyruvate
(Gibco BRL). The cells were then transfected with the plasmid
mzcytor17lig/pZP7X (Example 14) using a mammalian stable
Lipofectamine (GibcoBRL) transfection kit according to the
manufacturer's instructions.
[0381] One day after transfection, the cells were split 1:10 and
1:20 into the selection media (DMEM/FBS media with the addition of
1 .mu.M methotrexate (Sigma Chemical Co., St. Louis, Mo.)) in 150
mm plates. The media on the cells was replaced with fresh selection
media at day 5 post-transfection. Approximately 10 days
post-transfection, methotrexate resistant colonies were trypsinized
and the cells pooled and plated into large-scale culture flasks.
Once the cells were grown to approximately 90% confluence, they
were rinsed with PBS three times, and cultured with serum-free
ESTEP2 media (DMEM (Gibco BRL), 0.11 g/l Na Pyruvate, 3.7 g/l
NaHCO.sub.3, 2.5 mg/l insulin, 5 mg/l transferrin, pH7.0)
conditioned media. The conditioned media was collected three days
later, and put into a BaF3 proliferation assay using Alamar Blue,
described in Example 19 below.
Example 19
Mouse zcytor17lig does not Activate Human zcytor17 Receptor in BaF3
Assay Using Alamar Blue
[0382] Proliferation of BaF3/zcytor17, BaF3/zcytor17/OSMRbeta and
BaF3/zcytor17/WSX-1 cells (Example 4, and 5B) was assessed using
serum-free conditioned media from BHK cells expressing mouse
zcytor17lig (Example 18). BaF3/Zcytor17, BaF3/zcytor17/OSMRbeta and
BaF3/zcytor17/WSX-1 cells were spun down, washed and plated in
mIL-3 free media as described in Example 5B. Conditioned media from
BHK cells expressing mouse zcytor17lig (Example 18) was diluted
with mIL-3 free media to 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%,
0.75% and 0.375% concentrations. The proliferation assay was
performed as per Example 5B. The results of this assay were
negative, indicating that mouse zcytor17lig does not activate human
zcytor17, zcytor17/OSMRbeta, or zcytor17/WSX-1 receptor
complexes.
Example 20
Human zcytor17lig Activates Human, zcytor17/OSMRbeta Receptor, in
Luciferase Assay
A. Construction of BaF3/KZ134/zcytor17 Cell Line
[0383] The KZ134 plasmid was constructed with complementary
oligonucleotides ZC12,749 (SEQ ID NO:44) and ZC12,748 (SEQ ID
NO:45) that contain STAT transcription factor binding elements from
4 genes, which includes 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 RI
gene, (Seidel, H. et al., Proc. Natl. Acad. Sci. 92:3041-3045,
1995). These oligonucleotides contain Asp718-XhoI compatible ends
and were 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 was used to stably transfect BaF3 cells, using standard
transfection and selection methods, to make the BaF3/KZ134 cell
line.
[0384] A stable BaF3/KZ134 indicator cell line, expressing the
full-length zcytor17 receptor or zcytor17/OSMRbeta receptor was
constructed as per Example 4. Clones were diluted, plated and
selected using standard techniques. Clones were screened by
luciferase assay (see Example 20B, below) using the human
zcytor17lig conditioned media or purified zcytor17lig protein (see
Example 35, below) as an inducer. Clones with the highest
luciferase response (via STAT luciferase) and the lowest background
were selected. Stable transfectant cell lines were selected. The
cell lines were called BaF3/KZ134/zcytor17 or
BaF3/KZ134/zcytor17/OSMRbeta depending on the receptors transfected
into the cell line.
[0385] Similarly, BHK cell lines were also constructed using the
method described herein, and were used in luciferase assays
described herein. The cell lines were called BHK/KZ134/zcytor17 or
BHK/KZ134/zcytor17/OSMRbeta depending on the receptors transfected
into the cell line.
B. Human zcytor17lig Activates Human zcytor17 Receptor in
BaF3/KZ734/zcytor17/OSMRbeta or BHK/KZ134/zcytor17/OSMRbeta
Luciferase Assay
[0386] BaF3/KZ134/zcytor17 and BaF3/KZ134/zcytor17/OSMRbeta cells
were spun down and washed in mIL-3 free media. The cells were spun
and washed 3 times to ensure removal of mIL-3. Cells were then
counted in a hemacytometer. Cells were plated in a 96-well format
at about 30,000 cells per well in a volume of 100 .mu.l per well
using the mIL-3 free media. The same procedure was used for
untransfected BaF3/KZ134 cells for use as a control in the
subsequent assay. BHK/KZ134/zcytor17 or BHK/KZ134/zcytor17/OSMRbeta
cells were plated in a 96-well format at 15,000 cells per well in
100 .mu.l media. Parental BHK/KZ134 cells were used as a
control.
[0387] STAT activation of the BaF3/KZ134/Zcytor17,
BaF3/KZ134/zcytor17/OSMRbeta, BHK/KZ134/zcytor17, or
BHK/KZ134/zcytor17/OSMRbeta cells was assessed using (1)
conditioned media from BHK570 cells transfected with the human
zcytor17lig (Example 7), (2) conditioned media from BHK570 cells
transfected with the mouse zcytor17lig (Example 18), (3) purified
human zcytor17lig (Example 35), or (4) mIL-3 free media to measure
media-only control response. Conditioned media was diluted with
RPMI mIL-3 free media to 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%,
0.75% and 0.375% concentrations. Purified human zcytor17lig was
diluted to a concentration of 1200, 600, 300, 150, 75, 37.5, 18.75,
or 9.4 pM. 100 .mu.l of the diluted conditioned media or protein
was added to the BaF3/KZ134/Zcytor17, BaF3/KZ134/zcytor17/OSMRbeta,
BHK/KZ134/zcytor17, or BHK/KZ134/zcytor17/OSMRbeta cells. The assay
using the conditioned media was done in parallel on untransfected
BaF3/KZ134 or BHK/KZ134 cells as a control. The total assay volume
was 200 .mu.l. The assay plates were incubated at 37.degree. C., 5%
CO.sub.2 for 24 hours at which time the BaF3 cells were pelleted by
centrifugation at 2000 rpm for 10 min., and the media was aspirated
and 25 .mu.l of lysis buffer (Promega) was added. For the BHK cell
lines, the centrifugation step was not necessary as the cells are
adherant. After 10 minutes at room temperature, the plates were
measured for activation of the STAT reporter construct by reading
them on a luminometer (Labsystems Luminoskan, model RS) which added
40 .mu.l of luciferase assay substrate (Promega) at a five second
integration.
[0388] The results of this assay confirmed that the STAT reporter
response of the BaF3/KZ134/zcytor17/OSMRbeta and
BHK/KZ134/zcytor17/OSMRbeta cells to the human zcytor17lig when
compared to either the BaF3/KZ134/zcytor17 cells, the
BHK/KZ134/zcytor17 cells or the untransfected BaF3/KZ134 or
BHK/KZ134 control cells, showed that the response was mediated
through the zcytor17/OSMRbeta receptors. The results also showed
that the mouse zcytor17lig does not activate the STAT reporter
assay through the human receptor complex.
Example 21
Mouse zcytor17lig is Active in Mouse Bone Marrow Assay
A. Isolation of Non-adherent Low Density Marrow Cells:
[0389] Fresh mouse femur aspirate (marrow) is obtained from 6-10
week old male Balb/C or C57BL/6 mice. The marrow is then washed
with RPMI+10% FBS (JRH, Lenexa Kans.; Hyclone, Logan Utah) and
suspended in RPMI+10% FBS as a whole marrow cell suspension. The
whole marrow cell suspension is then subjected to a density
gradient (Nycoprep, 1.077, Animal; Gibco BRL) to enrich for low
density, mostly mononuclear, cells as follows: The whole marrow
cell suspension (About 8 ml) is carefully pipeted on top of about 5
ml Nycoprep gradient solution in a 15 ml conical tube, and then
centrifuged at 600.times.g for 20 minutes. The interface layer,
containing the low density mononuclear cells, is then removed,
washed with excess RPMI+10% FBS, and pelleted by centrifugation at
400.times.g for 5-10 minutes. This pellet is resuspended in
RPMI+10% FBS and plated in a T-75 flask at approximately 10.sup.6
cells/ml, and incubated at 37.degree. C. 5% CO.sub.2 for
approximately 2 hours. The resulting cells in suspension are
Non-Adherent Low Density (NA LD) Marrow Cells.
B. 96-Well Assay
[0390] NA LD Mouse Marrow Cells are plated at 25,000 to 45,000
cells/well in 96 well tissue culture plates in RPMI+10% FBS+1 ng/mL
mouse Stem Cell Factor (mSCF) (R&D Systems, Minneapolis,
Minn.), plus 5% conditioned medium from one of the following: (1)
BHK 570 cells expressing mouse zcytor17lig (Example 18), (2) BHK
570 cells expressing human zcytor17lig (Example 7), or (3) control
BHK 570 cells containing vector and not expressing either Ligand.
These cells are then subjected to a variety of cytokine treatments
to test for expansion or differentiation of hematopoietic cells
from the marrow. For testing, the plated NA LD mouse marrow cells
are subjected to human Interleukin-15 (hIL-15) (R&D Systems),
or one of a panel of other cytokines (R&D Systems). Serial
dilution of hIl-15, or the other cytokines, are tested, with 2-fold
serial dilution from about 50 ng/ml down to about 0.5 ng/ml
concentration. After 8 to 12 days the 96-well assays are scored for
cell proliferation by Alamar blue assay as described in Example
5B.
C. Results from the 96-well NA LD Mouse Marrow Assay
[0391] Conditioned media from the BHK cells expressing both mouse
and human zcytor17lig can promote the expansion of a population of
hematopoietic cells either alone or in synergy with other cytokines
in the NA LD mouse marrow in comparison to control BHK conditioned
medium. The population hematopoietic cells expanded by the mouse
zcytor17lig with or without other cytokines, and those
hematopoietic cells expanded by the human zcytor17lig with or
without other cytokines, are further propagated in cell culture.
These hematopoietic cells are stained with a Phycoerythrin labeled
anti-Pan NK cell antibody (PharMingen) and subjected to flow
cytometry analysis, which demonstrated that the expanded cells
stained positively for this natural killer (NK) cell marker.
Similarly, other specific hematopoietic cell markers can be used to
determine expansion of, for example, CD4+ or CD8+ T-cells, other
T-cell populations, B-cells, and other immune cell markers.
[0392] The same 96-well assay is run, using fresh human marrow
cells bought from Poietic Technologies, Gaithersburg, Md. Again, a
positive result shows that zcytor17lig alone or in synergy with
other cytokines, the mouse and human zcytor17lig can expand a
hematopoietic cell population that is stained positively for
specific cell markers as disclosed above.
Example 22
Constructs for Generating zcytor17lig Transgenic Mice
[0393] Construct for expressing human zcytor17lig from the MT-1
promoter Oligonucleotides are designed to generate a PCR fragment
containing a consensus Kozak sequence and the human zcytor17lig
coding region. These oligonucleotides are designed with an FseI
site at the 5' end and an AscI site at the 3' end to facilitate
cloning into (a) pMT12-8, our standard transgenic vector, or (b)
pKFO51, a lymphoid-specific transgenic vector (Example 22B).
[0394] PCR reactions are carried out with about 200 ng human
zcytor17lig template (SEQ ID NO:1) and oligonucleotides designed to
amplify the full-length or active portion of the zcytor17lig. PCR
reaction conditions are determined using methods known in the art.
PCR products are separated by agarose gel electrophoresis and
purified using a QiaQuick.TM. (Qiagen) gel extraction kit. The
isolated, correct sized DNA fragment is digested with FseI and AscI
(Boerhinger-Mannheim), ethanol precipitated and ligated into
pMT12-8 previously digested with FseI and AscI. The pMT12-8
plasmid, designed for expressing a gene of interest in liver and
other tissues in transgenic mice, contains an expression cassette
flanked by 10 kb of MT-1 5' DNA and 7 kb of MT-1 3' DNA. The
expression cassette comprises the MT-1 promoter, the rat insulin II
intron, a polylinker for the insertion of the desired clone, and
the human growth hormone (hGH) poly A sequence.
[0395] About one microliter of each ligation reaction is
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 are picked and grown in LB media
containing 100 .mu.g/ml ampicillin. Miniprep DNA is prepared from
the picked clones and screened for the human zcytorI 7 .mu.g insert
by restriction digestion with EcoRI alone, or FseI and AscI
combined, and subsequent agarose gel electrophoresis. Maxipreps of
the correct pMT-human zcytor17lig are performed. A SalI fragment
containing with 5' and 3' flanking sequences, the MT-1 promoter,
the rat insulin II intron, human zcytor17lig cDNA and the hGH poly
A sequence is prepared to be used for microinjection into
fertilized murine oocytes. Microinjection and production of
transgenic mice are produced as described in Hogan, B. et al.
Manipulating the Mouse Embryo, 2d ed., Cold Spring Harbor
Laboratory Press, NY, 1994.
B. Construct for Expressing Human zcytor17lig from the
Lymphoid-Specific E.mu.LCK Promoter
[0396] Oligonucleotides are designed to generate a PCR fragment
containing a consensus Kozak sequence and the human zcytor17lig
coding region. These oligonucleotides are designed with an FseI
site at the 5' end and an AscI site at the 3' end to facilitate
cloning into pKFO51, a lymphoid-specific transgenic vector.
[0397] PCR reactions are carried out with about 200 ng human
zcytor17lig template (SEQ ID NO:1) and oligonucleotides designed to
amplify the full-length or active portion of the zcytor17lig. A PCR
reaction is performed using methods known in the art. The isolated,
correct sized DNA fragment is digested with FseI and AscI
(Boerhinger-Mannheim), ethanol precipitated and ligated into pKFO51
previously digested with FseI and AscI. The pKFO51 transgenic
vector is derived from p1026X (Iritani, B. M., et al., EMBO J.
16:7019-31, 1997) and contains the T cell-specific lck proximal
promoter, the B/T cell-specific immunoglobulin .mu. heavy chain
enhancer, a polylinker for the insertion of the desired clone, and
a mutated hGH gene that encodes an inactive growth hormone protein
(providing 3' introns and a polyadenylation signal).
[0398] About one microliter of each ligation reaction is
electroporated, plated, clones picked and screened for the human
zcytor17 .mu.g insert by restriction digestion as described above.
A correct clone of pKFO51-zcytor17lig is verified by sequencing,
and a maxiprep of this clone is performed. A NotI fragment,
containing the lck proximal promoter and immunoglobulin .mu.
enhancer (E.mu.LCK), zcytor17lig cDNA, and the mutated hGH gene is
prepared to be used for microinjection into fertilized murine
oocytes.
C. Construct for Expressing Mouse zcytor17lig from the EF1 Alpha
Promoter
[0399] Primers ZC41,498 (SEQ ID NO:86) and ZC41,496 (SEQ ID NO:87)
were used to PCR a mouse testis cDNA library template. These PCR
reactions were successfully performed in approximately 50 .mu.l
volumes with or without 10% DMSO, using pfu turbo polymerase
(Stratagene) according to the manufacturer's recommendations; with
an additional application of a wax hot-start employing hot start
50s (Molecular Bioproducts, Inc. San Diego, Calif.). PCR
thermocycling was performed with a single cycle of 94.degree. C.
for 4 min; followed by 40 cycles of 94.degree. C.: 30 seconds,
48.degree. C.: 30 seconds, 72.degree. C.: 50 seconds; with
additional final 72.degree. C. extension for 7 minutes. The two PCR
reactions were pooled and purified using low melt agarose and
Gelase agarose digesting enzyme (Epicenter, Inc. Madison, Wis.)
according to the manufacturer's recommendations.
[0400] DNA sequenced PCR products revealed a murine zcytor17 cDNA
sequence (SEQ ID NO:90) which comprised an ORF identical to SEQ ID
NO:10, confirming that SEQ ID NO:10 encoded the mouse zcytor17lig
polypeptide. PCR primers, ZC41583 (SEQ ID NO:88) and ZC41584 (SEQ
ID NO:89), were then used to add FseI and AscI restriction sites
and a partial Kozak sequence to the mcytor17 .mu.g open reading
frame and termination codon (SEQ ID NO:92). A Robocycler 40
thermocycler (Stratagene) was used to run a temperature gradient of
annealing temperatures and cycling as follows. Pfu turbo polymerase
(Stratagene) was applied as described above, but only in 10% DMSO.
Cycling was performed with a single cycle of 94.degree. C. for 4
min; followed by 20 cycles of 94.degree. C.: 30 seconds, 65.degree.
C. to 51.degree. C. gradient: 30 seconds, 72.degree. C.: 1 minute;
and a single 72.degree. C. extension for 7 minutes. The template
for this second thermocycling reaction was 1 .mu.l of the initial
gel-purified mcytor17 .mu.g PCR product, above. Resulting PCR
product from the three lowest temperature reactions were pooled and
gel purified using the Gelase (Epicenter) method described above.
This purified fragment was then digested with FseI and AscI and
ligated into a pZP7X vector modified to have FseI and AscI sites in
its cloning site. This was sent to sequencing to confirm the
correct sequence. The cloned murine cDNA sequence is represented in
SEQ ID NO:90, and corresponding polypeptide sequence is shown in
SEQ ID NO:91 (which is identical to SEQ ID NO:11).
[0401] The isolated, correct sized DNA fragment digested with FseI
and AscI
[0402] (Boerhinger-Mannheim) was subcloned into a plasmid
containing EF1alpha promoter previously digested with FseI and
AscI. Maxipreps of the correct EF1alpha mouse zcytor17lig were
performed. The expression cassette contains the EF1alpha promoter
(with a deleted FseI site), the EF1alpha intron, SUR IRES like site
to facilitate expression, a polylinker flanked with rat insulin II
sites on the 5'end which adds FseI PmeI AscI sites for insertion of
the desired clone, and the human growth hormone (hGH) poly A
sequence. A 7.5 kb NotI fragment containing the EF1alpha promoter
expression cassette and mouse zcytor17lig was prepared to be used
for microinjection into fertilized murine oocytes. The EF1alpha
plsdmid was obtained from Louis-Marie of the Laboratoire de
Differenciation Cellulaire, as described in Taboit-Dameron et al.,
1999, Transgenic Research 8:223-235.
D. Construct for Expressing Mouse zcytor17lig from the
Lymphoid-Specific E.mu.LCK Promoter
[0403] Oligonucleotides were designed to generate a PCR fragment
containing a consensus Kozak sequence and the mouse zcytor17lig
coding region. These oligonucleotides were designed with an FseI
site at the 5' end and an AscI site at the 3' end to facilitate
cloning into pKFO51 (see Example 22B, above).
[0404] The isolated, correct sized zcytor17lig DNA fragment used in
EF1alpha constructs, digested with FseI and AscI
(Boerhinger-Mannheim), was subcloned into a plasmid containing
pKFO51, a lymphoid-specific transgenic vector. The pKFO51
transgenic vector is derived from p1026X (Iritani, B. M., et al.,
EMBO J. 16:7019-31, 1997) and contains the T cell-specific lck
proximal promoter, the B/T cell-specific immunoglobulin .mu. heavy
chain enhancer, a polylinker for the insertion of the desired
clone, and a mutated hGH gene that encodes an inactive growth
hormone protein (providing 3' introns and a polyadenylation
signal). A 6.5 kb NotI fragment, containing the lck proximal
promoter and immunoglobulin p enhancer (EpLCK), mouse zcytor17lig
cDNA, and the mutated hGH gene was prepared to be used for
microinjection into fertilized murine oocytes (Example 41).
Example 23
Construction of Mammalian Expression Vectors that Express
zcytor17lig-CEE
A. Construction of zCytor17Lig-CEE/pZMP21
[0405] An expression plasmid containing all or part of a
polynucleotide encoding human zCytor17lig was constructed via
homologous recombination. The plasmid was called
zcytor17Lig-CEE/pZMP21.
[0406] The construction of zCytor17Lig-CEE/pZMP21 was accomplished
by generating a zCytor17Lig-CEE fragment (SEQ ID NO:95) using PCR
amplification. The DNA template used for the production of the
zCytor17Lig-CEE fragment was zcytor17Lig/pZP7nx. The primers used
for the production of the zCytor17Lig-CEE fragment were: (1)
ZC41607 (SEQ ID NO:97) (sense sequence), which includes from the 5'
to the 3' end: 28 bp of the vector flanking sequence (5' of the
insert) and 21 bp corresponding to the 5' sequence of zCytor17Lig;
and (2) ZC41605 (SEQ ID NO:98) (anti-sense sequence), which
includes from the 5' to the 3' end: 37 bp of the vector flanking
sequence (3' of the insert), 3 bp of the stop codon, 21 bp encoding
a C-terminal EE tag, and 21 bp corresponding to the 3' end of
zCytor17Lig sequence. The fragment resulting from the above PCR
amplification is a copy of the template zCytor17Lig with the
addition of a C-terminal EE tag, yielding a final product
zCytor17Lig-CEE.
[0407] PCR reactions were run as follows: To a 100 .mu.l final
volume was added: 10 .mu.l of 10.times. Taq Polymerase Reaction
Buffer with 15 mM MgCl (Gibco), 1 .mu.l of Taq DNA Polymerase (5
units/.mu.l, Gibco), 3 .mu.l of 10 mM dNTPs, 78 .mu.l dH.sub.2O, 3
.mu.l of a 20 pmol/.mu.l stock of primer ZC41607 (SEQ ID NO:97) 3
.mu.l of a 20 pmol/.mu.l stock of primer ZC41605 (SEQ ID NO:98),
and 2 .mu.l of a 0.13 .mu.g/.mu.l stock of zCytor17lig template
DNA. A volume equal to 50 .mu.l of mineral oil was added to the
mixture. The reaction was heated to 94.degree. C. for 5 minutes,
followed by 35 cycles at 94.degree. C. for 1 minute; 55.degree. C.
for 2 minutes; 72.degree. C. for 3 minutes; followed by a 10 minute
extension at 72.degree. C. and held at 4.degree. C. until the
reaction was collected.
[0408] The plasmid pZMP21 was restriction digested with BglII
enzyme, cleaned with a QiaQuick PCR Purification Kit (Qiagen) using
a microcentrifuge protocol, and used for recombination with the PCR
fragment. Plasmid pZMP21 was constructed from pZMP20 which was
constructed from pZP9 (deposited at the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
and is designated No. 98668) with the yeast genetic elements taken
from pRS316 (deposited at the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. 20110-2209, and
designated No. 77145), an IRES element from poliovirus, and the
extracellular domain of CD8, truncated at the carboxyl terminal end
of the transmembrane domain. PZMP21 is a mammalian expression
vector containing an expression cassette having the MPSV promoter,
immunoglobulin signal peptide intron, multiple restriction sites
for insertion of coding sequences, a stop codon 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, enhancer and origin of replication, a DHFR gene,
the SV40 terminator, as well as the URA3 and CEN-ARS sequences
required for selection and replication in S. cerevisiae.
[0409] Fifty microliters of competent yeast cells (S. cerevisiae)
were independently combined with 100 ng of cut plasmid, 5 .mu.l of
previously described PCR mixture, and transferred to a 0.2 cm
electroporation cuvette. The yeast/DNA mixture was electropulsed at
0.75 kV (5 kV/cm), cc ohms, 25 .mu.F. Each cuvette had 600 .mu.l of
1.2 M sorbitol added, and the yeast was plated in one 100 .mu.l
aliquot and one 300 .mu.l aliquot onto two URA-D plates and
incubated at 30.degree. C. After about 72 hours, the Ura+ yeast
transformants from a single plate were resuspended in 1 ml H.sub.2O
and spun briefly to pellet the yeast cells. The cell pellet was
resuspended in 500 .mu.l of lysis buffer (2% Triton X-100, 1% SDS,
100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). The 500 .mu.l of the
lysis mixture was added to an Eppendorf tube containing 300 .mu.l
acid washed 600 .mu.m glass beads and 300 PI phenol-chloroform,
vortexed for 1 minute intervals two or three times, followed by a 5
minute spin in a Eppendorf centrifuge at maximum speed. Three
hundred microliters of the aqueous phase was transferred to a fresh
tube, and the DNA precipitated with 600 .mu.l 100% ethanol (EtOH),
followed by centrifugation for 10 minutes at 4.degree. C. The DNA
pellet was then washed with 500 .mu.l 70% EtOH, followed by
centrifugation for 1 minute at 4.degree. C. The DNA pellet was
resuspended in 30 .mu.l H.sub.2O.
[0410] Transformation of electrocompetent E. coli cells (MC1061)
was done with 5 .mu.l of the yeast DNA prep and 50 .mu.l of MC1061
cells. The cells were electropulsed at 2.0 kV, 25 PF and 400
ohms(.OMEGA.). Following electroporation, 600 .mu.l SOC (2% Bacto'
Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM
glucose) was added. The electroporated E. coli cells were plated in
a 200 .mu.l and a 50PI aliquot on two LB AMP plates (LB broth
(Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin). The plates
were incubated upside down for about 24 hours at 37.degree. C.
Three Ampicillin-resistant colonies were selected at random and
submitted for sequence analysis of the insert. Large scale plasmid
DNA was isolated from a sequence-confirmed clone using the Qiagen
Maxi kit (Qiagen) according to manufacturer's instructions.
B. Mammalian Expression of Human zcytor17lig
[0411] Full-length zCytor17Lig protein was produced in BHK cells
transfected with zCytor17Lig-CEE/pZMP21 (Example 23A). BHK 570
cells (ATCC CRL-10314) were plated in T75 tissue culture flasks and
allowed to grow to approximately 50 to 70% confluence at 37.degree.
C., 5% CO.sub.2, in growth media (SL7V4, 5% FBS, 1% pen/strep). The
cells were then transfected with zCytor17Lig-CEE/pZMP21 by
liposome-mediated transfection (using Lipofectamine.TM.; Life
Technologies), in serum free (SF) media (SL7V4). The plasmid (16
.mu.g) was diluted into 1.5 ml tubes to a total final volume of 640
.mu.l with SF media. Thirty-five microliters of the lipid mixture
was mixed with 605 .mu.l of SF medium, and the resulting mixture
was allowed to incubate approximately 15 minutes at room
temperature. Five milliliters of SF media was then added to the
DNA:lipid mixture. The cells were rinsed once with 10 ml of PBS,
the PBS was decanted, and the DNA:lipid mixture was added. The
cells are incubated at 37.degree. C. for five hours, then 15 ml of
media (SL7V4, 5% FBS, 1% pen/strep) was added to each plate. The
plates were incubated at 37.degree. C. overnight, and the DNA:lipid
media mixture was replaced with selection media (SL7V4, 5% FBS, 1%
pen/strep, 1 .mu.M methotrexate) the next day. Approximately 10
days post-transfection, methotrexate-resistant colonies from the
T75 transfection flask were trypsinized, and the cells were pooled
and plated into a T-162 flask and transferred to large-scale
culture.
Example 24
Expression of zcytor17 Soluble Receptor in E. coli
A. Construction of Expression Vector pCMH01 that Expresses
huzcytor17/MBP-6H Fusion Polypeptide
[0412] An expression plasmid containing a polynucleotide encoding a
zcytor17 soluble receptor fused C-terminally to maltose binding
protein (MBP) was constructed via homologous recombination. The
fusion polypeptide contains an N-terminal approximately 388 amino
acid MBP portion fused to any of the zcytor17 soluble receptors
described herein. A fragment of zcytor17 cDNA (SEQ ID NO:4) was
isolated using PCR as described herein. Two primers were used in
the production of the zcytor17 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 zcytor17,
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 zcytor17. Two .mu.l of the 100 PI PCR
reaction was run on a 1.0% agarose gel with 1.times.TBE buffer for
analysis, and the expected approximately fragment was seen. The
remaining PCR reaction was combined with the second PCR tube and
precipitated with 400 .mu.l of absolute ethanol. The precipitated
DNA used for recombining into the Sma1 cut recipient vector pTAP170
to produce the construct encoding the MBP-zcytor17 fusion, as
described below.
[0413] Plasmid pTAP170 was 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 (NEB) 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 pTAP170 was constructed using yeast homologous
recombination. 100 ng of EcoR1 cut pMAL-c2 was recombined with 1
.mu.g Pvu1 cut pRS316, 1 g linker, and 1 .mu.g Sca1/EcoR1 cut
pRS316. The linker consisted of oligos zc19,372 (SEQ ID NO:172)
(100 pmole): zc19,351 (SEQ ID NO:173) (1 pmole): zc19,352 (SEQ ID
NO:174) (1 pmole), and zc19,371 (SEQ ID NO:175) (100 pmole)
combined in a PCR reaction. Conditions were as follows: 10 cycles
of 94.degree. C. for 30 seconds, 50.degree. C. for 30 seconds, and
72.degree. C. for 30 seconds; followed by 4.degree. C. soak. PCR
products were concentrated via 100% ethanol precipitation.
[0414] One hundred microliters of competent yeast cells (S.
cerevisiae) were combined with 10 .mu.l of a mixture containing
approximately 1 .mu.g of the human zcytor17 insert, and 100 ng of
SmaI digested pTAP170 vector, and transferred to a 0.2 cm
electroporation cuvette. The yeast/DNA mixture was electropulsed at
0.75 kV (5 kV/cm), infinite ohms, 25 .mu.F. To each cuvette was
added 600 .mu.l of 1.2 M sorbitol. The yeast was then plated in two
300 .mu.l aliquots onto two -URA D plates and incubated at
30.degree. C.
[0415] After about 48 hours, the Ura+ yeast transformants from a
single plate were picked, DNA was isolated, and transformed into
electrocompetent E. coli cells (e.g., MC1061, Casadaban et. al. J.
Mot. Biol. 138, 179-207), and plated on MM/CA+KAN 25 .mu.g/L plates
(Pryor and Leiting, Protein Expression and Purification 10:309-319,
1997) using standard procedures. Cells were grown in MM/CA with 25
.mu.g/ml Kanomyacin for two hours, shaking, at 37.degree. C. One ml
of the culture was induced with 1 mM IPTG. Two to four hours later
the 250 .mu.l of each culture was 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 were vortexed for one minute and heated to 65.degree. C.
for 10 minutes. 20 .mu.l were loaded per lane on a 4%-12% PAGE gel
(NOVEX). Gels were run in 1.times.MES buffer. The positive clones
were designated pCMH01 and subjected to sequence analysis.
[0416] One microliter of sequencing DNA was used to transform
strain BL21. The cells were electropulsed at 2.0 kV, 25.degree. F.
and 400 ohms. Following electroporation, 0.6 ml MM/CA with 25
.mu.g/L Kanomyacin. Cells were grown in MM/CA and induced with ITPG
as described above. The positive clones were used to grow up for
protein purification of the huzcytor17/MBP-6H fusion protein using
standard techniques.
B. Purification of huzcytor17/MBP-6H Soluble Receptor from E. coli
Fermentation
[0417] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for the purification
of recombinant huzcytor17/MBP-6H soluble receptor polypeptide. E.
coli cells containing the pCMH01 construct and expressing
huzcytor17/MBP-6H soluble receptor polypeptide were constructed
using standard molecular biology methods and cultured in SuperBroth
II (12 g/L Casien, 24 g/L Yeast Extract, 11.4 g/L di-potassium
phosphate, 1.7 g/L Mono-potassium phosphate; Becton Dickenson,
Cockeysville, Md.). The resulting cells were harvested and frozen
in 0.5% glycerol. Twenty grams of the frozen cells were used for
protein purification.
[0418] Thawed cells were resuspended in 500 mL Amylose
Equilibration buffer (20 mM Tris, 100 mM NaCl, pH 8.0). A French
Press cell breaking system (Constant Systems Ltd., Warwick, UK)
with a temperature setting of -7.degree. C. to -10.degree. C. and
30K PSI was used to lyse the cells. The resuspended cells were
checked for breakage by A.sub.600 readings before and after cycling
through the French Press. The lysed cell suspension was pelleted at
10,000G for 30 minutes. Supernatant was harvested from the debris
pellet for protein purification.
[0419] Twenty-five milliliters of Amylose resin (New England
Biolabs, Beverly, Mass.) was poured into a Bio-Rad, 2.5 cm
D.times.10 cm H glass column. The column was packed and
equilibrated by gravity with 10 column volumes (CVs) of Amylose
Equilibration buffer. The harvested cell supernatant was batch
loaded to the Amylose resin, overnight with rocking. The loaded
resin was returned to the glass column, washed with 10 CVs Amylose
Equilibration buffer, and eluted by gravity with .about.2 CVs
Amylose Elution buffer (Amylose Equilibration buffer, 10 mM
Maltose, Fluka Biochemical, Switzerland). Ten 5 ml fractions were
collected over the elution profile and assayed for absorbance at
280 and 320 nM. The Amylose resin was regenerated with 1 CV of
distilled H.sub.2O, 5 CVs of 0.1% (w/v) SDS (Sigma), 5 CVs of
distilled H.sub.2O, 5 CVs of Amylose Equilibration buffer, and
finally 1 CV of Amylose Storage buffer (Amylose Equilibration
buffer, 0.02% (w/v) Sodium Azide). The regenerated resin was stored
at 4.degree. C.
[0420] Elution profile fractions of interest were pooled and
dialyzed in a 10K dialysis chamber (Slide-A-Lyzer, Pierce
Immunochemical) against 4 changes of 4 L PBS pH 7.4 (Sigma) over an
8 hour time period. Following dialysis, the material harvested
represented the purified huzcytor17/MBP-6H polypeptide. The
purified huzcytor17/MBP-6H polypeptide was filter sterilized and
analyzed via SDS-PAGE Coomassie staining for an appropriate
molecular weight product. The concentration of the
huzcytor17/MBP-6H polypeptide was determined by BCA analysis to be
0.76 mg/ml.
[0421] Purified huzcytor17/MBP-6H polypeptide was appropriately
formulated for the immunization of rabbits and sent to R & R
Research and Development (Stanwood, Wash.) for polyclonal antibody
production (Example 25, below).
Example 25
Human zcytor17 Receptor Polyclonal Antibody
A. Preparation and Purification
[0422] Polyclonal antibodies were prepared by immunizing 2 female
New Zealand white rabbits with the purified recombinant protein
huzcytor17/MBP-6H (Example 24). The rabbits were each given an
initial intraperitoneal (IP) injection of 200 .mu.g of purified
protein in Complete Freund's Adjuvant followed by booster IP
injections of 100 .mu.g protein in Incomplete Freund's Adjuvant
every three weeks. Seven to ten days after the administration of
the second booster injection (3 total injections), the animals were
bled and the serum was collected. The animals were then boosted and
bled every three weeks.
[0423] The huzcytor17/MBP-6H specific rabbit serum was pre-adsorbed
of anti-MBP antibodies using a CNBr-SEPHAROSE 4B protein column
(Pharmacia LKB) that was prepared using 10 mg of non-specific
purified recombinant MBP-fusion protein per gram of CNBr-SEPHAROSE.
The huzcytor17/MBP-6H-specific polyclonal antibodies were affinity
purified from the pre-adsorbed rabbit serum using a CNBr-SEPHAROSE
4B protein column (Pharmacia LKB) that was prepared using 10 mg of
the specific antigen purified recombinant protein
huzcytor17/MBP-6H. Following purification, the polyclonal
antibodies were dialyzed with 4 changes of 20 times the antibody
volume of PBS over a time period of at least 8 hours. Human
zcytor17-specific antibodies were characterized by ELISA using 500
ng/ml of the purified recombinant protein huzcytor17/MBP-6H as
antibody target. The lower limit of detection (LLD) of the rabbit
anti-huzcytor17/MBP-6H affinity purified antibody is 500 pg/ml on
its specific purified recombinant antigen huzcytor17/MBP-6H.
B. SDS-PAGE and Western Blotting Analysis of Rabbit Anti-human
Zcytor17 MBP-6H Antibody
[0424] Rabbit anti-human ZcytoR17 MBP-6H antibody was tested by
SDS-PAGE (NuPage 4-12%, Invitrogen, Carlsbad, Calif.) with
coomassie staining method and Western blotting using goat
anti-rabbit IgG-HRP. Either purified protein (200-25 ng) or
conditioned media containing zcytor17 was electrophoresed using an
Invitrogen Novex's Xcell II mini-cell, and transferred to
nitrocellulose (0.2 mm; Invitrogen, Carlsbad, Calif.) at room
temperature using Novex's Xcell blot module with stirring according
to directions provided in the instrument manual. The transfer was
run at 300 mA for one hour in a buffer containing 25 mM Tris base,
200 mM glycine, and 20% methanol. The filter was then blocked with
Western A buffer (in house, 50 mM Tris, pH 7.4, 5 mM EDTA, pH 8.0,
0.05% Igepal CA-630, 150 mM NaCl, and 0.25% gelatin) overnight with
gentle rocking at 4.degree. C. The nitrocellulose was quickly
rinsed, then the rabbit anti-human zcytoR17 MBP-6H (1:1000) was
added in Western A buffer. The blot was incubated for 1.5 hours at
room temperature with gentle rocking. The blot was rinsed 3 times
for 5 minutes each in Western A, then goat anti-rabbit IgG HRP
antibody (1:1000) was added in Western A buffer. The blot was
incubated for 1.25 hours at room temperature with gentle rocking.
The blot was rinsed 3 times for 5 minutes each in Western A, then
quickly rinsed in H.sub.20. The blot was developed using
commercially available chemiluminescent substrate reagents (ECL
Western blotting detection reagents 1 and 2 mixed 1:1; reagents
obtained from Amersham Pharmacia Biotech, Buckinghamshire, England)
and the blot was exposed to X-ray film for up to 15 minutes.
[0425] The rabbit anti-human zcytor17 MBP-6H was able to detect
human zcytor17 present in conditioned media as well as zcytoR17
purified protein as a band at 120 kDa under reducing
conditions.
Example 26
Tissue Distribution of Mouse zcytor17 in Tissue Panels Using
PCR
[0426] A panel of cDNAs from murine tissues was screened for mouse
zcytor17 expression using PCR. The panel was made in-house and
contained 94 marathon cDNA and cDNA samples from various normal and
cancerous murine tissues and cell lines are shown in Table 6,
below. The cDNAs came from in-house libraries or marathon cDNAs
from 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 ZC 10,651 (SEQ ID NO:46) and ZC10,565 (SEQ ID
NO:47) and then diluted based on the intensity of the transferrin
band. 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:48) and 3' alpha tubulin specific oligo primer
ZC17,574 (SEQ ID NO:49) or 3' G3PDH specific oligo primer ZC17,600
(SEQ ID NO:50); 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 ZC38,065 (SEQ ID NO:51) and ZC38,068 (SEQ ID
NO:52), TaKaRa Ex Taq.TM. (TAKARA Shuzo Co LTD, Biomedicals Group,
Japan), and Rediload dye (Research Genetics, Inc., Huntsville,
Ala.). The amplification was carried out as follows: 1 cycle at
94.degree. C. for 5 minutes; 5 cycles of 94.degree. C. for 30
seconds, 68.degree. C. for 30 seconds; 35 cycles of 94.degree. C.
for 30 seconds, 56.degree. C. for 30 seconds and 72.degree. C. for
30 seconds, followed by 1 cycle at 72.degree. C. for 5 minutes.
About 10 .mu.l of the PCR reaction product was subjected to
standard Agarose gel electrophoresis using a 4% agarose gel. The
correct predicted DNA fragment size was observed in brain, CD90+
cells, dendritic, embryo, MEWt#2, Tuvak-prostate cell line,
salivary gland, skin and testis.
[0427] The DNA fragment for skin and testis were excised and
purified using a Gel Extraction Kit (Qiagen, Chatsworth, Calif.)
according to manufacturer's instructions. Fragments were confirmed
by sequencing to show that they were indeed mouse zcytor17.
TABLE-US-00006 TABLE 6 Tissue/Cell line #samples Tissue/Cell line
#samples 229 1 7F2 1 Adipocytes-Amplified 1 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 27
Human Zcytor17 Expression in Various Tissues Using Real-Time
Quantitative RT/PCR
A. Primers and Probes for Human Zcytor17, OSMRbeta and Zcytor17lig
for Conventional and Quantitative RT-PCR
[0428] Real-time quantitative RT-PCR using the ABI PRISM 7900
Sequence Detection System (PE Applied Biosystems, Inc., Foster
City, Calif.) has been previously described (See, Heid, C. A. et
al., Genome Research 6:986-994, 1996; Gibson, U. E. M. et al.,
Genome Research 6:995-1001, 1996; Sundaresan, S. et al.,
Endocrinology 139:4756-4764, 1998). This method incorporates use of
a gene specific probe containing both reporter and quencher
fluorescent dyes. When the probe is intact the reporter dye
emission is negated due to the close proximity of the quencher dye.
During PCR extension using additional gene-specific forward and
reverse primers, the probe is cleaved by 5' nuclease activity of
Taq polymerase which releases the reporter dye from the probe
resulting in an increase in fluorescent emission.
[0429] The primers and probes used for real-time quantitative
RT-PCR analyses of human Zcytor17, OSMRbeta and Zcytor17ligand
expression were designed using the primer design software Primer
Express.TM. (PE Applied Biosystems, Foster City, Calif.). Primers
for human Zcytor17 were designed spanning an intron-exon junction
to eliminate possible amplification of genomic DNA. The forward
primer, ZC37,877 (SEQ ID NO:53) and the reverse primer, ZC37,876
(SEQ ID NO:54) were used in a PCR reaction at a 200 nM
concentration to synthesize a 73 bp product. The corresponding
Zcytor17 TaqMan.RTM. probe, designated ZC37,776 (SEQ ID NO:55) was
synthesized and labeled by PE Applied Biosystems and used in each
PCR reaction at a concentration of 200 nM. The ZC37,776 (SEQ ID
NO:55) probe was labeled at the 5'end with a reporter fluorescent
dye (6-carboxy-fluorescein) (FAM) (PE Applied Biosystems) and at
the 3' end with a fluorescent quencher dye
(6-carboxy-tetramethyl-rhodamine) (TAMRA) (Epoch Biosciences,
Bothell, Wash.).
[0430] Primers for human OSMRbeta were designed spanning an
intron-exon junction to eliminate possible amplification of genomic
DNA. The forward primer, ZC43,891 (SEQ ID NO:137) and the reverse
primer, ZC43,900 (SEQ ID NO:138) were used in a PCR reaction
(below) at a 200 nM concentration. The corresponding OSMRbeta
TaqMan.RTM. probe, designated ZC43,896 (SEQ ID NO: 139) was
synthesized and labeled by PE Applied Biosystems and used in each
PCR reaction at a concentration of 200 nM. The ZC43,896 (SEQ ID
NO:139) probe was labeled at the 5'end with a reporter fluorescent
dye (6-carboxy-fluorescein) (FAM) (PE Applied Biosystems) and at
the 3' end with a non-fluorescent quencher dye (ECLIPSE) (Epoch
Biosciences).
[0431] Primers for human Zcytor17ligand were designed spanning an
intron-exon junction to eliminate possible amplification of genomic
DNA. The forward primer, ZC43,280 (SEQ ID NO:140) and the reverse
primer, ZC43,281 (SEQ ID NO:141) were used in a PCR reaction
(below) at about 200 nM concentration. The corresponding
Zcytor17ligand TaqMan.RTM. probe, designated ZC43,275 (SEQ ID
NO:142) was synthesized and labeled by PE Applied Biosystems and
used in each PCR reaction at a concentration of 200 nM. The
ZC43,275 (SEQ ID NO:142) probe was labeled at the 5'end with a
reporter fluorescent dye (6-carboxy-fluorescein) (FAM) (PE Applied
Biosystems) and at the 3' end with a non-fluorescent quencher dye
(ECLIPSE) (Epoch Biosciences).
[0432] As a control to test the integrity and quality of RNA
samples tested, all RNA samples were screened for either rRNA or
GUS using primer and probe sets either ordered from PE Applied
Biosystems (rRNA kit) or designed in-house (GUS). The rRNA kit
contained the forward primer (SEQ ID NO:56), the rRNA reverse
primer (SEQ ID NO:57), and the rRNA TaqMan.RTM. probe (SEQ ID
NO:58). The rRNA probe was labeled at the 5'end with a reporter
fluorescent dye VIC (PE Applied Biosystems) and at the 3' end with
the quencher fluorescent dye TAMRA (PE Applied Biosystems). The GUS
primers and probe were generated in-house and used in each PCR
reaction at 200 nM and 100 nM, respectively. The forward primer was
ZC40,574 (SEQ ID NO:143) and the reverse primer was ZC40,575 (SEQ
ID NO:144). The GUS probe ZC43,017 (SEQ ID NO:145) was labeled at
the 5'end with a reporter fluorescent dye (Yakima-Yellow) (Epoch
Biosciences) and at the 3'end with a non-fluorescent quencher dye
(ECLIPSE) (Epoch Biosciences). The rRNA and GUS results also serve
as an endogenous control and allow for the normalization of the
Zcytor17 mRNA expression results seen in the test samples.
[0433] For conventional non-quantitative RT-PCR, primers were
designed using the primer design software Primer Express.TM. (PE
Applied Biosystems, Foster City, Calif.). The human zcytor17
primers generate an approximately 1000 base pair product and are as
follows: forward primer ZC28,917 (SEQ ID NO:83), and reverse primer
ZC28,480 (SEQ ID NO:146). The human OSMRbeta primers generate a 202
base pair product and are as follows: forward primer ZC41,653(SEQ
ID NO:147) and reverse primer ZC41,655 (SEQ ID NO:148). The human
Zcytor17ligand primers generate a 305 base pair product and are as
follows: forward primer ZC41,703 (SEQ ID NO:149) and reverse primer
ZC41,704 (SEQ ID NO:150).
B. Primers and Probes for Murine Zcytor17, OSMRbeta and
Zcytor17ligand for Conventional and Quantitative RT-PCR
[0434] The primers and probes used for real-time quantitative
RT-PCR analyses of murine Zcytor17, OSMRbeta and Zcytor17lig
expression were designed using the primer design software Primer
Express.TM. (PE Applied Biosystems, Foster City, Calif.). Primers
for murine Zcytor17 were designed spanning an intron-exon junction
to eliminate possible amplification of genomic DNA. The forward
primer, ZC43,272 (SEQ ID NO:151) and the reverse primer, ZC43,273
(SEQ ID NO:152) were used in the PCR reactions (below) at 300 nM
concentration. The corresponding Zcytor17 TaqMan.RTM. probe,
designated ZC43,478 (SEQ ID NO:153) was synthesized and labeled by
PE Applied Biosystems. The ZC43,478 (SEQ ID NO:153) probe was
labeled at the 5'end with a reporter fluorescent dye
(6-carboxy-fluorescein) (FAM) (PE Applied Biosystems) and at the 3'
end with a quencher fluorescent dye
(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems).
The ZC43,478 (SEQ ID NO:153) probe was used in the PCR reactions at
a concentration of 100 nM.
[0435] Primers for murine Zcytor17ligand were designed spanning an
intron-exon junction to eliminate possible amplification of genomic
DNA. The forward primer, ZC43,278 (SEQ ID NO:154) and the reverse
primer, ZC43,279 (SEQ ID NO:155) were used in the PCR reactions at
500 nM concentration. The corresponding Zcytor17ligand TaqMan.RTM.
probe, designated ZC43,276 (SEQ ID NO:156) was synthesized and
labeled by PE Applied Biosystems. The ZC43,478 (SEQ ID NO:153)
probe was labeled at the 5'end with a reporter fluorescent dye
(6-carboxy-fluorescein) (FAM) (PE Applied Biosystems) and at the 3'
end with a non-fluorescent quencher dye (ECLIPSE) (Epoch
Biosciences). The ZC43,276 (SEQ ID NO:156) probe was used in the
PCR reactions (below) at a concentration of 200 nM.
[0436] Primers for murine OSMRbeta were designed spanning an
intron-exon junction to eliminate possible amplification of genomic
DNA. The forward primer, ZC43,045 (SEQ ID NO:157) and the reverse
primer, ZC43,046 (SEQ ID NO:158) were used in the PCR reactions at
a 300 nM concentration. The corresponding OSMRbeta TaqManQ probe,
designated ZC43,141(SEQ ID NO: 159) was synthesized and labeled by
Epoch Biosciences. The ZC43,141 (SEQ ID NO:159) probe was labeled
at the 5'end with a reporter fluorescent dye
(6-carboxy-fluorescein) (FAM) (PE Applied Biosystems) and at the 3'
end with a non-fluorescent quencher dye (ECLIPSE) (Epoch
Biosciences). The ZC43,141 (SEQ ID NO:159) probe was used in the
PCR reactions (below) at a concentration of 100 nM.
[0437] As a control to test the integrity and quality of RNA
samples tested, all RNA samples were screened for either murine GUS
or transferrin receptor using primers and probes designed using the
primer design program Primer Express.TM. (PE Applied Biosystems
Inc., Foster City, Calif.). The murine GUS primers are as follows:
forward primer, ZC43,004 (SEQ ID NO:160), reverse primer, ZC43,005
(SEQ ID NO:161), and TaqMan.RTM. probe ZC43,018 (SEQ ID NO:162).
The murine GUS probe ZC43,018 (SEQ ID NO:162) was labeled at the
5'end with a reporter fluorescent dye Yakima-Yellow (Epoch
Biosciences) and at the 3' end with the non-fluorescent quencher
dye ECLIPSE (Epoch Biosciences). The murine GUS primers were used
in the PCR reactions at 300 nM and the probe, ZC43,018 (SEQ ID
NO:162), was used at 100 nM. In some cases murine Transferrin
Receptor was used instead of GUS as the endogenous control. The
transferrin receptor forward primer, ZC40,269 (SEQ ID NO:163) and
the reverse primer, ZC40,268 (SEQ ID NO:164) were used at 300 nM.
The transferrin receptor probe, ZC40,298 (SEQ ID NO:165) was used
in PCR at 100 nM and was labeled at the 5'end with a reporter
fluorescent dye VIC (PE Applied Biosystems) and at the 3'end with a
fluorescent quencher dye (TAMRA) (PE Applied Biosystems). The
murine GUS and transferrin receptor results also serve as an
endogenous control and allow for the normalization of the Zcytor17,
OSMRbeta and Zcytor17ligand mRNA expression results seen in the
test samples.
[0438] For conventional semi-quantitative RT-PCR, primers were
designed using the primer design software Primer Express.TM. (PE
Applied Biosystems). The murine Zcytor17 primers generate a 276
base pair product and are as follows: forward primer ZC43,140 (SEQ
ID NO:166), and reverse primer ZC43,139 (SEQ ID NO:167). The murine
OSMRbeta primers generate a 575 base pair product and are as
follows: forward primer ZC41,608 (SEQ ID NO:168) and reverse primer
ZC41,609 (SEQ ID NO:169). The murine Zcytor17ligand primers
generate a 657 bp product and are as follows: forward primer
ZC41,502 (SEQ ID NO:170) and reverse primer ZC41,500 (SEQ ID NO:
171).
C. Protocols for Realtime Quantitative RT-PCR and Conventional
Semi-Quantitative RT-PCR
[0439] Relative levels of Zcytor17, OSMRbeta and Zcytor17ligand
mRNA were determined by analyzing total RNA samples using the
one-step RT-PCR method (PE Applied Biosystems). Total RNA from
Zcytor17- and OSMRbeta-transfected BAF cells (human) or BHK cells
(murine) was isolated by standard methods and used to generate a
standard curve used for quantitation of Zcytor17 and OSMRbeta. The
curve consisted of 10-fold serial dilutions ranging from 100-0.01
ng/.mu.l with each standard curve point analyzed in triplicate.
Similarly, for Zcytor17ligand, activated CD4+ T cell RNA
(previously shown to make Zcytor17ligand) was used to generate a
standard curve in the same 100-0.01 ng/.mu.l range. Total RNA from
human or murine cells was analyzed in triplicate for either human
or murine Zcytor17, OSMRbeta and Zcytor17ligand transcript levels
and for one of the following endogenous control genes: rRNA, GUS or
transferrin receptor. In a total volume of 10 .mu.l, each RNA
sample was subjected to a One-Step RT-PCR reaction containing:
approximately 50-100 ng of total RNA in a preformulated 2.times.
master mix containing an internal control dye
(ROX)(carboxy-x-rhodamine) and Thermo-Start.RTM. DNA Polymerase
(Abgene, Surrey, UK); appropriate primers for the gene of interest
(see parts A and B of current example); the appropriate probe (see
parts A and B for concentration); Superscript reverse transcriptase
(50 U/.mu.l) (PE Applied Biosystems), and an appropriate volume of
RNase-free water. PCR thermal cycling conditions were as follows:
an initial reverse transcription (RT) step of one cycle at
48.degree. C. for 30 minutes; followed by a Thermo-Start.RTM.
enzyme activation step of one cycle at 95.degree. C. for 10
minutes; followed by 40 cycles of amplification at 95.degree. C.
for 15 seconds and 60.degree. C. for 1 minute. Relative Zcytor17,
OSMRbeta and Zcytor17ligand RNA levels were determined by using the
Standard Curve Method as described by the manufacturer, PE
Biosystems (User Bulletin #2: ABI Prism 7700 Sequence Detection
System, Relative Quantitation of Gene Expression, Dec. 11, 1997).
The rRNA, GUS or Transferrin Receptor measurements were used to
normalize the levels of the gene of interest.
[0440] The semi-quantitative RT-PCR reactions used the `Superscript
One-Step RT-PCR System with Platinum Taq` (Invitrogen, Carlsbad,
Calif.). Each 25 .mu.l reaction consisted of the following: 12.5
.mu.l of 2.times. Reaction Buffer, 0.5 .mu.l (20 pmol/.mu.l) of
forward primer, 0.5 .mu.l (20 pmol/.mu.l) of reverse primer, 0.4
.mu.l RT/Taq polymerase mix, 5.0 .mu.l of Rediload Gel Loading
Buffer (Invitrogen), 5.1 .mu.l RNase-free water, and 1.0 .mu.l
total RNA (100 ng/.mu.l). The amplification was carried out as
follows: one cycle at 45.degree. C. for 30 minutes followed by
35-38 cycles of 94.degree. C., 20 seconds; Variable annealing temp
(See Table 7 below), 20 seconds; 72.degree. C., 45 seconds; then
ended with a final extension at 72.degree. C. for 5 minutes. Eight
to ten microliters of the PCR reaction product was subjected to
standard agarose gel electrophoresis using a 2% agarose gel.
TABLE-US-00007 TABLE 7 Murine Zcytor17 58.degree. C. anneal temp
Murine OSMRbeta 60.degree. C. anneal temp Murine 52.degree. C.
anneal temp Zcytor17ligand Human Zcytor17 55.degree. C. anneal temp
Human OSMRbeta 59.degree. C. anneal temp Human 59.degree. C. anneal
temp Zcytor17ligand
D. Isolation of RNA from Human and Murine PBMC Subsets and Cell
Lines
[0441] Blood was drawn from several anonymous donors and peripheral
blood mononuclear cells (PBMC) isolated using standard Ficoll
gradient methodology. Monocytes were then isolated using the
Monocyte Isolation Kit and the Magnetic Cell Separation System
(Miltenyi Biotec, Auburn, Calif.). The monocytes were then plated
onto ultra-low adherence 24-well plates in endotoxin-free media.
They were either unstimulated or treated with recombinant human
IFNg (R&D Systems Inc.) at 10 ng/ml. Cells were collected at 24
and 48 hours. In similar manner, CD4+ and CD8+ T cells were
isolated from PBMC's using the anti-CD4 or anti-CD8 magnetic beads
from Miltenyi Biotec. Cells were then activated for 4 or 16 hours
in tissue culture plates coated with 0.5 .mu.g/ml anti-CD3
antibodies in media containing 5 .mu.g/ml anti-CD28 antibodies. NK
cells were also isolated from PBMC's using Miltenyi's anti-CD56
coated magnetic beads. Some of the NK cells were collected at time
zero for RNA and the others were plated in media containing Phorbol
Myristate Acetate (PMA) (5 ng/ml) and ionomycin (0.5 .mu.g/ml) for
24 hours. Additionally, several human monocyte-like cell lines,
U937, THP-1 and HL-60, were collected in either their resting or
activated states. U937 cells were activated overnight with PMA (10
ng/ml). HL-60's were activated overnight with PMA (10 ng/ml) or for
72 and 96 hours with IFNg (10 ng/ml) to drive them down a monocytic
pathway. THP-1 cells were activated overnight with a combination of
LPS (10 ng/ml) and IFNg (10 ng/ml). RNA was prepared from all
primary cells using the RNeasy Midiprep.TM. Kit (Qiagen, Valencia,
Calif.) as per manufacturer's instructions. Carryover DNA was
removed using the DNA-Free.TM. kit (Ambion, Inc., Austin, Tex.).
RNA concentration was determined using standard spectrophotometry
and RNA quality determined using the Bioanalyzer 2100 (Agilent
Technologies, Palo Alto, Calif.).
[0442] Murine T Cell RNA was collected using a variety of methods
well-known in the art. Primary splenic CD4+ and CD8+ T cells were
isolated from the spleens of C57B1/6 mice using antibody-coated
magnetic beads and the Magnetic Cell Separation System from
Miltenyi Biotec. The CD4+ and CD8+ T cells were then activated by
culturing the cells in 24-well plates coated with anti-CD3
antibodies (500 ng/ml) in media containing anti-CD28 antibodies at
5 .mu.g/ml. Cells were harvested for RNA at 0, 4 and 16 hours.
Similarly, CD4+ T cells were isolated and then skewed towards a Th1
or Th2 phenotype using the following protocol. Since C57B1/6 T
cells are already skewed in the Th1 direction, all that was
required was to activate for 6 hours with 0.5 .mu.g/ml PMA and 10
ng/ml ionomycin. `Th2` skewing was obtained by plating naive CD4+ T
cells with 2.5 .mu.g/ml anti-CD28, 10 ng/ml mIL-2 (R&D Systems
Inc.) and 25 ng/ml mIL-4 (R&D Systems) into plates coated with
0.5 .mu.g/ml anti-CD3. After 2 days in culture, cells were
resuspended in media containing 10 ng/ml mIL-2 (R&D Systems)
and 25 ng/ml mIL-4. Cells were cultured for an additional three
days then activated with PMA and ionomycin for 6 hours.
[0443] One additional set of Th1 and Th2 skewed T cells was derived
using the T Cell Receptor Transgenic DO11.10 T cell line. All cells
were plated into anti-CD3 and anti-CD28 coated plates. The `Th1 `
cells were plated in media containing mIL-12 (1 ng/ml) and
anti-IL-4 (10 .mu.g/ml). The `Th2` cells were plated in media
containing mIL-4 (10 ng/ml) and anti-IFNg (10 .mu.g/ml). After 24
hours, all cultures were given mIL-2 (10 ng/ml). After two more
days, the media on the cells was changed and new media containing
the aforementioned cytokines was added and cells were cultured an
additional 4 days before being harvested.
[0444] All of the murine T cell RNA was prepared using the RNeasy
Midiprep.TM. Kit (Qiagen) and contaminating DNA was removed using
the DNA-free.TM. kit from Ambion.
E. Isolation of RNA from the Murine Models of Pancreatitis and
Irritable Bowel Disease
[0445] To induce a condition similar to human Irritable Bowel
Disease (IBD), the hybrid mouse strain C57B16/129S6F1 was used.
Mice were divided into 4 groups with an average size of six mice
per group. Group 1 was given no Dextran Sulfate Sodium (DSS) and
was sacrificed on day 14. Group 2 received 2% DSS for two days
prior to being sacrificed. Group 3 received 2% DSS for seven days
prior to sacrifice. Group 4 received 2% DSS for seven days then
allowed to recover for seven days and was sacrificed on day 14. On
the day of sacrifice, the distal colon sections were removed and
placed in RNAlater.TM. (Ambion). The colon sections were
homogenized using standard techniques and RNA was isolated using
the RNeasy Midiprep.TM. Kit (Qiagen). Contaminating DNA was removed
by DNA-free.TM. (Ambion) treatment as per manufacturer's
instructions.
[0446] In a different study, acute pancreatitis was induced in male
CD-1 mice by caerulein injection. Mice were divided into three
groups (n=8 mice/group). Group 1 animals were given seven i.p.
injections (1 injection per hour) with Vehicle (saline), and then
sacrificed at 12 and 24 hours following the first injection. Groups
2 and 3 were given seven i.p. injections of caerulein (Sigma)
(Catalog#C-9026) at a dose of 50 .mu.g/kg/hr for six hours (1
injection per hour). Group 2 was sacrificed at 12 hrs after the
first injection and Group 3 was sacrificed at 24 hrs following the
first injection. Pancreases were removed at the time of sacrifice
and snap frozen for RNA isolation. Tissues were homogenized and RNA
was isolated using the Qiagen RNeasy Midiprep.TM. Kit.
[0447] In yet another study, murine Zcytor17ligand transgenic mice
were generated and observed for phenotypic changes (see Example
41). Piloerection and hair loss was observed in many of the
transgenic mice. Four transgenic mice were sacrificed and skin
samples from both normal and hairless areas were removed and snap
frozen for later RNA isolation. Skin sections from two
non-transgenic control mice were collected as well. Skin samples
were homogenized and then digested with Proteinase K (Qiagen)
(Catalog# 19133) for 20 minutes at 60.degree. C. RNA was then
isolated using the Qiagen RNeasy Midiprep.TM. Kit following
manufacturer's instructions. Carryover DNA was removed using
DNA-free.TM. kit from Ambion.
F. Results of Quantitative and Semi-Quantitative RT-PCR for Human
Zcytor17, OSMRbeta and Zcytor17ligand
[0448] Zcytor17 and OSMRbeta expression was examined by
quantitative RT-PCR in four sets of primary human monocytes that
were either in their resting state or activated with IFNg for 24 or
48 hours. Zcytor17 expression was below detection in the
unstimulated cells but increased dramatically after the 24-hour
activation with IFNg, and was the highest after 48 hours of
activation. In all cases OSMRbeta was below detection.
Zcytor17ligand was not tested in these samples.
[0449] In the primary T cells, Zcytor17 was below detection in both
the resting CD4+ and CD8+ subsets. After a four-hour activation,
however, expression of Zcytor17 went up in both subsets and then
decreased to a slightly lower level at the 16 hour time point.
OSMRbeta was below detection in these samples. Zcytor17ligand
expression was examined using semi-quantitative RT-PCR. No
expression was detected in the unstimulated CD4+ and CD8+ T cells.
However, after the four hour activation, high levels of
Zcytor17ligand were detected. This level dropped somewhat at the 16
hour time point.
[0450] Expression of Zcytor17 was not examined in NK cells. OSMRb
was below detection in these samples. Zcytor17ligand expression was
below detection in the resting NK cells, however there was a faint
signal generated by the activated NK cells suggesting that these
cells may make Zcytor17ligand under certain conditions.
[0451] In the human monocyte-like cell lines, U937, THP-1 and
HL-60, OSMRbeta expression was below detection in all of the
resting and activated samples except for activated THP-1 samples
where a faint signal was detected. Zcytor17 expression was high in
both the U937 and THP-1 resting cell lines and showed a strong
upregulation following activation. Expression in U937's was the
highest of any cell type. In the HL-60's, Zcytor17 was expressed at
moderate levels in the unstimulated cells and decreased upon
stimulation with PMA. However, the expression of Zcytor17 was
dramatically upregulated in the HL-60's when stimulated with IFNg
for 72 and 96 hours. All of the human expression data is summarized
in Table 8 below. TABLE-US-00008 TABLE 8 Primary Human Activation
Monocytes Status Zcytor17 OSMRbeta Zcytor17Ligand Human Unstim - -
Monocytes Human Act. 24 hr + - Monocytes IFNg Human Act. 48 hr ++ -
Monocytes IFNg Human CD4+ Unstim - - - Human CD4+ Act 4 hr ++ - ++
Human CD4+ Act. 16 hr + - + Human CD8+ Unstim - - - Human CD8+ Act
4 hr ++ - ++ Human CD8+ Act. 16 hr + - + Human NK Unstim - - Cells
Human NK Act 24 hr - + Cells U937 Unstim ++ - - U937 Act. 16 hr +++
- - THP-1 Unstim ++ - - THP-1 Act. 16 hr +++ + - HL-60 Unstim ++ -
- HL-60 Act. 16 hr + - - PMA HL-60 Act. 72 hr +++ - - IFNg HL-60
Act. 96 hr +++ - - IFNg
G. Results of Quantitative and Semi-Quantitative RT-PCR for Murine
Zcytor17, OSMRbeta and Zcytor17ligand
[0452] Murine Zcytor17, OSMRbeta and Zcytor17ligand expression
levels were examined in several murine T cells populations and the
results are summarized in Table 9 below. Murine Zcytor17 expression
was tested by semi-quantitative RT-PCR and shown to be at low
levels on both resting and activated primary CD4+ T cells.
Expression of Zcytor17 was detected on resting CD8+ T cells and
then seemed to drop upon activation with anti-CD3 and anti-CD28
antibodies at both the 4- and 16-hour time points. OSMRbeta
expression was measured by quantitative RT-PCR and shown to be
expressed in resting and activated CD4+ and CD8+ T cells. The
expression of OSMRbeta went up after a 4-hour activation and then
returned to the unstimulated levels by 16 hours in both the CD4+
and CD8+ T cells. Zcytor17ligand was detected by quantitative
RT-PCR and shown to be expressed at very low levels in unstimulated
CD4+ T cells. However, following a 4-hour activation,
Zcytor17ligand expression was dramatically upregulated and then
dropped slightly by the 16-hour time point. In CD8+ T cells, no
Zcytor17ligand was detected in the unstimulated cells. There was
some Zcytor17ligand expression at the 4-hour time point, but by 16
hours expression levels had dropped back below detection.
[0453] In the DO11.10 T cells, Zcytor17 expression was detected in
the naive and Th2 skewed cells, but not in the Th1 skewed cells.
OSMRbeta expression was at low levels in the naive DO11.10 cells.
There was a dramatic increase in OSMRbeta expression levels in the
Th1 skewed cells and a moderate increase of expression in the
Th2-skewed cells. The Zcytor17ligand expression in these cells was
shown to be predominantly by the Th2 skewed subset. Low levels were
detected in the Th1 subset and no expression was detected in the
naive cells. These results are summarized in the Table 9 below.
[0454] In the primary CD4+ T cells that were skewed in either the
Th1 or Th2 direction, Zcyto17 wasn't examined. OSMRbeta expression
was detected in all three samples with the highest levels found in
the Th2 sample. Similar to the DO11.10 results, Zcytor17ligand
expression was detected at high levels in the Th2 skewed subset,
with a small amount detected in the Th1 subset and levels were
below detection in the unstimulated cells. These results are
summarized in the Table 9 below. TABLE-US-00009 TABLE 9 Murine T
Cells Zcytor17 OSMRbeta Zcytor17ligand CD4+ T Cells Unstimulated +
+ +/- CD4+ T Cells 4 hr Activation + ++ ++ CD4+ T Cells 16 hr + + +
Activation CD8+ T Cells Unstimulated + + - CD8+ T Cells 4 hr
Activation +/- ++ + CD8+ T Cells 16 hr - + - Activation DO11.10
Naive + + - DO11.10 Th1 - +++ + DO11.10 Th2 + ++ ++ CD4+ T Cells
Unstimulated ++ - CD4+ T Cells - Th1 Skewed +++ + CD4+ T Cells -
Th2 Skewed ++ +++
[0455] In the Zcytor17ligand transgenic skin samples, Zcytor17,
OSMRbeta and Zcytor17ligand expression levels were determined using
quantitative RT-PCR. Zcytor17 was shown to be present in all
samples at roughly equivalent levels. There were dramatically
higher levels of OSMRbeta expression in the non-transgenic control
animals than the transgenic samples. Zcytor17ligand expression was
below detection in the non-transgenic control animals with moderate
to high levels of expression in the transgenic animals. The results
are summarized in Table 10 below. TABLE-US-00010 TABLE 10 Murine
Zcytor17ligand Skin Transgenic Skin Phenotype Zcytor17 OSMRbeta
Zcytor17ligand Wild Type Mouse Normal + +++ - Wild Type Mouse
Normal + +++ - Transgenic #1 Normal + + + Transgenic #1 Hair Loss +
+ + Transgenic #2 Normal + + + Transgenic #2 Hair Loss + + +
Transgenic #3 Normal + + + Transgenic #3 Hair Loss + + + Transgenic
#4 Normal + + +++ Transgenic #4 Hair Loss + + +++
[0456] In a different experiment, Zcytor17, OSMRbeta and
Zcytor17ligand expression levels were measured by quantitative
RT-PCR in the pancreases of mice subjected to acute pancreatitis.
Zcytor17 expression was below detection in all of the samples.
OSMRbeta expression was seen at low levels in the normal control
samples (Group 1), but showed a strong upregulation at the 12-hour
time point (Group 2) and slightly lower levels at the 24-hour time
point (Group 3). Zcytor17ligand expression was below detection in
the control animals, but showed high levels in both of the
caerulein injected groups. The data is summarized in Table 11
below. TABLE-US-00011 TABLE 11 Pancreatitis Model Description
Zcytor17 OSMRbeta Zcytor17ligand Group 1 Normal Control - + - Group
2 12 hr Post - +++ ++ Injection Group 3 24 hr Post - ++ ++
Injection
[0457] In another experiment, Zcytor17 and OSMRbeta expression
levels were examined in the distal colons of mice subjected to DSS
treatment. In this murine model of Inflammatory Bowel Disease,
expression levels of both genes were determined by quantitative
RT-PCR and are summarized in Table 12 below. Zcytor17 expression
levels increased with the severity of the disease, with low levels
of expression in the Group 1 normal animals and increasing amounts
seen Groups 2 and 3. In the Group 4 animals, the Zcytor17 levels
had returned to more normal levels. Unlike Zcytor17 expression,
OSMRbeta levels were the highest in the control animals and levels
actually decreased in all three DSS treated groups. TABLE-US-00012
TABLE 12 IBD Model Description SAC Day Zcytor17 OSMRbeta Group 1
Normal Control 14 + ++ Group 2 DSS-Treated 2 days 2 ++ + Group 3
DSS-Treated 7 days 7 +++ + Group 4 DSS-Treated 7 days 14 + +
Example 28
Human Zcytor17lig Tissue Distribution Expression based on RT-PCR
Analysis of Multiple Tissue First-Strand cDNAs
[0458] Gene expression of the zcytor17lig was examined using
commercially available normalized multiple tissue first-strand cDNA
panels (OriGene Technologies, Inc. Rockville, Md.; BD Biosciences
Clontech, Palo Alto, Calif.). These included the OriGene "Human
Tissue Rapid-Scan.TM. Panel" (Cat. #CHSCA-101, containing 22
different tissues, bone marrow, and plasma blood leucocytes) and
the BD Biosciences Clontech "Human Blood Fractions MTC.TM. Panel"
(Cat. #K1428-1, containing 9 different blood fractions).
[0459] PCR reactions were set up using the zcytor17lig specific
oligo primers ZC41,458 (SEQ ID NO:60), and ZC41,457 (SEQ ID NO:61),
which yield a 139 bp product, and ZC41,459 (SEQ ID NO: 62), and
ZC41,460 (SEQ ID NO:63), which yield a 92 bp product, Qiagen
HotStarTaq DNA polymerase and buffer (Qiagen, Inc., Valencia,
Calif.), dH.sub.2O, and RediLoad.TM. dye (Research Genetics, Inc.,
Huntville, Ala.). The PCR cycler conditions were as follows: an
initial 1 cycle 15 minute denaturation at 95.degree. C., 35 cycles
of a 45 second denaturation at 95.degree. C., 1 minute annealing at
53.degree. C. or 56.degree. C. and 1 minute and 15 seconds
extension at 72.degree. C., followed by a final 1 cycle extension
of 7 minutes at 72.degree. C. The reactions were separated by
electrophoresis on a 2% agarose gel (EM Science, Gibbstown, N.J.)
and visualized by staining with ethidium bromide.
[0460] A DNA fragment of the correct size was observed in the
following human adult tissues using the OriGene "Human Tissue
Rapid-Scan.TM. Panel": testis, plasma blood leucocytes (PBL), and
bone marrow.
[0461] A DNA fragment of the correct size was observed in the
following human blood fractions using the BD Biosciences Clontech
"Human Blood Fractions MTC.TM. Panel": activated mononuclear cells
(B- & T-cells and monocytes), activated CD8+ cells
(T-suppressor/cytotoxic), activated CD4+ cells (T-helper/inducer)
and faintly in resting CD8+ cells.
Example 29
Cloning the Human Oncostatin M Receptor
[0462] The OncostatinM beta receptor (OSMRbeta) is a type I
cytokine receptor with structural similarity to IL12R-B2. ZcytoR17
has structural similarity to IL12R-B1. The OSMRbeta and zcytor17
were tested to see whether they could interact as subunits in a
cytokine signaling complex, and whether together they could act as
a signaling receptor, or soluble receptor antagonist, for
zcytor17lig.
[0463] To isolate OSMRbeta, oligonucleotide PCR primers ZC39982
(SEQ ID NO:64) and ZC39983 (SEQ ID NO:65) were designed to amplify
the full length coding region of the human OncostatinM beta cDNA
sequence (SEQ ID NO:6) (Genbank Accession No. U60805; Mosley B, JBC
Volume 271, Number 50, Issue of Dec. 20, 1996 pp. 32635-32643).
[0464] PCR reactions were run on an array of cDNA library templates
using a robust polymerase, Advantage II (Clonetech, PaloAlto,
Calif.), in order to identify a source of the cDNA. The template
DNA used was from amplified cDNA plasmid libraries each containing
5 million independent cDNA clones. Reactions were assembled as per
manufacturer's instructions using 400 fmol/.mu.l of each
oligonucleotide and 2-20 ng/.mu.l purified plasmid library DNA as
template. The cDNA libraries were derived from the following human
tissues and cell lines: fetal brain, prostate smooth muscle, bone
marrow, RPM11588, thyroid, WI-38, testis, stimulated peripheral
blood mononuclear cells, stimulated CD3+ cells, THP-1, activated
tonsil, HACAT and fetal liver. Reactions were performed on a
thermocycler machine using the following conditions: 30 cycles of
95.degree. C. for 20 seconds, 68.degree. C. for 3 minutes. At the
conclusion of 30 cycles an additional single extension cycle of 8
minutes at 68.degree. C. was run. PCR products were visualized by
TAE agarose, gel electrophoresis in the presence of ethidium
bromide followed by UV illumination. The most abundant product was
found to be from a prostate smooth muscle cDNA library. The PCR
reaction using prostate smooth muscle template and oligonucleotides
ZC39982 (SEQ ID NO:64) and ZC39983 (SEQ ID NO:65) was repeated
using a less robust but higher fidelity thermostable DNA polymerase
"turboPFu", (Stratagene, La Jolla, Calif.). Thirty amplification
cycles were run with the following conditions: denaturing at
94.degree. C., 30 seconds, annealing at 63.degree. C. 45 seconds,
extension at 72.degree. C. 3.5 minutes. A single band product was
gel purified on a 0.8% TAE, agarose gel.
[0465] This DNA was then amplified again using primers ZC39980 (SEQ
ID NO:66) and ZC39981 (SEQ ID NO:67) designed to include
restriction enzyme recognition sequences to allow the cloning of
this cDNA into a mammalian expression vector.
[0466] The PCR reaction was performed using "TurboPfu" and the
purified PCR product for 15 cycles of: 95.degree. C. 1 minute,
64.degree. C. 1 minute 20 seconds, 72.degree. C. 4.5 minutes. The
PCR reaction was then digested with EcoR1 and Xho1 (Invitrogen,
Carlsbad, Calif.) and gel purified as described above. A mammalian
expression vector, pZ7NX, was prepared by digesting with EcoR1 and
Xho1 and the PCR product was ligated to this vector and
electroporated into E. coli DH10b cells. Several bacterial colonies
were isolated and sequenced. One clone was correct with the
exception of a single non-conservative mutation. In order to change
this base to match the expected sequence, an oligonucleotide
spanning mutation and a neighboring Pst1 restriction site was used
in a PCR reaction with "TurboPfu" using the pZP7Nx-h. OncostatinM R
plasmid previously sequenced as a template. The PCR amplified DNA
was digested with Pst1 and Xho1 and cloned back into the pZP7Nx-h
OncostatinM R plasmid in place of the Pst1/Xho1 fragment containing
the offending mutation. This new plasmid was sequenced over the
recently amplified Pst1 to Xho1 region to confirm the correction
and make sure no other errors were created in the amplification
process. This analysis confirmed sequence that matched the expected
sequence over the coding region. The sequence is shown in SEQ ID
NO:6, and corresponding amino acid sequence shown in SEQ ID
NO:7.
Example 30
Constructs for Generating a Human Zcytor17/OncostatinM Receptor
(OSMRbeta) Heterodimer
[0467] A system for construction, expression and purification of
such soluble heterodimeric receptors is known in the art, and has
been adapted to the receptor pair, human oncostatin M receptor
(OSMRbeta) and human zcytor17. For this construct, the
polynucleotide for the soluble receptor for OSMRbeta is shown in
SEQ ID NO:68 and corresponding polypeptide is shown in SEQ ID
NO:69; and the polynucleotide for the soluble receptor for human
zcytor17 is shown in SEQ ID NO:70 and corresponding polypeptide is
shown in SEQ ID NO:71.
[0468] To construct a cell line expressing a secreted soluble
hzcytor17/human OSMRbeta heterodimer, a construct was made so that
the resulting heterodimeric soluble receptor comprises the
extracellular domain of human OSMRbeta fused to the heavy chain of
IgG gamma1 (Fc4) (SEQ ID NO:37) with a Glu-Glu tag (SEQ ID NO:35)
at the C-terminus; while the extracellular domain of zcytoR17 is
fused to Fc4 (SEQ ID NO:37) with a His tag (SEQ ID NO:72) at the
C-terminus. For both of the hzcytor17 and human OSMRbeta arms of
the heterodimer a Gly-Ser spacer of 12 amino acids (SEQ ID NO:73)
was engineered between the extracellular portion of the receptor
and the N-terminus of Fc4.
A. Construction of Human Soluble OSMRbeta/Fc4-CEE
[0469] For construction of the human soluble OSMRbeta/Fc4-CEE
portion of the heterodimer the extracellular portion of human
OSMRbeta was isolated using PCR with oligos ZC14063 (SEQ ID NO:48)
and ZC41557 (SEQ ID NO:74) under PCR reaction conditions as
follows: 30 cycles of 95.degree. C. for 60 sec., 57.degree. C. for
30 sec., and 72.degree. C. for 100 sec.; and 72.degree. C. for 7
min. PCR products were purified using QIAquick PCR Purification Kit
(Qiagen), digested with EcoRI and BglII (Boerhinger-Mannheim),
separated by gel electrophoresis and purified using a QIAquick gel
extraction kit (Qiagen).
[0470] The expression cassette, plasmid backbone and Fc4-GluGlu tag
portion of the chimera were contained within a previously made in
house plasmid vector. The plasmid vector was digested with EcoR1
and BamH1 (Boerhinger-Mannheim), separated by gel electrophoresis
and purified using a QlAquick gel extraction kit (Qiagen). The
digested and purified fragments of human OSMRbeta and Fc4-cEE
containing plasmid were ligated together using T4 DNA Ligase (Life
Technologies, Bethesda, Md.) using standard ligation methods.
Minipreps of the resulting ligation were screened for an EcoRI/Sma1
insert of the correct size (772 bp) for the soluble OSMRbeta and
positive minipreps were sequenced to confirm accuracy of the PCR
reaction. This new plasmid construction is termed
pZP9-ONCOMR-Fc4CEE.
B. Construction of Human Soluble Zcytor17/Fc4-CHIS
[0471] For construction of the hzcytor17/Fc4-CHIS portion of the
heterodimer, the extracellular portion of human zcytor17 was
isolated by digestion of a plasmid previously containing
Zcytor17-Fc4 soluble receptor. The plasmid was first digested with
Sal1 (New England Biolabs, Beverly, Mass.) after which the reaction
was serially phenol chloroform extracted and ethanol precipitated.
The digested DNA was then treated with T4 DNA Polymerase
(Boerhinger-Mannheim), to fill in the 5' overhangs created by the
SalI digestion, leaving the DNA ends blunt, after which the
reaction was serially phenol chloroform extracted and ethanol
precipitated. The blunted DNA was then further digested with BglII
to cut at the 3' end.), separated by gel electrophoresis and
purified using a QIAquick gel extraction kit (Qiagen) as per
manufacturer's instruction. The resulting DNA fragment containing
the sequence coding for the extracellular domain of zcytoR17 was
ligated into an Fc4-CHIS tag containing mammalian expression vector
prepared as follows.
[0472] The expression cassette, plasmid backbone and Fc4-CHIS tag
portion of the chimera were contained within a previously made in
house plasmid vector. This plasmid vector was digested with EcoR1
(Boerhinger-Mannheim) after which the reaction was serially phenol
chloroform extracted and ethanol precipitated. The digested DNA was
then treated with T4 DNA Polymerase (Boerhinger-Mannheim), to fill
in the 5' overhangs created by the EcoR1 digestion, leaving the DNA
ends blunt, after which the reaction was serially phenol chloroform
extracted and ethanol precipitated. The blunted DNA was then
further digested with BamH1 (Boerhinger-Mannheim) to cut at the 3'
end, separated by gel electrophoresis and purified using a QIAquick
gel extraction kit (Qiagen). The digested and purified fragments of
human zcytor17 and Fc4-CHIS containing plasmid were ligated
together using T4 DNA Ligase (Life Technologies, Bethesda, Md.)
using standard ligation methods.
[0473] Minipreps of the resulting ligation were screened by PCR
using the zcytor17 specific sense primer ZC29180 (SEQ ID NO:22) and
the Fc4 specific antisense primer ZC29232 (SEQ ID NO:75) with the
following PCR reaction conditions: 30 cycles of 94.degree. C. for
60 sec., 68.degree. C. for 150 sec; and 72.degree. C. for 7 min. An
expected product size of 848 bp confirmed the correct assembly of
the plasmid termed pZEM228 hzcytor17/Fc4HIS.
[0474] A second zcytor17-Fc4 construction was created for use in
generating homodimer protein from COS cells. Briefly the coding
region for the full fusion protein was isolated by digestion of a
plasmid previously containing Zcytor17-Fc4 soluble receptor with
SalI (Boerhinger-Mannheim). The reaction was serially phenol
chloroform extracted and ethanol precipitated. The digested DNA was
then treated with T4 DNA Polymerase (Boerhinger-Mannheim), to fill
in the 5' overhangs created by the EcoR1 digestion, leaving the DNA
ends blunt, after which the reaction was serially phenol chloroform
extracted and ethanol precipitated. The blunted DNA was then
further digested with NotI (Boerhinger-Mannheim) to cut at the 3'
end, separated by gel electrophoresis and purified using a QIAquick
gel extraction kit (Qiagen). A mammalian expression vector
containing a CMV driven expression cassette was digested to
generate compatible ends and the 2 fragments were ligated together.
Minipreps of the resulting ligation were screened by PCR using the
vector specific sense primer ZC14063 (SEQ ID NO:48) and the
zcytor17 specific antisense primer ZC27899 (SEQ ID NO:19) with the
following PCR reaction conditions: 30 cycles of 94.degree. C. for
30 sec., 64.degree. C. for 30 sec; 70.degree. C. for 90 sec; and
72.degree. C. for 7 min. An expected product size of approximately
1000 bp confirmed the correct assembly of the plasmid termed
pZP7NX-hzcytor17-Fc4. This plasmid was subsequently transfected
into COS cells using Lipofectamine (Gibco/BRL), as per
manufacturer's instructions. The cells were conditioned for 60
hours in DMEM+5% FBS (Gibco/BRL) after which the protein was
purified over a protein G-sepharose 4B chromatography column and
made available for in vitro bioassays, for example, such as those
described herein.
C. Generating a Human Zcytor17/OncostatinM Receptor (OSMRbeta)
[0475] About 16 .mu.g each of the pZP9-ONCOMR-Fc4CEE and pZEM228
hzcytor17/Fc4HIS were co-transfected into BHK-570 (ATCC No.
CRL-10314) cells using lipofectamine (Gibco/BRL), as per
manufacturer's instructions. The transfected cells were selected
for 10 days in DMEM+5% FBS (Gibco/BRL) containing 0.5 mg/ml G418
(Gibco/BRL) and 250 nM methyltrexate (MTX)(Sigma, St. Louis, Mo.)
for 10 days.
[0476] The resulting pool of doubly-selected cells was used to
generate the heterodimeric protein. Three cell Factories (Nunc,
Denmark) of this pool were used to generate 10 L of serum free
conditioned medium. This conditioned media was passed over a 1 ml
protein-A column and eluted in (10) 750 microliter fractions. Four
of these fractions found to have the highest concentration were
pooled and dialyzed (10 kD MW cutoff) against PBS. The desired
heterodimeric soluble zcytor17/OSMRbeta protein complex was
isolated from other media components by passing the pool over a
Nickel column and washing the column with various concentrations of
Imidazole. The soluble zcytor17/OSMRbeta protein eluted at
intermediate concentrations of Imidazole, while hzcytor17/Fc4HIS
homodimer eluted at higher concentrations of Imidazole.
Example 31
Tissue Distribution of Human zcytor17 in Tissue Panels Using
Northern Blot and PCR
A. Human zcytor17 Tissue Distribution Using Northern Blot
[0477] Human Multiple Tissue Northern Blots (Human 12-lane MTN Blot
I and II, and Human Immune System MTN Blot II; Human Endocrine MTN,
Human Fetal MTN Blot II, Human Multiple Tissue Array) (Clontech) as
well as in house blots containing various tissues were probed to
determine the tissue distribution of human zcytor17 expression. The
in-house prepared blots included the following tissue and cell line
mRNA: SK-Hep-1 cells, THP1 cells, Adrenal gland (Clontech); Kidney
(Clontech), Liver (Clontech and Invitrogen); Spinal cord
(Clontech), Testis (Clontech), Human CD4+ T-cells, Human CD8+
T-cells, Human CD19+ T-cells, human mixed lymphocyte reaction
(MLR), THP1 cell line (ATCC No. TIB-202), U937 cell line, p388D1
mouse lymphoblast cell line (ATCC No. CCL-46) with or without
stimulation by Tonomycin; and WI-38 human embryonic lung cell line
(ATCC No. CRL-2221) with or without stimulation by Ionomycin.
[0478] An approximately 500 bp PCR derived probe for zcytor17 (SE
ID NO:4) was amplified using oligonucleotides ZC28,575 (SEQ ID
NO:77) and ZC27,899 (SEQ ID NO:19) as primers. The PCR
amplification was carried out as follows: 30 cycles of 94.degree.
C. for 1 minute, 65.degree. C. for 1 minute, and 72.degree. C. for
1 minute; followed by 1 cycle at 72.degree. C. for 7 minutes. The
PCR product was visualized by agarose gel electrophoresis and the
approximately 500 bp PCR product was gel purified as described
herein. The probe was radioactively labeled using the PRIME IT
II.TM. Random Primer Labeling Kit (Stratagene) according to the
manufacturer's instructions. The probe was purified using a
NUCTRAP.TM. push column (Stratagene). EXPRESSHYB.TM. (Clontech)
solution was used for the prehybridization and as a hybridizing
solution for the Northern blots. Prehybridization was carried out
at 68.degree. C. for 2 hours. Hybridization took place overnight at
68.degree. C. with about 1.5.times.10.sup.6 cpm/ml of labeled
probe. The blots were washed three times at room temperature in
2.times.SSC, 0.05% SDS, followed by 1 wash for 10 minutes in
2.times.SSC, 0.1% SDS at 50.degree. C. Several faint bands were
seen after several days exposure. An approximately 9 kb transcript
was seen in trachea, skeletal muscle and thymus; an approximately 2
kb transcript was seen in PBL, HPV, U937 and THP-1 cells; and
[0479] about a 1.2 kb transcript was seen in placenta, bone marrow
and thyroid, and HPV and U937 cells. In all the tissues listed
above, the signal intensity was faint. There appeared to be little
expression in most normal tissues, suggesting that zcytor17
expression may be dependent on activation of the cell or tissues in
which it is expressed.
B. Tissue Distribution in Tissue Panels Using PCR
[0480] A panel of cDNAs from human tissues was screened for
zcytor17 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 as shown below in Table 13.
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:78) and ZC21196 (SEQ ID NO:79) 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:48) and 3' alpha tubulin specific oligo primer
ZC17,574 (SEQ ID NO:49) or 3' G3PDH specific oligo primer ZC17,600
(SEQ ID NO:50); 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. The PCR
reactions were set up using oligos ZC26,358 (SEQ ID NO:80) and
ZC26,359 (SEQ ID NO:81), TaKaRa Ex Taq.TM. (TAKARA Shuzo Co LTD,
Biomedicals Group, Japan), 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 30 seconds, 66.3.degree. C. for 30 seconds and
72.degree. C. for 30 seconds, followed by 1 cycle at 72.degree. C.
for 5 minutes. About 10 .mu.l of the PCR reaction product was
subjected to standard agarose gel electrophoresis using a 4%
agarose gel. The correct predicted DNA fragment size was observed
in lymph node, prostate, thyroid, HPV (prostate epithelia), HPVS
(prostate epithelia, selected), lung tumor, uterus tumor reactions,
along with the genomic DNA reaction.
[0481] The DNA fragment for prostate tissue (2 samples), HPV
(prostate epithelia), HPVS (prostate epithelia, selected), and
genomic were excised and purified using a Gel Extraction Kit
(Qiagen, Chatsworth, Calif.) according to manufacturer's
instructions. Fragments were confirmed by sequencing to show that
they were indeed zcytor17. TABLE-US-00013 TABLE 13 Tissue/Cell line
#samples Tissue/Cell line #samples Adrenal gland 1 Bone marrow 3
Bladder 1 Fetal brain 3 Bone Marrow 1 Islet 2 Brain 1 Prostate 3
Cervix 1 RPMI #1788 (ATCC # CCL-156) 2 Colon 1 Testis 4 Fetal brain
1 Thyroid 2 Fetal heart 1 WI38 (ATCC # CCL-75 2 Fetal kidney 1 ARIP
(ATCC # CRL-1674 - rat) 1 Fetal liver 1 HaCat - human keratinocytes
1 Fetal lung 1 HPV (ATCC # CRL-2221) 1 Fetal muscle 1 Adrenal gland
1 Fetal skin 1 Prostate SM 2 Heart 2 CD3+ selected PBMC's Ionomycin
+ PMA 1 stimulated K562 (ATCC # CCL-243) 1 HPVS (ATCC # CRL-2221) -
1 selected Kidney 1 Heart 1 Liver 1 Pituitary 1 Lung 1 Placenta 2
Lymph node 1 Salivary gland 1 Melanoma 1 HL60 (ATCC # CCL-240) 3
Pancreas 1 Platelet 1 Pituitary 1 HBL-100 1 Placenta 1 Renal
mesangial 1 Prostate 1 T-cell 1 Rectum 1 Neutrophil 1 Salivary
Gland 1 MPC 1 Skeletal muscle 1 Hut-102 (ATCC # TIB-162) 1 Small
intestine 1 Endothelial 1 Spinal cord 1 HepG2 (ATCC # HB-8065) 1
Spleen 1 Fibroblast 1 Stomach 1 E. Histo 1 Testis 2 Thymus 1
Thyroid 1 Trachea 1 Uterus 1 Esophagus tumor 1 Gastric tumor 1
Kidney tumor 1 Liver tumor 1 Lung tumor 1 Ovarian tumor 1 Rectal
tumor 1 Uterus tumor 1
C. Expression Analysis of zcytorR17 by PCR and Northern
[0482] Annotation of the cell types and growth conditions that
affect expression of the receptor is a useful means of elucidating
its function and predicting a source of ligand. To that end a wide
variety of tissue and cell types were surveyed by PCR. The
thermostable polymerase Advantage II.TM. (Clontech, La Jolla,
Calif.) was used with the oligonucleotide primers ZC29,180 (SEQ ID
NO:22) and ZC29,179 (SEQ ID NO:82) and 1-10 ng of the various cDNA
templates listed below for 30 amplification cycles of (94.degree.
C., 30 sec.; 66.degree. C., 20 sec.; 68.degree. C., 1 min. 30
sec.). Following this, 20% of each reaction was run out on 0.8%
agarose, TAE/ethidium bromide gels and visualized with UV light.
Samples were then scored on the basis of band intensity. See Table
14 below. TABLE-US-00014 TABLE 14 Cells and Conditions Score 0-5
Hel stimulated with PMA 0 U937 3 MCF-7 0 HuH7 1 Human follicle 0
HT-29 0 HEPG2 0 HepG2 stimulated with IL6 0 Human dermal
endothelial 0 Human venous endothelial 0 Human CD4+ 0 BEWO 0 Human
CD19+ 1 Human PBMC stimulated with PHA, PMA, Ionomycin, IL2, 0 IL4,
TNF.alpha. 24 hours Human PBMC stimulated with LPS, PWM,
IFN.gamma., 0 TNF.alpha., 24 hours Human PBMC all of the above
conditions for 48 hours 4 HUVEC p.2 4 RPMI1788 0 TF1 0 Monkey
spleen T cells stimulated with PMA, Ionomycin 0 Human prostate
epithelia HPV transformed 5 Human tonsils, inflamed 0 HACAT 0 Human
chondrocyte 1 Human synoviacyte 1 THP1 5 REH 0
[0483] Of the strong positive PCR signals, two were from the human
monocyte cell lines U937 and THP1.
[0484] These two cell lines along with a prostate epithelia line
were selected for further analysis by Northern blot. Previous
attempts at visualizing a transcript by northern analysis using
mRNA from various tissues yielded weak and diffuse signals in the
surprisingly large size range of 7-10 kb making this data difficult
to interpret. A denaturing formaldehyde/MOPS/0.8% agarose gel was
prepared (RNA Methodologies, Farrell, RE Academic Press) and 2
.mu.g of polyA+ mRNA was run for each sample along side an RNA
ladder (Life Technologies, Bethesda, Md.). The gel was then
transferred to Hybond nylon (Amersham, Buckinghamshire, UK), UV
crosslinked, and hybridized in ExpressHyb solution (Clontech,
LaJolla, Calif.) at 68.degree. C. overnight using a probe to human
zcytoR17 generated by PCR with the oligos ZC28,575 (SEQ ID NO:77),
and ZC27,899 (SEQ ID NO:19) and labeled with a Megaprime .sup.32P
kit (Amersham). The northern blot was subsequently washed with
0.2.times.SSC+01% SDS at 65 C for 15 minutes and exposed to film
for 7 days with intensifying screens. A prominent 8 kb band was
seen in both the prostate epithelia and U937 lanes while a fainter
band was present in the THP1 lane.
[0485] To optimize the cDNA used as a hybridization probe, four
different regions of the full-length human zcytoR17 sequence were
amplified by PCR, labeled and hybridized as described above to
southern blots containing genomic and amplified cDNA library DNA.
The four probes, herein designated probes A-D, were amplified using
the following primer pairs: (A) ZC28,575 (SEQ ID NO:77), ZC27,899
(SEQ ID NO:19); (B) ZC27,895 (SEQ ID NO:20), ZC28,917 (SEQ ID
NO:83); (C) ZC28,916 (SEQ ID NO:84), ZC28,918 (SEQ ID NO:85); and
(D) ZC28,916 (SEQ ID NO:84), ZC29,122 (SEQ ID NO:21). Human genomic
DNA along with amplified cDNA libraries demonstrated to contain
zcytor17 by PCR were digested with EcoR1 and Xho1 to liberate
inserts and run out on duplicate TAE/0.8% agarose gels, denatured
with 0.5M NaOH, 1.5 M NaCl, blotted to Hybond, UV crosslinked and
each hybridized with a distinct probe. Probe B was found to have
the least nonspecific binding and strongest signal. Thus, Probe B
was used for all subsequent hybridizations.
[0486] Given that the THP1 cells are an excellent model of
circulating monocytes and expressed zcytor17 at low levels we
treated them with a variety of compounds in an effort to increase
expression of zcytoR17. The cells were grown to a density of
2e5/ml, washed and resuspended in various stimulating media, grown
for four or thirty hours, and harvested for RNA preparations. Each
media was supplemented with one of the following drugs or pairs of
cytokines: LPS 2 ug/ml (Sigma Chemicals, St. Louis, Mo.),
hTNF.alpha. 2 ng/ml (R&D Systems, Minneapolis, Minn.), hGM-CSF
2 ng/ml (R&D Systems, Minneapolis, Minn.), hTFN.gamma. 50 ng/ml
(R&D Systems, Minneapolis, Minn.), hMCSF 1 ng/ml (R&D
Systems, Minneapolis, Minn.), hIL6 1 ng/ml (R&D Systems,
Minneapolis, Minn.), hIL1.beta. 2 ng/ml (R&D Systems,
Minneapolis, Minn.), hIFN.gamma. 50 ng/ml+hIL4 0.5 ng/ml (R&D
Systems, Minneapolis, Minn.), hIFN.gamma. 50 ng/ml+hIL10 1 ng/ml
(R&D Systems, Minneapolis, Minn.), PMA 10 ng/ml (Calbiochem,
San Diego, Calif.) and an untreated control. At the end of the
culture period Total RNA was prepared using an RNAeasy Midi-kit
(Qiagen, Valencia, Calif.). Poly A+ RNA was selected from the total
RNA using an MPG kit (CPG, Lincoln Park, N.J.). Two micrograms of
polyA+ RNA from each condition was run on formaldehyde/MOPS/agarose
gels, transferred to nylon and UV crosslinked as described above.
These northern blots were then hybridized, as above, to probe B at
68.degree. C. overnight, washed at high stringency with
0.2.times.SSC, 0.1% SDS at 65 C, exposed to film overnight then
exposed to phosphor screens for signal quantitation. A dominant 8
kb mRNA as well a relatively weaker 2.8 kb band were seen in all
lanes. A 20-fold increase in zcytor17 mRNA was seen in RNA from
cells treated with hIFN.gamma. for 30 hours, this effect was
slightly muted with simultaneous treatment with IL4. Minor 3 fold
increases in mRNA were seen in RNA from cells treated with LPS,
TNF.alpha. and GM-CSF while MCSF, IL6, and IL1.beta. had no effect
on zcytor17 mRNA levels. Taken together this data suggests a role
for the zcytor17 receptor and its ligand in monocyte macrophage
biology and by extension any number of disease processes in which
these cells participate.
Example 32
Tissue Distribution of Human zcytor17 in Tissue Panels Using
Northern Blot and PCR
[0487] A human zcytor17lig cDNA fragment was obtained using PCR
with gene specific primers: Sense primer ZC41438 (SEQ ID NO:93) and
antisense primer ZC41437 (SEQ ID NO:94) and template human
zcytor17lig cDNA (SEQ ID NO:90) This fragment was purified using
standard methods and about 25 ng labeled with .sup.32P alpha dCTP
using the Prime-It RmT random primer labeling kit (Stratagene) and
hybridized in Ultrahyb, (Ambion) and used to expose Biomax
film/intensifying screens per the manufacturer's recommendations in
each case. New, previously unused blots Including the Clontech
Human 12 lane MTN, the human brain MTN II, and the human brain MTN
blot IV, the human immune system MTN II, and the human MTE array
II, from Clontech were hybridized overnight at 42.degree. C. per
the Ambion ultrahyb method. Non-specific radioactive counts were
washed off using 0.1SSC/0.5% SDS at 55.degree. C. The positive
blots included the human 12 lane MTN (Clontech). Of the 12 tissues
examined, only placenta was positive for an approximately 1.2 KB
transcript.
Example 33
Construction of Mammalian Expression Vectors that Express Human
zcytor17lig-CEE
A. Construction of zCytor17Lig-CEE/pZMP21
[0488] An expression plasmid containing all or part of a
polynucleotide encoding zCytor17Lig-CEE (SEQ ID NO:95) was
constructed via homologous recombination. The plasmid was called
zCytor17Lig-CEE/pZMP21.
[0489] The construction of zCytor17Lig-CEE/pZMP21 was accomplished
by generating a zCytor17Lig-CEE fragment using PCR amplification.
The DNA template used for the production of the zCytor17Lig-CEE
fragment was zCytor17Lig/pZP7nx. The primers used for the
production of the zCytor17Lig-CEE fragment were: (1) ZC41,607 (SEQ
ID NO:97) (sense sequence), which includes from the 5' to the 3'
end: 28 bp of the vector flanking sequence (5' of the insert) and
21 bp corresponding to the 5' sequence of zCytor17Lig; and (2)
ZC41,605 (SEQ ID NO:98) (anti-sense sequence), which includes from
the 5' to the 3' end: 37 bp of the vector flanking sequence (3' of
the insert), 3 bp of the stop codon, 21 bp encoding a C-terminal EE
tag, and 21 bp corresponding to the 3' end of zCytor17Lig sequence.
The fragment resulting from the above PCR amplification is a copy
of the template zCytor17Lig with the addition of a C-terminal EE
tag, yielding a final product zCytor17Lig-CEE.
[0490] PCR reactions were run as follows: To a 100 .mu.P final
volume was added: 10 .mu.l of 10.times.Taq Polymerase Reaction
Buffer with 15 mM MgCl (Gibco), 1 .mu.l of Taq DNA Polymerase (5
units/.mu.l, Gibco), 3 .mu.l of 10 mM dNTPs, 78 .mu.l dH.sub.2O, 3
.mu.l of a 20 pmol/.mu.l stock of primer ZC41,607 (SEQ ID NO:97) 3
.mu.l of a 20 pmol/.mu.l stock of primer ZC41,605 (SEQ ID NO:98),
and 2 .mu.l of a 0.13 .mu.g/.mu.l stock of zCytor17lig template
DNA. A volume equal to 50 .mu.l of mineral oil was added to the
mixture. The reaction was heated to 94.degree. C. for 5 minutes,
followed by 35 cycles at 94.degree. C. for 1 minute; 55.degree. C.
for 2 minutes; 72.degree. C. for 3 minutes; followed by a 10 minute
extension at 72.degree. C. and held at 4.degree. C. until the
reaction was collected.
[0491] The plasmid pZMP21 was restriction digested with BglII
enzyme, cleaned with a QiaQuick PCR Purification Kit (Qiagen) using
a microcentrifuge protocol, and used for recombination with the PCR
fragment. Plasmid pZMP21 was constructed from pZMP20 which was
constructed from pZP9 (deposited at the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
and is designated No. 98668) with the yeast genetic elements from
pRS316 (deposited at the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209, and designated No.
77145), an IRES element from poliovirus, and the extracellular
domain of CD8, truncated at the carboxyl terminal end of the
transmembrane domain. PZMP21 is a mammalian expression vector
containing an expression cassette having the MPSV promoter,
immunoglobulin signal peptide intron, multiple restriction sites
for insertion of coding sequences, a stop codon 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, enhancer and origin of replication, a DHFR gene,
the SV40 terminator, as well as the URA3 and CEN-ARS sequences
required for selection and replication in S. cerevisiae.
[0492] Fifty microliters of competent yeast cells (S. cerevisiae)
were independently combined with 100 ng of cut plasmid, 5 .mu.l of
previously described PCR mixture, and transferred to a 0.2 cm
electroporation cuvette. The yeast/DNA mixture was electropulsed at
0.75 kV (5 kV/cm), cc ohms, 25 .mu.F. Each cuvette had 600 .mu.l of
1.2 M sorbitol added, and the yeast was plated in one 100 .mu.l
aliquot and one 300 .mu.l aliquot onto two URA-D plates and
incubated at 30.degree. C. After about 72 hours, the Ura+ yeast
transformants from a single plate were resuspended in 1 ml H.sub.2O
and spun briefly to pellet the yeast cells. The cell pellet was
resuspended in 500 .mu.l of lysis buffer (2% Triton X-100, 1% SDS,
100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). The 500 .mu.l of the
lysis mixture was added to an Eppendorf tube containing 300 .mu.l
acid washed 600 .mu.m glass beads and 300 .mu.l phenol-chloroform,
vortexed for 1 minute intervals two or three times, followed by a 5
minute spin in a Eppendorf centrifuge at maximum speed. Three
hundred microliters of the aqueous phase was transferred to a fresh
tube, and the DNA precipitated with 600 .mu.l 100% ethanol (EtOH),
followed by centrifugation for 10 minutes at 4.degree. C. The DNA
pellet was then washed with 500 .mu.l 70% EtOH, followed by
centrifugation for 1 minute at 4.degree. C. The DNA pellet was
resuspended in 30 .mu.l H.sub.2O.
[0493] Transformation of electrocompetent E. coli cells (MC1061)
was done with 5 .mu.l of the yeast DNA prep and 50 .mu.l of MC1061
cells. The cells were electropulsed at 2.0 kV, 25 .mu.F and 400
ohms(.OMEGA.). Following electroporation, 600 .mu.l SOC (2% Bacto'
Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM
glucose) was added. The electroporated E. coli cells were plated in
a 200 .mu.l and a 50 .mu.l aliquot on two LB AMP plates (LB broth
(Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin). The plates
were incubated upside down for about 24 hours at 37.degree. C.
Three Ampicillin-resistant colonies were selected at random and
submitted for sequence analysis of the insert. Large-scale plasmid
DNA was isolated from a sequence-confirmed clone using the Qiagen
Maxi kit (Qiagen) according to manufacturer's instructions.
B. Construction of Mouse zCytor17Lig(m)-CEE/pZMP21
[0494] An expression plasmid containing the entire polynucleotide
encoding murine zCytor17Lig-CEE (SEQ ID NO:104 and SEQ ID NO:105)
was also constructed via homologous recombination using the method
described in Example 33A above. The primers used were: (1) ZC41643
(SEQ ID NO:106) (forward, 5' to 3' sense) having a 28 bp vector
overlap 5' of the insertion point; 21 bp of the 5' end of
zcytor17lig(m) and (2) ZC41641 (SEQ ID NO:107) (reverse, 5' to 3'
anti-sense) having a 37 bp vector overlap 3' of the insertion
point; 3 bp stop codon; 21 bp C-terminal EE tag; 24 bp of the 3'
end of zCytor17Lig(m)-CEE. The plasmid was called
zcytor17lig(m)-CEE/pZMP21. The polynucleotide sequence of
zcytor17lig(m)-CEE is shown in SEQ ID NO:104, and corresponding
polypeptide sequence is shown in SEQ ID NO:105.
Example 34
Transfection and Expression of zcytor17lig-CEE Polypeptides
A. Expression of Human zCytor17Lig-CEE/pZMP2] in 293T Cells
[0495] Zcytor17Lig-CEE was expressed transiently in 293T cells
(Stanford University School of Medicine, Stanford, Calif.; ATCC No.
SD-3515) to generate initial purified protein. The day before the
transfection, 293T cells were seeded at 6.5.times.10.sup.4
cells/cm.sup.2 in 30 T162 culture flasks with a total volume of 30
ml of culture media (SL7V4+5% FBS+1% Pen/Strep) per flask. The
cells were allowed to incubate for 24 hours at 37.degree. C.
[0496] A DNA/Liposome mixture was prepared as follows: Two 50 ml
conical tubes were filled with 25 mLs of transfection media
(SL7V4+1% Pen/Strep) and 1.13 mg of zCytor17Lig-CEE/pZMP21 (Example
33) was added to each. A separate set of two 50 ml conical tubes
were filled with 22 ml of transfection media (above) and 3 ml of
liposomes (Lipofectamine, Gibco) was added to each. For each set of
tubes, one tube of DNA was added to one tube of liposomes and the
DNA/liposome mix was incubated for 30 minutes. The two 50 ml
conical tubes containing the DNA/liposome mixtures were pooled
(about 100 ml) and 300 ml of transfection media was added.
[0497] The 30 flasks of the 293T cells were decanted, washed
1.times. with about 15 ml of PBS, and 12.5 ml of the diluted
DNA/liposome mixture was added to each flask. The flasks were
incubated for 3 hours at 37.degree. C. After the incubation period,
25 ml of culture media (above) were added to each T162 flask. The
transfection media was harvested after approximately 96 hours and
was used for protein purification (Example 35).
B. Expression of Mouse zCytor17Lig-CEE(m)/pZMP2 in 293T Cells
[0498] Mouse zCytor17Lig(m)-CEE was expressed transiently in 293T
cells as described in Example 34A and cultured media was used for
protein purification (Example 35).
Example 35
Purification of Zcytor17lig-CEE from 293T Cells
[0499] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying both
mouse and human Zcytor17lig containing C-terminal Glu-Glu (EE) tags
(SEQ ID NO:103). Conditioned media from 293T cells expressing
Zcytor17lig-CEE (Example 34) was purified. Total target protein
concentrations of the conditioned media were determined via
SDS-PAGE and Western blot analysis with the anti-EE antibody.
[0500] A 5.5 ml column of anti-EE Poros 50 A (PE BioSystems,
Framingham, Mass.) (prepared as described below) was poured in a
Waters AP-1, 1 cm.times.7 cm glass column (Waters, Milford, Mass.).
The column was flow packed and equilibrated on a BioCad Sprint (PE
BioSystems, Framingham, Mass.) with phosphate buffered saline (PBS)
pH 7.4. The conditioned media was adjusted with NaCl to 0.3 M and
the pH adjusted to 7.2. The conditioned media was then loaded on
the column overnight with about 3 ml/minute flow rate. The column
was washed with 10 column volumes (CVs) of PBS pH 7.4, and again
washed with 3CVs 5.times. Sigma PBS pH 7.4. It was step eluted with
0.5 M Acetate, 0.5 M NaCl, pH 2.5 at 3 ml/minute. The fraction
tubes contained 1 ml Tris base (no pH adjustment) to neutralize the
elution immediately. The column was again washed for 2CVs with
5.times. Sigma PBS, pH 7.4 to neutralize the column and then
equilibrated in PBS (pH 7.4). Two 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 5.times.PBS and the acid elution peak fractions were
analyzed for the target protein via SDS-PAGE Silver staining and
Western Blotting with the primary antibody anti-EE and secondary
antibody, anti mouse-HRP conjugated. The acid elution fractions of
interest were pooled and concentrated from 38 ml to 0.8 ml using a
5000 Dalton molecular weight cutoff membrane spin concentrator
(Millipore, Bedford, Mass.) according to the manufacturer's
instructions.
[0501] To separate Zcytor17lig-CEE from aggregated material and any
other contaminating co-purifying proteins, the pooled concentrated
fractions were subjected to size exclusion chromatography on a
1.6.times.60 cm (120 ml) Superdex 75 (Pharmacia, Piscataway, N.J.)
column equilibrated and loaded in PBS at a flow rate of 1.0 ml/min
using a BioCad Sprint. Three milliliter 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 Zcytor17lig-CEE
protein.
[0502] On Western blotted, Coomassie Blue and Silver stained
SDS-PAGE gels, the Zcytor17lig-CEE 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.
[0503] To prepare PorosA50 anti-EE, a 65 ml bed volume of Poros A50
(PE Biosystems) was washed with 100 ml of water and then 0.1 M
triethanolamine, pH 8.2 (TEA, ICN, Aurora, Ohio), 1 M
Na.sub.2S0.sub.4, pH 8.8 containing 0.02% sodium azide using a
vacuum flask filter unit. The EE monoclonal antibody solution, at a
concentration of 2 mg/ml in a volume of 300 ml, was mixed with the
washed resin in a volume of 250 ml. After an overnight incubation
at room temperature, the unbound antibody was removed by washing
the resin with 5 volumes of 200 mM TEA, 1 M Na.sub.2S0.sub.4, pH
8.8 containing 0.02% sodium azide as described above. The resin was
resuspended in 2 volumes of TEA, 1 M Na.sub.2S0.sub.4, pH 8.8
containing 0.02% sodium azide and transferred to a suitable
container. Three ml of 25 mg/ml (68 mM) Disuccinimidyl suberate (in
DMSO supplied by Pierce, Rockford, Ill.) is added and the solution
is incubated for three hours at room temperature. Nonspecific sites
on the resin were then blocked by incubating for 10 min at room
temperature with 5 volumes of 20 mM ethanolamine (Sigma, St. Louis,
Mo.) in 200 mM TEA, pH 8.8 using the vacuum flask filter unit. The
resin is washed with PBS, pH 7.4, followed by 0.1 M Glycine, pH 3
and then neutralized with 10.times.PBS. After washing with
distilled water, the final coupled anti-EE Poros-A 50 resin was
stored at 4.degree. C. in 20% Ethanol.
Example 36
N-Terminal Sequencing of Human and Mouse Zcytor17lig
A. N-Terminal Sequencing of Human Zcytor17lig
[0504] Standard automated N-terminal polypeptide sequencing (Edman
degradation) was performed using reagents from Applied Biosystems.
N-terminal sequence analysis was performed on a Model 494 Protein
Sequencer System (Applied Biosystems, Inc., Foster City, Calif.).
Data analysis was performed with Model 610A Data Analysis System
for Protein Sequencing, version 2.1a (Applied Biosystems).
[0505] A purified human zcytor17lig-CEE sample (Example 35) was
supplied. The sample was loaded onto a prepared glass fiber filter
for n-terminal sequencing. The glass fiber filter was prepared by
precycling it with Biobrene.TM..
[0506] N-terminal sequence analysis of the secreted human
zcytor17lig polypeptide did not verify the predicted cleavage site
of the signal sequence but resulted in a mature start at residue
27(Leu) in SEQ ID NO:2 of the human zcytor17lig precursor
sequence.
B. N-Terminal Sequencing of Human Zcytor17lig
[0507] Standard automated N-terminal polypeptide sequencing (Edman
degradation) was performed using reagents from Applied Biosystems.
N-terminal sequence analysis was performed on a Model 494 Protein
Sequencer System (Applied Biosystems, Inc., Foster City, Calif.).
Data analysis was performed with Model 610A Data Analysis System
for Protein Sequencing, version 2.1a (Applied Biosystems).
[0508] A purified mouse zcytor17lig-CEE sample was supplied as
captured on Protein G Sepharose/anti-EE beads (Example 35). The
beads were placed in reducing SDS PAGE sample buffer and on a
boiling water bath before running on SDS PAGE, using a Novex SDS
PAGE system (4-12% Bis-Tris MES NuPAGE; Invitrogen) as per
manufacturer's instructions. The gel was electrotransferred to a
Novex PVDF membrane (Invitrogen), and Coomassie blue stained
(Sigma, St. Louis, Mo.) using standard methods. Corresponding
anti-EE Western blots were performed to identify the zcytor17lig
band for N-terminal protein sequencing. The mouse anti-EE IgG HRP
conjugated antibody used was produced in house.
[0509] N-terminal sequence analysis of the secreted mouse
zcytor17lig polypeptide verified the predicted cleavage site of the
signal sequence resulting in a mature start at 31 (Ala) in
reference to SEQ ID NO:11 and SEQ ID NO:91 of the mouse zcytor17lig
precursor sequence.
Example 37
Cos Cell Binding Assay
[0510] A binding assay was used to test the binding of the
zcytor17lig to receptors comprising zcytor17 receptor, such as the
zcytor17 receptor or receptor heterodimers and trimers comprising
zcytor17 receptor (e.g., zcytor17/OSMR, zcytor17/WSX-1, or
zcytor17/OSMR/WSX-1, or other Class I cytokine receptor subunits).
Zcytor17 receptor plasmid DNA was transfected into COS cells and
transfected COS cells were used to assess binding of the
zcytor17lig to receptors comprising zcytor17 receptor as described
below.
A. COS Cell Transfections
[0511] The COS cell transfection was performed as follows: Mix 800
ng receptor plasmid DNA in the following combinations:
pZp7pX/zcytor17 alone; pZp7Z/WSX-1 alone; pZp7NX/OSMR alone;
pZp7pX/zcytor17+pZp7NX/OSMR; pZp7pX/zcytor17+pZp7Z/WSX-1;
pZp7NX/OSMR+pZp7Z/WSX-1; pZp7pX/zcytor17+pZp7NX/OSMR+pZp7Z/WSX-1)
and 4 ul Lipofectamine.TM. in 80 .mu.l 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), incubate
at room temperature for 30 minutes and then add 320 .mu.l serum
free DMEM media. Add this 400 ul mixture onto 2.times.10.sup.5 COS
cells/well plated on 12-well tissue culture plate
(fibronectin-coated) and incubate for 5 hours at 37.degree. C. Add
500 ul 20% FBS DMEM media (100 ml FBS, 55 mg sodium pyruvate and
146 mg L-glutamine in 500 ml DMEM) and incubate overnight.
B. Binding Assay
[0512] The binding assay was performed as follows: media was rinsed
off cells with PBS+0.1% BSA, and then cells were blocked for 60
minutes with the same solution. The cells were then incubated for 1
hour in PBS+0.1% BSA with 1.0 .mu.g/ml zcytor17ligCEE purified
protein. Cells were then washed with PBS+0.1% BSA and incubated for
another hour with 1:1000 diluted mouse anti-GluGlu antibody. Again
cells were washed with PBS+0.1% BSA, then incubated for 1 hour with
1:200 diluted goat anti-mouse-HRP conjugated antibody.
[0513] Positive binding was detected with fluorescein tyramide
reagent diluted 1:50 in dilution buffer (EN kit) and incubated for
4-6 minutes, and washed with PBS+0.1% BSA. Cells were fixed for 15
minutes with 1.8% Formaldehyde in PBS, then washed with PBS+0.1%
BSA. Cells were preserved with Vectashield Mounting Media (Vector
Labs Burlingame, Calif.) diluted 1:5 in PBS. Cells were visualized
using a FITC filter on fluorescent microscope.
[0514] Positive binding was detected for cells transfected with
zcytor17 only, zcytor17+OSMRbeta, zcytor17+WSX-1, and
zcytor17+OSMRbeta+WSX-1. No binding was detected for cells
transfected with WSX-1+OSMRbeta, with OSMRbeta only, or with WSX-1
only.
Example 38
Mouse zcytor17lig Activates Mouse zcytor17/OSMRbeta Receptor-In
Luciferase Assay
A. Cloning of Full-Length Mouse zcytor17 and Mouse OSMRbeta for
Expression
[0515] A mouse testes cDNA library was screened for a full-length
clone of mouse zcytoR17. The library was plated at 65,500 cfu/plate
on 24 LB+Amp plates. Filter lifts were prepared using Hybond N
(Amersham-Pharmacia Biotech, Inc., Piscataway, N.J.) on a total of
approximately 1.6 million colonies. The filters were marked with a
hot needle for orientation and then denatured for 6 minutes in 0.5
M NaOH and 1.5 M Tris-HCl, pH 7.2. The filters were then
neutralized in 1.5 M NaCl and 0.5 M Tris-HCl, pH 7.2 for 6 minutes.
The DNA was affixed to the filters using a UV crosslinker
(Stratalinker.RTM., Stratagene, La Jolla, Calif.) at 1200 joules.
The filters were then left to dry overnight at room
temperature.
[0516] The next day, the filters were pre-washed at 65.degree. C.
in pre-wash buffer consisting of 0.25.times.SSC, 0.25% SDS and 1 mM
EDTA. Cell debris was manually removed using Kimwipes.RTM.
(Kimberly-Clark) and the solution was changed 3 times over a period
of 1 hour. Filters were air dried and stored at room temperature
until needed. The filters were then prehybridized for approximately
3 hours at 63.degree. C. in 20 ml of ExpressHyb.TM. Hybridization
Solution (Clontech, Palo Alto, Calif.).
[0517] Probe B (Example 31) was generated by PCR from human
zcytor17 template using oligonucleotide primers ZC27,895 (SEQ ID
NO:20) and ZC28,917 (SEQ ID NO:83) and was radioactively labeled
with .sup.32P using a commercially available kit (Megaprime DNA
Labeling System; Amersham Pharmacia Biotech, Piscataway, N.J.)
according to the manufacturer's instructions. The probe was
purified using a Stratagene.TM. push column (NucTrap.RTM. column;
Stratagene, La Jolla, Calif.). The probe was denatured at
100.degree. C. for 15 min and added to ExpressHyb.TM.. Filters were
hybridized in 15 ml hybridizing solution containing
1.6.times.10.sup.6 cpm/ml of probe at 63.degree. C. overnight.
Filters were washed at 55.degree. C. in 2.times.SSC, 0.1% SDS and 1
mM EDTA and exposed to X-ray film at -80.degree. C. for 41/2 days.
Thirteen positives were picked from the plates as plugs and placed
in 1 ml LB+amp in 1.7 ml tubes. Tubes were placed at 4.degree. C.
overnight. These 13 positives were subjected to two further rounds
of purification. The tertiary plates were outgrown at 37.degree. C.
after filter lifts were taken and single colonies were picked and
sent to sequencing. Three of these were determined to contain
sequence of the mouse ortholog of zcytoR17.
[0518] In addition, a PCR product was generated using CTLL-2 cDNA
as a template and oligonucleotides ZC38,239 (SEQ ID NO:123) and
ZC38,245 (SEQ ID NO:124) as primers. CTLL-2 is a mouse cytotoxic T
lymphocyte cell line (ATCC No. TIB-214). This PCR reaction was run
as follows: 1 cycle at 95.degree. C. for 1 minute, 30 cycles at
95.degree. C. for 15 seconds, 68.degree. C. for 3 minutes, then
68.degree. C. for 10 minutes; 4.degree. C. soak. The PCR reaction
used approximately 0.5 ng of cDNA, 20 pmoles of each
oligonucleotide, and 1 .mu.l of Advantage II polymerase mix
(ClonTech). About 6% of the PCR product was used as a template in a
new PCR reaction, as above, except with oligonucleotides ZC38,239
(SEQ ID NO:123) and ZC38,238 (SEQ ID NO:125). This PCR reaction was
run as follows: 30 cycles at 94.degree. C. for 45 seconds,
65.degree. C. for 45 seconds, 72.degree. C. for 1 minute, then
72.degree. C. for 7 minutes; 10.degree. C. soak. Most of the PCR
reaction was loaded on a 1.0% agarose gel and the predominant band
at approximately 360 bp was excised, the DNA fragment was eluted,
and DNA sequencing was performed.
[0519] The sequence of the mouse zcytor17 polynucleotide is shown
in SEQ ID NO:126 and the corresponding amino acid sequence shown in
SEQ ID NO:127. In addition, a truncated soluble form of the mouse
zcytor17 polynucleotide is shown in SEQ ID NO:128 and the
corresponding amino acid sequence shown in SEQ ID NO:129.
[0520] To obtain a full-length mouse OSMRbeta cDNA, 5' and 3' PCR
products were isolated and joined using an internal BamHI site. The
PCR primers were designed using the nucleotide sequence SEQ ID
NO:134 and include EcoRI and XbaI restriction sites for cloning
purposes. The genomic mouse OSMRbeta nucleic acid sequence is shown
in SEQ ID NO:134, wherein the coding sequence encompasses residues
780 to 3692 encoding a mouse OSMRbeta 970 amino acid polypeptide,
which is shown in SEQ ID NO:135. A degenerate nucleic acid sequence
which encodes the polypeptide of SEQ ID NO:135 is shown in SEQ ID
NO:136.
[0521] A 5' PCR product was generated using an in-house 3T3-L1
(differentiated mouse adipocyte) cDNA library as a template and
oligonucleotides ZC41,764 (SEQ ID NO:130) and ZC41,598 (SEQ ID
NO:131) as primers. This 5' PCR reaction was run as follows: 30
cycles at 95.degree. C. for 45 seconds, 55.degree. C. for 45
seconds, 72.degree. C. for 1 minute 30 seconds, then 72.degree. C.
for 7 minutes; 4.degree. C. soak. The PCR reaction used
approximately 3 .mu.g of plasmid prepared from the cDNA library, 20
pmoles of each oligonucleotide, and five units of Pwo DNA
polymerase (Roche). About 90% of the 5' PCR product was digested
with EcoRI and BamHI and gel purified on a 1.0% agarose gel. The
approximately 1446 bp band was excised and used for ligation (see
below).
[0522] A 3' PCR product was generated using a mouse placenta
in-house cDNA library as a template and oligonucleotides ZC41,948
(SEQ ID NO:132) and ZC41,766 (SEQ ID NO:133) as primers. This 3'
PCR reaction was run as follows: 30 cycles at 95.degree. C. for 45
seconds, 55.degree. C. for 45 seconds, 72.degree. C. for 1 minute
30 seconds, then 72.degree. C. for 7 minutes; 4.degree. C. soak.
The PCR reaction used approximately 3 .mu.g of plasmid prepared
from the cDNA library, 20 pmoles of each oligonucleotide, and five
units of Pwo DNA polymerase (Roche). About 90% of the 3' PCR
product was digested with BamHI and XbaI and gel purified on a 1.0%
agarose gel. The approximately 2200 bp band was excised and used
for ligation along with the 5' PCR product (described above) to the
expression vector pZP-5Z digested with EcoRI and XbaI. The
three-part ligation was performed with the 5' EcoRI to BamHI
fragment above, the 3' BamHI to XbaI fragment, and the expression
vector pZP-5Z digested with EcoRI and XbaI. This generated a pZP-5Z
plasmid containing a full-length cDNA for mouse OSMRbeta
(nucleotides 780 to 3692 of SEQ ID NO:134), designated
pZP-5Z/OSMRbeta. The full length mouse OSMRbeta cDNA in
pZP5Z/OSMRbeta has two amino acid insertions from SEQ ID NO:135.
There is a duplication of amino acid Glycine at position 370 and a
duplication of amino acid Glutamic Acid at position 526. Plasmid
pZP-5Z is a mammalian expression vector containing an expression
cassette having the CMV promoter, multiple restriction sites for
insertion of coding sequences, 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, enhancer and origin of replication, a zeocin resistance
gene and the SV40 terminator.
[0523] The resulting transformants were sequenced to confirm the
mouse OSMRbeta cDNA sequence.
B. Construction of BaF3/KZ134/zcytor17m,
BaF3/KZ134/zcytor17m/OSMRbetam, BHK/KZ734/zcytor117m, and
BHK/KZ734/zcytor17 m/OSMRbetam Cell Lines
[0524] Stable BaF3/KZ134 and BHK/KZ134 cell lines (Example 20) were
transfected with an expression plasmid encoding full-length mouse
zcytor17, pZP-7P/zcytor17m (Example 38A), to create
BaF3/KZ134/zcytor17m and BHK/KZ134/zcytor17m cells, respectively.
The mouse OSMRbeta expression plasmid, pZP-5Z/OSMRbetam (Example
38A), was then transfected into these cells to create
BaF3/KZ134/zcytor17 m/OSMRbetam and BHK/KZ134/zcytor17 m/OSMRbetam
cell lines, respectively. Methods were as described in Example 4
with the exception that Baf3/KZ134/zcytor17m and
BHK/KZ134/zcytor17m were selected with, in addition to Geneticin, 2
ug/ml puromycin while Baf3/KZ134/zcytor17m/OSMRbetam and
BHK/KZ134/zcytor17m/OSMRbetam were selected with, in addition to
Geneticin, 2 ug/ml puromycin and 200 ug/ml zeocin.
[0525] Clones were diluted, plated and selected using standard
techniques. Clones were screened by luciferase assay (see Example
20, above) using the mouse zcytor17lig conditioned media or
purified mouse zcytor17lig protein (Example 35) as an inducer.
Clones with the highest luciferase response (via STAT luciferase)
and the lowest background were selected. Stable transfectant cell
lines were selected.
[0526] C. Mouse Zcytor17lig Activates Mouse zcytor17 Receptor in
BaF3/KZ134/zcytor17m/OSMRbetam or BHK/KZ134/zcytor17m/OSMRbetam
Luciferase Assay
[0527] Cell lines were plated for luciferase assays as described in
Example 20 above. STAT activation of the BaF3/KZ134/Zcytor17m,
BaF3/KZ134/zcytor17 m/OSMRbetam, BHK/KZ134/zcytor17m, or
BHK/KZ134/zcytor17m/OSMRbetam cells was assessed using (1)
conditioned media from BHK570 cells transfected with the human
zcytor17lig (Example 7), (2) conditioned media from BHK570 cells
transfected with the mouse zcytor17lig (Example 18), (3) purified
mouse and human zcytor17lig (Example 35), and (4) mIL-3 free media
to measure media-only control response. Luciferase assays were
performed as described in Example 20.
[0528] The results of this assay confirm the STAT reporter response
of the BaF3/KZ134/zcytor17 m/OSMRbetam and BHK/KZ134/zcytor17
m/OSMRbetam cells to the mouse zcytor17lig when compared to either
the BaF3/KZ134/zcytor17m cells, the BHK/KZ134/zcytor17m cells or
the untransfected BaF3/KZ134 or BHK/KZ134 control cells, and show
that the response is mediated through the mouse zcytor17/OSMRbeta
receptors. The results also show that the human zcytor17lig does
not activate the STAT reporter assay through the mouse receptor
complex.
Example 39
Human zcytor17 Ligand Binding to zcytor17 and zcytor17/OSMRbeta by
Flow Cytometry
[0529] The biotinylation of human zcytor17L was done as follows:
100 .mu.L of zcytor17 at 5.26 mg/mL was combined with 30 .mu.L of
10 mg/mL EZ-link Sulfo-NHS-LC-biotin (Pierce, Rockford, Ill.)
dissolved in ddH.sub.2O. This solution was incubated on a rocker
for 30 minutes at room temperature. After biotinylation the
solution was dialyzed in PBS using a Slide-A-Lyzer dialysis
cassette.
[0530] To test the binding properties of human zcytor17 ligand to
different receptor combinations both BHK and BAF3 cells were
transfected with expression plasmids using standard techniques
well-known in the art. These plasmids were transfected into both
cell lines in the following combinations: zcytor17 alone, OSMRbeta
alone, and both zcytor17 and OSMRbeta. Transfection was performed
as detailed above. Untransfected BHK and BAF3 cells were used as
controls. Cells were stained by FACS as follows: 2E5 cells were
stained with either: 2.0 .mu.g/mL, 100 ng/mL, 10 ng/mL, 1.0 ng/mL,
100 pg/mL, 10 pg/mL, 1.0 pg/mL of biotinylated zcytor17L or left
unstained for 30 minutes on ice in FACS buffer (PBS+2% BSA+2% NHS
(Gemini)+2% NGS). Cells were washed 1.5 times and then stained with
SA-PE (Jackson Immuno Laboratories) at 1:250 for 30 minutes on ice.
Cells were then washed 1.5 times with FACS buffer and resuspended
in FACS buffer and analyzed by FACS on a BD FACSCaliber using
CellQuest software (Becton Dickinson, Mountain View, Calif.).
[0531] Both BHK and BAF3 cells showed that zcytor17 ligand bound to
both zcytor17 alone and in combination with OSMRbeta with the
binding to the zcytor17/OSMRbeta heterodimer being slightly
stronger. No binding was seen in either cell lines expressing
OSMRbeta alone. The zcytor17 ligand bound in a concentration
dependent manner. The mean fluorescent intensity (MFI) values for
the BHK binding are shown below in Table 15. TABLE-US-00015 TABLE
15 zcytor17 .mu.g/mL 2.0 0.100 0.010 0.001 0.0001 0.00001 0.000001
0.0 BHK C17 + OSMRbeta 3780 2126 328 53 17 15 14 13 BHK-C17 3032
1600 244 39 16 15 14 15 BHK-OSMRbeta 13 X X X X X X 0 BHK-WT 15 14
13 X X X X 13 zcytor17 .mu.g/mL 10.0 3.33 1.11 0.37 0.12 0.04 0.00
BAF3-C17 + OSMRbeta 531 508 489 441 364 247 7 BAF3-OSMRbeta 6 5 5 5
5 5 11 BAF3-WT 13 13 12 12 12 12 13 zcytor17 ng/mL 100.0 10.0 1.0
0.0 BAF3-C17 347 72 17 7
Example 40
Gene Expression Array Analysis of Human Zcytor17lig Treated
Cells
[0532] RNA was isolated from human zcytor17lig treated A549
cells,
[0533] zcytor17lig treated SK-LU-1 cells, and untreated control
cells using a RNeasy Midi Kit (Qiagen, Valencia, Calif.) according
to the manufactures instructions.
[0534] Gene expression profiling of the cells treated with
zcytor17lig and the
[0535] respective control cells was carried out using GEArray Q
series cDNA expression arrays (SuperArray Inc., Bethesda, Md.). The
Q Series cDNA expression arrays contain up to 96 cDNA fragments
associated with a specific biological pathway, or genes with
similar functions or structural features. Comparison of arrays from
treated and control cells allows for a determination of the up and
down regulation of specific genes. Probe labeling, hybridization
and detection were carried out according to the manufactures
instructions. Chemiluminscent signal detection and data acquisition
was carried out on a Lumi-Imager workstation (Roche, Indianapolis,
Ind.). The resulting image data was analyzed using ImageQuant 5.2
(Amersham Biosciences, Inc., Piscataway, N.J.) and GEArray Analyzer
1.2 (SuperArray Inc., Bethesda, Md.) software.
[0536] Analysis of the results from the Human Interleukin and
Receptor Q
[0537] series HS-014N arrays, showed, after normalization, an
approximate 4.7 fold increase of IL13RA2 signal in the zcytor17lig
treated human SK-LU-1 cells and an approximate 2.2 fold increase of
the IL13RA2 signal in the zcytor17lig treated human A549 cells.
[0538] These results indicate that zcytor17lig significantly up
regulated
[0539] IL13RA2 in the SK-LU-1 and A549 cells. Both of these are
established cell lines derived from human lung carcinomas (Blobel
et al., Virchows Arch B Cell Pathol Incl Mol Pathol, 1984;
45(4):407-29). More specifically, A549 is characterized as a human
pulmonary epithelial cell line (Lin, et al., J Pharm Pharmacol,
2002 September; 54(9):1271-8; Martinez et al., Toxicol Sci., 2002
October; 69(2):409-23).
[0540] Interleukin-13 (IL13), a cytokine secreted by activated T
lymphocytes, has been demonstrated to be both necessary and
sufficient for the expression of allergic asthma and for use in
experimental models of asthma, which include airway
hyperresponsiveness, eosinophil recruitment, and mucus
overproduction (Wills-Karp et al., Science, 1998;282:2258-2261). It
has been shown, that selective neutralization of IL13 will
ameliorate the asthma phenotype (Grunig et al., Science, 1998;
282:2261-2263). It has also been reported that IL13 is involved in
the up regulation of mucin gene MUC8 expression in human nasal
polyp epithelium and cultured nasal epithelium (Kimm et al., Acta
Otolaryngol., 2002; September; 122(6):638-643; Seong et al.,
Acta.sub.--Otolaryngol., 2002; June; 122(4):401-407). MUC8, a major
airway mucin glycoprotein, is implicated as playing a role in the
pathogenesis of mucus hypersecretion in chronic sinusitis with
polps (Seong et al., Acta Otolaryngol., 2002; June;
122(4):401-407).
[0541] Functionally, IL13 signals through a receptor complex
consisting of the interleukin-13 receptor alpha-1 chain (IL13RA1)
and IL-4 receptor alpha (IL4RA) (Daines and Hershey, J Biol. Chem.,
2002; 22(12):10387-10393). It has also been shown, that the
interleukin-13 receptor alpha-2 (IL13RA2) binds IL13 with high
affinity, but by itself (Daines and Hershey, J Biol. Chem., 2002;
22(12):10387-10393). This receptor lacks, however, the cytoplasmic
domain necessary for signaling and, therefore, is considered to be
a decoy receptor. It has been shown that IL13RA2 is predominately
an intracellular molecule that can be quickly mobilized from
intracellular stores and surface expressed following cellular
treatment with interferon (IFN)-gamma. The surface expression of
IL13RA2 after IFN-gamma treatment does not involve protein
synthesis and results in diminished IL13 signaling (Daines and
Hershey, J Biol. Chem., 2002; 22(12):10387-10393).
[0542] The results of the gene expression array analysis for
zcytor17lig indicate the action of zcytor17lig to be novel to that
of IFN-gamma in that the zcytor17lig treatment of lung epithelial
derived cell lines resulted in a significant increase of IL13RA2
gene expression. It is conceivable, therefore, that zcytor17lig
treatment would be beneficial in cases where long-term up
regulation of IL13RA2 expression and down regulation of IL13 is
desired such as in asthma, airway hyperactivity (AHR), and mucin
regulation, including chronic sinusitis with polps.
Example 41
Murine zcytor17lig Transgenic Mice
[0543] To evaluate the in vivo effects of zcytor17lig
overexpression, multiple founders of transgenic mice expressing the
murine form of the gene were generated, driven by two different
promoters: the lymphocyte-specific promoter E.mu./lck, and the
ubiquitous promoter, EF1.alpha. (Example 22). Serum protein levels
range from approximately 20-300 ng/ml. The E.mu./lck promoter
generated mice with higher levels of serum protein than those in
the EF1.alpha.-zcytor17lig transgenic mice.
[0544] The zcytor17lig transgenic mice developed a skin phenotype
around 4-8 weeks of age. The fur of the transgenic mice became
"ruffled," with obvious piloerection and mild to severe hair loss,
usually on their backs, sides of the torso, and around their eyes.
This phenotype was consistently found in mice with detectable
levels of zcytor17lig protein in their serum. Among the founders,
100% incidence rate among the mice expressing the E.mu./lck-driven
gene, and a 50% incidence in the EF1.alpha.-zcytor17lig transgenic
mice was noted, correlating well with the relative levels of
zcytor17lig that was detected in their serum. The transgenic skin
appeared to be pruritic, as evidenced by the scratching behavior of
the mice, sometimes excessive enough to induce excoriation and
lesions of the skin, which usually became infected (with at least
Staphylococcus aureus). The mice were originally identified with
metal ear tags, but in most cases, the ear tags were forcibly
removed by the mice themselves. This often resulted in severe
damage to the external ear. These wounded ears often did not heal
properly, as reflected in the presence of long-lasting pustules and
crusting, and a seeping, expanding wound would that developed in
many of the animals, behind and between their ears. Some of the
transgenic mice also developed scabby wounds on their shoulders and
neck. Skin lesions were observed in a subset of the animals,
generally evolving on areas of skin where hair loss had already
been apparent, and were often exacerbated by the scratching
behavior of the mice.
[0545] RealTime quantitative RT-PCR was used to detect zcytor17lig
RNA transcripts in transgenic (but not non-transgenic) skin
samples, with the E.mu./lck transgenic skin expressing more
zcytor17lig RNA than skin from EF1.alpha.-zcytor17lig transgenic
mice. The genes encoding the zcytor17 receptor subunits, zcytor17
and OSM-Rbeta, were expressed in the skin of both non-transgenic
and zcytor17lig-transgenic mice.
[0546] An examination of the lymphoid tissues from a subset of the
E.mu./lck-transgenic founders by flow cytometry revealed a
significant increase in the proportion of activated T cells in the
spleen and lymph nodes of these mice. Two of the four mice analyzed
had severely enlarged cervical lymph nodes, possibly due to the
presence of lesions on their necks. A subtle increase in spleen
weight and a slight increase in monocytes and neutrophils
circulating in the blood of the transgenic mice was observed. There
was no increase in a variety of cytokines tested, nor were there
changes in the circulating serum amyloid A levels in these mice.
The effects on the immune cells in the transgenic mice may be a
direct or an indirect result of zcytor17lig, or are secondary
effects of the skin lesions.
[0547] Histopathology was performed on many tissues other than
skin, including liver, thymus, spleen, kidney, and testes, and no
significant abnormalities in these organs were noted. Analysis of
the transgenic skin, however, did reveal a number of alterations,
which varied greatly depending upon the source and location of skin
(e.g., normal, hairless, or lesional). In many cases, the ears of
the transgenic mice had a thickened epidermis as compared to the
non-transgenic controls (e.g., approximately 4 layers versus 2
layers), and the underlying tissues contained low to moderate
numbers of inflammatory cells, which were primarily mononuclear
with occasional neutrophils. The epidermis over the abdomen
appeared multifocally slightly thicker in the transgenic, but there
was no apparent increase in inflammatory cells in the underlying
dermis or subcutis. In the hairless portions of skin from these
mice, there were dilated hair follicles that contained some debris
but no hair shafts (e.g., the hairs fell out by the roots). In the
lesioned areas, there was severe thickening of the epidermis
(acanthosis), increased keratin on the surface of the skin
(hyperkeratosis), scattered ulcers of varying size and significant
numbers of inflammatory cells in the dermis (mainly neutrophils,
with varying numbers of macrophages and lymphocytes). The dermis
also contained numerous mast cells bordering the lesions. Some of
the hair shafts in the lesioned areas of the transgenic skin were
in the active stage (anagen), in contrast to many of the hair
shafts in "normal" areas which were in the involuting (catagen) to
inactive (telogen) stage.
[0548] The phenotype of the zcytor17lig transgenic mice strongly
resembles that of atopic dermatitis (AD) patients, and mouse models
of AD. AD is a common chronic inflammatory disease that is
characterized by hyperactivated cytokines of the helper T cell
subset 2 (Th2). Zcytor17lig is preferentially expressed by Th2 vs.
Th1 cells, which lends further credence to this comparison.
Although the exact etiology of AD is unknown, multiple factors have
been implicated, including hyperactive Th2 immune responses,
autoimmunity, infection, allergens, and genetic predisposition. Key
features of the disease include xerosis (dryness of the skin),
pruritus (itchiness of the skin), conjunctivitis, inflammatory skin
lesions, Staphylococcus aureus infection, elevated blood
eosinophilia, elevation of serum IgE and IgG1, and chronic
dermatitis with T cell, mast cell, macrophage and eosinophil
infiltration. Colonization or infection with S. aureus has been
recognized to exacerbate AD and perpetuate chronicity of this skin
disease.
[0549] AD is often found in patients with asthma and allergic
rhinitis, and is frequently the initial manifestation of allergic
disease. About 20% of the population in Western countries suffer
from these allergic diseases, and the incidence of AD in developed
countries is rising for unknown reasons. AD typically begins in
childhood and can often persist through adolescence into adulthood.
Current treatments for AD include topical corticosteroids, oral
cyclosporin A, non-corticosteroid immunosuppressants such as
tacrolimus (FK506 in ointment form), and interferon-gamma. Despite
the variety of treatments for AD, many patients' symptoms do not
improve, or they have adverse reactions to medications, requiring
the search for other, more effective therapeutic agents.
[0550] Epithelial cells, which express the heterodimeric receptor
for zcytor17lig (zcytoRI7 and OSM-Rbeta), are located at the sites
(e.g., skin, gut, lung, etc.) of allergen entry into the body and
interact closely with dendritic cells (professional antigen
presenting cells) in situ. Dendritic cells play an important role
in the pathogenesis of allergic diseases, and it is possible that
zcytor17lig can interact with its receptor on epithelial cells in
the skin and lung and influence immune responses in these organs.
Zcytor17lig and its receptor(s) may therefore contribute to the
pathogenesis of allergic diseases such as AD and asthma.
Furthermore, the phenotype of the zcytor17lig transgenic mice
suggests that this ligand may play a role in wound healing, since
the mice seem unable to repair damage to their ears, and often bear
long-lasting lesions on their backs and sides. An antagonist of
zcytor17lig might therefore represent a viable therapeutic for
these and other indications.
Example 42
Luciferase Assay on Human Transformed Epithelial Cell Lines via
Transient Infection with an Adenoviral STAT/SRE Reporter Gene
[0551] A wide variety of human transformed epithelial cell lines
(see Table 16 below) were seeded in 96-well flat-bottom plates at
10,000 cell/well in regular growth media as specified for each cell
type. The following day, the cells were infected with an adenovirus
reporter construct, KZ136, at a multiplicity of infection of 5000.
The KZ136 reporter contains the STAT elements in addition to a
serum response element. The total volume was 100 ul/well using DMEM
supplemented with 2 mM L-glutamine (GibcoBRL), 1 mM Sodium Pyruvate
(GibcoBRL) and 1.times. Insulin-Transferrin-Selenium supplement
(GibcoBRL) (hereinafter referred to as serum-free media). Cells
were cultured overnight.
[0552] The following day, the media was removed and replaced with
100 .mu.l of induction media. The induction media was human
zcytor17 ligand diluted in serum-free media at 100 ng/ml, 50 ng/ml,
25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.125 ng/ml and 1.56 ng/ml. A
positive control of 20% FBS was used to validate the assay and to
ensure the infection by adenovirus was successful. The cells were
induced for 5 hours at which time the media was aspirated. The
cells were then washed in 50 .mu.l/well of PBS, and subsequently
lysed in 30 .mu.l/well of 1.times. cell lysis buffer (Promega).
After a 10-minute incubation at room temperature, 25 .mu.l/well of
lysate was transferred to opaque white 96-well plates. The plates
were then read on the Luminometer using 5-second integration with
40 .mu.l/well injection of luciferase substrate (Promega).
[0553] The results revealed the ability of multiple epithelial cell
lines to respond to zcytor17 ligand as shown in Table 16 below.
TABLE-US-00016 TABLE 16 Cell Line Species Tissue Morphology Disease
Fold Induction A549 Human Lung Epithelial Carcinoma 2x Sk-Lu-1
Human Lung Epithelial Adenocarcinoma 6x WI-38 Human Embryonic Lung
Fibroblast Negative MRC-5 Human Lung Fibroblast Negative DU 145
Human Prostate Epithelial Carcinoma 10x PZ-HPV-7 Human Prostate
Epithelial Transformed with HPV 5x PC-3 Human Prostate Epithelial
Adenocarcinoma Negative U2OS Human Bone Epithelial Osteosarcoma
15.5x SaOS2 Human Bone Epithelial Osteosarcoma 22x MG-63 Human Bone
Fibroblast Osteosarcoma Negative 143B Human Bone Fibroblast
Osteosarcoma 3.5x HOS Human Bone Fibroblast and Epithelial 8x
TRBMeC Human Vascular Bone Marrow Epithelial 2x HT144 Human Skin
Fibroblast Melanoma 5x C32 Human Skin Melanoma Negative Sk-Mel-2
Human Skin Polygonal Melanoma 2.7x WM-115 Human Skin Epithelial
Melanoma 2x HCT-116 Human Colon Epithelial Carcinoma Negative HT-29
Human Colon Epithelial Carcinoma Negative CaCo2 Human Colon
Epithelial Adenocarcinoma 3x HBL-100 Human Breast Epithelial 1.5x
ME-180 Human Cervix Epithelial Carcinoma Negative HeLa 299 Human
Cervix Epithelial Adenocarcinoma Negative SK-N-SH Human Brain
Epithelial Neuroblastoma Negative U138 MG Human Brain Polygonal
Glioblastoma Negative HepG2 Human Liver Epithelial Carcinoma
Negative Chang liver Human Liver Epithelial Negative Sk-Hep-1 Human
Liver Epithelial Adenocarcinoma 4x Int 407 Human Intestine
Epithelial Negative 3a-Sub E Human Placenta Negative
Example 43
Cytokine Production by Human Epithelial Cell Lines Cultured with
Human zcytor17 Ligand
[0554] Human disease-state epithelial cell lines (A549, human lung
epithelial carcinoma; SkLu1, human lung epithelial adenocarcinoma;
DU145, human prostate epithelial carcinoma; PZ-HPV-7, human
prostate epithelial HPV transformed; U20S, human bone epithelial
osteosarcoma) were screened for cytokine production in response to
zcytor17 ligand in vitro. These cell lines have both zcytor17 and
OSMR-beta, identified by RT-PCR, and respond to human zcytor17
ligand when assayed with the adenoviral luciferase reporter
construct, KZ136 (Example 42). Cytokine production by these cell
lines was determined in response to human zcytor17 ligand in a
series of three experiments.
A. Cytokine Production by Human Disease-State Epithelial Cell Lines
Cultured with Human zcytor17lig
[0555] Cells were plated at a density of 4.5.times.10.sup.5 cells
per well in a 6 well plate (Costar) and cultured in respective
growth media. The cells were cultured with test reagents; 100 ng/mL
zcytor17 ligand, 10 ng/mL Interferon gamma (IFN gamma) (R&D
Systems, Minneapolis, Minn.), 10 ng/mL Tumor Necrosis Factor alpha
(TNF alpha) (R&D Systems, Minneapolis, Minn.), 10 ng/mL
IL-1beta (R&D Systems, Minneapolis, Minn.) or 100 ug/mL
Lipopolysaccharide (LPS) (Sigma). Supernatants were harvested at 24
and 48 hours and assayed for cytokines; GM-CSF
(Granulocyte-Macrophage Colony-Stimulating Factor), IL-1b, IL-6,
IL-8, MCP-1 (Macrophage Chemoattractant Protein-1) and TNFa.
Multiplex Antibody Bead kits from BioSource International
(Camarillo, Calif.) were used to measure cytokines in samples.
Assays were read on a Luminex-100 instrument (Luminex, Austin,
Tex.) and data was analyzed using MasterPlex software (MiraiBio,
Alameda, Calif.). Cytokine production (pg/mL) for each cell line in
the 24-hour samples is shown below in Table 17. TABLE-US-00017
TABLE 17 A549 SkLu1 DU145 U2OS PZ-HPV-7 GM-CSF pg/mL zcytor17L
18.80 10.26 16.19 13.26 14.10 IFN-g 16.19 13.36 11.56 16.26 11.81
IL-1b 104.60 126.44 76.77 338.25 27.32 TNFa 106.67 33.20 58.50
107.09 33.79 LPS 17.64 10.62 11.81 25.47 18.34 control 14.81 8.56
13.26 21.67 13.96 IL-1b pg/mL zcytor17L 26.90 30.17 28.77 29.07
28.00 IFN-g 29.07 35.33 21.96 26.90 26.73 IL-1b 1332.88 1256.17
979.02 1107.35 998.60 TNFa 31.11 33.28 35.33 31.24 25.66 LPS 33.28
28.77 29.07 31.11 31.24 control 28.77 28.77 26.73 31.24 29.07 IL-6
pg/mL zcytor17L 20.09 26.89 193.05 19.37 17.30 IFN-g 17.52 33.64
217.58 27.02 17.63 IL-1b 175.44 5920.19 2375.29 304.08 18.44 TNFa
354.16 1002.51 1612.17 103.58 18.33 LPS 18.06 35.65 162.18 22.42
17.30 control 17.63 27.80 71.23 19.32 17.19 IL-8 pg/mL zcytor17L
86.33 150.81 150.61 45.92 6.81 IFN-g 24.07 72.82 163.31 81.78 1.35
IL-1b 1726.24 4083.12 4407.79 5308.83 124.17 TNFa 3068.68 3811.75
2539.39 3324.02 69.65 LPS 20.28 167.13 230.39 115.08 7.95 control
14.92 109.78 107.27 93.44 9.49 MCP-1 pg/mL zcytor17L 8.97 187.29
26.84 105.15 7.20 IFN-g 7.30 267.99 17.05 88.68 7.71 IL-1b 8.11
8039.84 88.78 3723.81 4.70 TNFa 8.50 7100.37 153.26 3826.80 2.80
LPS 9.40 185.83 22.65 61.62 5.61 control 8.16 167.93 13.68 47.78
5.61 TNFa pg/mL zcytor17L 16.23 17.52 16.67 15.80 17.09 IFN-g 15.80
17.09 15.80 16.65 15.80 IL-1b 16.66 17.09 15.80 17.95 16.23 TNFa
1639.92 1648.83 2975.07 1348.33 3554.82 LPS 16.87 15.80 15.37 17.09
17.52 control 16.23 15.80 15.80 17.09 16.66
[0556] All cell lines tested produced GM-CSF and IL-8 in response
to stimulation with control cytokines IL-1b and TNFa. Most cell
lines produced IL-6 and MCP-1 in response to IL-1b and TNFa
stimulation. Zcytor17 ligand stimulated IL-6 production in the
DU145 cell line compared to control (193 pg/mL vs. 71 pg/mL).
Zcytor17 ligand stimulated 3 of 5 cell lines to produce IL-8 with
the greatest effect seen in A549 cells (5 fold), and reduced IL-8
production in U20S cells by 2 fold. There was a slight effect on
MCP-1 production by DU145 and U20S cells when cultured with
zcytor17 ligand.
B. Cytokine Production by Normal Human Epithelial Cell Lines
Cultured with Human zcytor17lig
[0557] In addition to the human epithelial cell lines, normal human
bronchial epithelial cells (NHBE, Clonetics) were also tested.
Cells were plated at a density of 1.times.10.sup.5 cells per well
in a 24 well plate and cultured with test reagents; 1000 ng/mL, 100
ng/mL and 10 ng/mL zcytor17 ligand (A760F), 10 ng/mL TNFa, 10 ng/mL
OSM, 10 ng/mL IFNa, 10 ng/mL TGFb or 10 ng/mL Lymphotactin.
Supernatants were harvested at 24 and 48 hours and assayed for
cytokines; IL-6, IL-8, MCP-1, MIP-1a, RANTES and Eotaxin. Cytokines
were assayed as previously described. Cytokine production (pg/mL)
for each cell line in the 48-hour samples is shown below in Table
18. TABLE-US-00018 TABLE 18 A549 DU145 SkLu1 U2OS NHBE IL-6 pg/ml
r17L 24.5 56.3 32.1 25.2 64.5 1000 ng/ml r171L 100 ng/ml 25.0 65.0
31.0 25.4 50.2 r17L 10 ng/ml 24.8 51.8 30.2 25.3 54.3 TNFa 272.9
355.4 437.5 36.1 299.3 OSM 26.4 73.5 112.4 25.6 80.4 IFNa 24.6
109.3 33.7 26.4 52.4 TGFb 24.4 102.6 42.7 27.8 268.9 control 24.5
36.3 29.9 25.2 47.9 IL-8 pg/ml r17L 35.0 243.3 45.6 18.6 402.0 1000
ng/ml r171L 100 ng/ml 31.0 290.7 40.1 21.3 296.0 r17L 10 ng/ml 30.4
240.4 33.4 18.9 361.8 TNFa 2809.3 2520.9 1385.2 784.9 1486.3 OSM
37.8 60.6 68.0 22.5 494.6 IFNa 18.9 315.3 39.5 33.1 231.6 TGFb 9.9
77.5 19.6 88.9 246.9 control 10.9 238.0 38.0 39.7 315.8 MCP-1 pg/ml
r17L nd nd 149.1 81.0 nd 1000 ng/ml r171L 100 ng/ml nd nd 130.6
81.9 nd r17L 10 ng/ml nd nd 111.7 49.1 nd TNFa nd 22.1 2862.6
1104.7 nd OSM nd 17.2 448.2 85.8 nd IFNa nd nd 131.7 10.5 nd TGFb
nd 1.7 54.5 27.6 nd control nd nd 113.0 1.7 nd nd = not
detected
[0558] DU145 cells produced IL-6 in response to zcytor17 ligand,
repeating the previous results in Example 43A. However, only A549
and U20S had similar IL-8 responses as seen Example 43A. SkLu1 and
U2OS cells both produced MCP-1 in response to zcytor17 ligand.
Cytokine production by NHBE cells was marginal compared to
controls.
C. Cytokine Production by Human Disease-State Epithelial Cell Lines
Co-Cultured with Human zcytor17lig and IFN Gamma
[0559] Cells were plated at a density of 2.times.10.sup.5 cells per
well in 24 well plate and co-cultured with 10 ng/mL IFN
gamma+/-zcytor17 ligand at 100 ng/mL, 10 ng/mL or 1 ng/mL.
Supernatants were collected at 24 and 48 hours and assayed for IL-8
and MCP-1 as described above. Cytokine production (pg/mL) for each
cell line in the 24-hour samples is shown below in Table 19.
TABLE-US-00019 TABLE 19 IL-8 pg/ml MCP-1 pg/ml A549 10 ng/mL IFNg +
100 ng/mL r17L 86.7 nd 10 ng/mL IFNg + 10 ng/mL r17L 75.1 nd 10
ng/mL IFNg + 1 ng/mL r17L 63.6 nd 10 ng/ml IFNg 35.4 nd control
36.6 nd DU145 10 ng/mL IFNg + 100 ng/mL r17L 102.3 nd 10 ng/mL IFNg
+ 10 ng/mL r17L 92.9 nd 10 ng/mL IFNg + 1 ng/mL r17L 79.9 nd 10
ng/ml IFNg 70.7 nd control 79.4 nd SkLu1 10 ng/mL IFNg + 100 ng/mL
r17L 152.2 604.9 10 ng/mL IFNg + 10 ng/mL r17L 194.4 870.7 10 ng/mL
IFNg + 1 ng/mL r17L 138.7 585.4 10 ng/ml IFNg 170.8 652.6 control
203.0 292.3 U2OS 10 ng/mL IFNg + 100 ng/mL r17L 106.8 357.0 10
ng/mL IFNg + 10 ng/mL r17L 108.2 347.7 10 ng/mL IFNg + 1 ng/mL r17L
109.9 293.3 10 ng/ml IFNg 118.8 159.8 control 146.8 7.0
[0560] A549 cells produced IL-8 in response to zcytor17 ligand,
however there was no effect of co-culturing cells with the addition
of IFN gamma. U20S cells made 20 fold more MCP-1 when cultured with
IFNg and 50 fold more MCP-1 when cultured with IFN gamma+zcytor17
ligand.
Example 44
Zcytor17lig Effects on .sup.3H-TdR Incorporation in DU145 Prostate
Epithelial Carcinoma Cells
[0561] Cells were seeded in 96-well tissue clusters (Falcon) at a
density of 25,000/well in MEM (Life Technologies) growth medium
supplemented with glutamine, pyruvate, non-essential amino acids
(Life Technologies) and 10% fetal bovine serum (Hyclone). At
confluence (24 hours later), cells were switched to growth arrest
media by substituting 0.1% BSA (Life Technologies) for serum. After
48 hours to achieve cell synchronization, the growth-arrest medium
was replaced with fresh medium. Then, human recombinant zcytor17lig
(test reagent) was added at various concentrations (from 0.24 to 60
ng/mL) (see Table 16 below), to test for the effect of the protein
on basal DNA replication. Some wells received 2.5% FBS (Hyclone) in
addition to zcytor17Ligand, in order to test effect of the protein
on elevated levels of TdR incorporation. FBS 10% and 20 ng/ml
Platelet Derived Growth Factor-BB (PDGF-BB) (R&D) were used as
positive control.
[0562] Eighteen hours following addition of zCytor17Ligand and the
rest of the test reagents, cells were pulsed with 250 nCi/mL
[.sup.3H]-thymidine (NEN) for 4 hours. Following the 4-hour pulse,
media were discarded and 100 .mu.L trypsin solution (Life
Technologies) was added in each well to dislodge the cells. The
radioactivity incorporated by DU145 was determined by harvesting
the cells with a Packard Filtermate 196 cell harvester and by
counting the incorporated label using a Packard TopCount NXT
microplate scintillation counter.
[0563] As can be seen in Table 20 below, zcytor17lig induced
thymidine incorporation in quiescent cells (in 0.1% BSA) in a
concentration-dependent manner. This effect reached 2.5-fold of the
BSA control at the highest concentration used, 60 ng/mL. In
addition, this effect of zcytor17lig was also detectable when the
baseline incorporation was elevated by the addition of 2.5% FBS (in
this series as potent a mitogen as 10% FBS). These results
therefore indicate that under both basal and stimulated conditions
zcytor17lig can act as a mitogenic factor for the DU145 carcinoma
cells.
[0564] Table 16 shows the effects of zcytor17lig on thymidine
incorporation by DU145 cells. Results are expressed in cpm/well and
numbers are the mean.+-.st.dev of triplicate wells. TABLE-US-00020
TABLE 20 0.1% BSA 2.5% FBS BSA Control 1139 .+-. 336 4228 .+-. 600
Zcytor17lig (0.24 ng/mL) 1430 .+-. 136 4894 .+-. 1037 Zcytor17lig
(0.74 ng/mL) 1657 .+-. 32 5038 .+-. 810 Zcytor17lig (2.22 ng/mL)
1646 .+-. 57 5162 .+-. 808 Zcytor17lig (6.67 ng/mL) 2226 .+-. 189
6385 .+-. 1613 Zcytor17lig (20 ng/mL) 2168 .+-. 108 5880 .+-. 1085
Zcytor17lig (60 ng/mL) 2512 .+-. 111 6165 .+-. 417 PDGF-BB (20
ng/mL) 4094 .+-. 202 5927 .+-. 360
Example 45
Expression of huzcytor17Ligand in E. coli
A. Construction of Expression Vector pRPS01 that Expresses
huzcytor17Lig/MBP-6H Fusion Polypeptide
[0565] An expression plasmid containing a polynucleotide encoding a
huzcytor17lig fused C-terminally to Maltose Binding Protein (MBP)
was constructed via homologous recombination. The fusion
polypeptide contains an N-terminal approximately 388 amino acid MBP
portion fused to the huzcytor17Lig described herein. A fragment of
huzcytor17lig cDNA was isolated using the PCR method as described
herein. Two primers were used in the production of the zcytor17lig
fragment in a standard PCR reaction: (1) one containing 40 bp of
the vector flanking sequence and 20 bp corresponding to the amino
terminus of the huzcytor17lig, and (2) another containing 40 bp of
the 3' end corresponding to the flanking vector sequence and 20 bp
corresponding to the carboxyl terminus of the huzcytor17lig. Two
microliters of the 100 .mu.l PCR reaction was run on a 1.0% agarose
gel with 1.times.TBE buffer for analysis, and the expected
molecular weight fragment was observed. The remaining PCR reaction
was combined with the second PCR tube and precipitated with 400
.mu.l of absolute ethanol. The precipitated DNA was used for
recombination into the Sma1 cut recipient vector pTAP98 to produce
the construct encoding the MBP-huzcytor17lig fusion, as described
below.
[0566] The vector pTAP98 was constructed using yeast homologous
recombination. One hundred nanograms of EcoRI cut pMAL-c2 was
recombined with 1 .mu.g Pvu1 cut pRS316, 1 .mu.g linker, and 1 g
Sca1/EcoR1 cut pRS316 were combined in a PCR reaction. PCR products
were concentrated via 100% ethanol precipitation. The competent
yeast cell (S. cerevisiae) strain, SF838-9Da, was combined with 10
.mu.l of a mixture containing approximately 1 .mu.g of the
huzcytor17lig PCR product (above) and 100 ng of SmaI digested
pTAP98 vector, and electroporated at 0.75 kV, 25.degree. F. and
.infin. ohms. The resulting reaction mixture was plated onto URA-D
plates and incubated at 30.degree. C.
[0567] After 48 hours, the Ura+ yeast transformants from a single
plate were selected. DNA was isolated and transformed into
electrocompetent E. coli cells (e.g., MC1061, Casadaban et. al. J.
Mol. Biol 138, 179-207). The resulting E. coli cells were plated on
MM/CA+AMP 100 mg/L plates (Pryor and Leiting, Protein Expression
and Purification 10:309-319, 1997) using standard procedures. Four
individual clones were harvested from the plates and inoculated
into MM/CA with 100 .mu.g/ml Ampicillin for two hours at 37.degree.
C. One milliliter of each of the culture was induced with 1 mM
IPTG. Approximately 2-4 hours later, 250 .mu.l of each induced
culture was 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 were vortexed for
one minute and heated to 65.degree. C. for 10 minutes. Twenty
microliters of each sample was loaded per lane on a 4%-12% PAGE gel
(NOVEX). Gels were run in 1.times.MES buffer. The positive clones
were designated pRPS01 and subjected to sequence analysis.
[0568] One microliter of sequencing DNA was used to transform
electrocompetent E. coli cell strain MC1061. The cells were
electropulsed at 2.0 kV, 25 .mu.F and 400 ohms. Following
electroporation, cells were rescued 0.6 ml SOC and grown on LB+Amp
plates at 37.degree. C. overnight, with 100 mg/L Ampicillin. Four
cultures were induced with ITPG and screened for positives as
described above. The positive clones were expanded for protein
purification of the huzcytor17lig/MBP-6H fusion protein using
standard techniques.
B. Purification of huzcytor17Lig/MBP-6H from E. coli
Fermentation
[0569] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used to purify recombinant
huzcytor17Lig/MBP-6H polypeptide. E. coli cells containing the
pRPS01 construct and expressing huzcytor17Lig/MBP-6H, were
constructed using standard molecular biology methods and cultured
in 50.0 g/L SuperBroth II (12 g/L Casien, 24 g/L Yeast Extract,
11.4 g/L di-potassium phosphate, 1.7 g/L Mono-potassium phosphate;
Becton Dickenson, Cockeysville, Md.), 5 g/L glycerol and 5 mL/L 1M
Magnesium Sulfate. Twenty grams of cells were harvested and frozen
for protein purification.
[0570] The thawed cells were resuspended in 500 mL Amylose
Equilibration buffer (20 mM Tris, 100 mM NaCl, pH 8.0). A French
Press cell breaking system (Constant Systems Ltd., Warwick, UK)
with a temperature setting of -7.degree. C. to -10.degree. C. and
30K PSI was used to lyse the cells. The resuspended cells were
assayed for breakage by A.sub.600 readings before and after cycling
through the French Press. The processed cell suspension was
pelleted at 10,000G for 30 minutes to remove the cellular debris
and the supernatant was harvested for protein purification.
[0571] A 25 ml column of Amylose resin (New England Biolabs,
Beverly, Mass.) (prepared as described below) was poured into a
Bio-Rad, 2.5 cm D.times.10 cm H glass column. The column was packed
and equilibrated by gravity with 10 column volumes (CVs) of Amylose
Equilibration buffer. The processed cell supernatant was batch
loaded to the Amylose resin overnight, with rocking. The resin was
returned to the Bio-Rad column and washed with 10 CV's of Amylose
Equilibration buffer by gravity. The column was eluted with
.about.2 CV of Amylose Elution buffer (Amylose Equilibration
buffer+10 mM Maltose, Fluka Biochemical, Switzerland) by gravity.
Ten 5 mL fractions were collected over the elution profile and
assayed for Absorbance at 280 and 320 nM. The Amylose resin was
regenerated with 1 CV of distilled H.sub.2O, 5 CVs of 0.1% (w/v)
SDS (Sigma), 5 CVs of distilled H.sub.2O, 5 CVs of Amylose
Equilibration buffer and finally 1 CV of Amylose Storage buffer
(Amylose Equilibration buffer+0.02% Sodium Azide). The regenerated
column was stored at 4.degree. C.
[0572] Elution profile fractions of interest were pooled and
dialyzed in a 10K dialysis chamber (Slide-A-Lyzer, Pierce
Immunochemicals) against 4.times.4 L PBS pH 7.4 (Sigma) over an 8
hour time period to remove low molecular weight contaminants,
buffer exchange and desalt. Following dialysis, the material
harvested represented the purified huzcytor17Lig/MBP-6H
polypeptide. The purified huzcytor17Lig/MBP-6H polypeptide was
filter sterilized and analyzed via SDS-PAGE Coomassie staining for
an appropriate molecular weight product. The concentration of the
huzCytor17Lig/MBP-6H polypeptide was determined by BCA analysis to
be 1.28 mg/mL.
Example 46
Human zcytor17lig Polyclonal Antibody
A. Preparation and Purification
[0573] Polyclonal antibodies were prepared by immunizing 2 female
New Zealand white rabbits with the purified recombinant protein
hzcytor17L/MBP-6H (Example 45). The rabbits were each given an
initial intraperitoneal (IP) injection of 200 .mu.g of purified
protein in Complete Freund's Adjuvant followed by booster IP
injections of 100 .mu.g protein in Incomplete Freund's Adjuvant
every three weeks. Seven to ten days after the administration of
the second booster injection (3 total injections), the animals were
bled and the serum was collected. The animals were then boosted and
bled every three weeks.
[0574] The hzcytor17L/MBP-6H specific rabbit serum was pre-adsorbed
of anti-MBP antibodies using a CNBr-SEPHAROSE 4B protein column
(Pharmacia LKB) that was prepared using 10 mg of non-specific
purified recombinant MBP-fusion protein per gram of CNBr-SEPHAROSE.
The hzcytor17L/MBP-6H-specific polyclonal antibodies were affinity
purified from the pre-adsorbed rabbit serum using a CNBr-SEPHAROSE
4B protein column (Pharmacia LKB) that was prepared using 10 mg of
the specific antigen purified recombinant protein
hzcytor17L/MBP-6H. Following purification, the polyclonal
antibodies were dialyzed with 4 changes of 20 times the antibody
volume of PBS over a time period of at least 8 hours.
Hzcytor17-Ligand-specific antibodies were characterized by ELISA
using 500 ng/ml of the purified recombinant proteins
hzcytor17L/MBP-6H or hzcytor17L-CEE produced in a baculovirus
expression system as antibody targets. The lower limit of detection
(LLD) of the rabbit anti-hzcytor17L/MBP-6H affinity purified
antibody is 100 pg/ml on its specific purified recombinant antigen
hzcytor17L/MBP-6H and 500 pg/ml on purified recombinant
hzcytor17L-CEE produced in a baculovirus expression system.
B. SDS-PAGE and Western Blotting Analysis of Rabbit Anti-Human
ZcytoR17lig MBP-6H Antibody
[0575] Rabbit Anti-human ZcytoR17lig MBP-6H antibody was tested by
SDS-PAGE (NuPage 4-12%, Invitrogen, Carlsbad, Calif.) with
coomassie staining method and Western blotting using goat
anti-rabbit IgG-HRP. Human and mouse zcytor17lig purified protein
(200-25 ng) was electrophoresed using an Invitrogen Novex's Xcell
II mini-cell, and transferred to nitrocellulose (0.2 mm;
Invitrogen, Carlsbad, Calif.) at room temperature using Novex's
Xcell blot module with stirring according to directions provided in
the instrument manual. The transfer was run at 300 mA for one hour
in a buffer containing 25 mM Tris base, 200 mM glycine, and 20%
methanol. The filter was then blocked with Western A buffer (in
house, 50 mM Tris, pH 7.4, 5 mM EDTA, pH 8.0, 0.05% Igepal CA-630,
150 mM NaCl, and 0.25% gelatin) overnight with gentle rocking at
4.degree. C. The nitrocellulose was quickly rinsed, then the rabbit
anti-human zcytoR17lig MBP-6H (1:1000) was added in Western A
buffer. The blot was incubated for 1.5 hours at room temperature
with gentle rocking. The blot was rinsed 3 times for 5 minutes each
in Western A, then goat anti-rabbit IgG HRP antibody (1:5000) was
added in Western A buffer. The blot was incubated for 1 hour at
room temperature with gentle rocking. The blot was rinsed 3 times
for 5 minutes each in Western A, then quickly rinsed in H.sub.20.
The blot was developed using commercially available
chemiluminescent substrate reagents (ECL Western blotting detection
reagents 1 and 2 mixed 1:1; reagents obtained from Amersham
Pharmacia Biotech, Buckinghamshire, England) and the blot was
exposed to x-ray film for up to 5 minutes.
[0576] The purified human zcytor17lig appeared as a large band at
about 30 kDa and a smaller band at about 20 kDa under reduced
conditions. The mouse zcytor17lig was not detected by the rabbit
anti-human zcytor17lig antibody.
Example 47
Zcytor17lig Effects on U937 Monocyte Adhesion to Transformed Bone
Marrow Endothelial Cell (TRBMEC) Monolayer
[0577] Transformed Bone Marrow Endothelial Cells (TRBMEC) were
seeded in 96-well tissue clusters (Falcon) at a density of
25,000/well in medium M131 (Cascade Biologics) supplemented with
Microvascular Growth Supplement (MVGS) (Cascade Biologics). At
confluence (24 hours later), cells were switched to M199
(Gibco-Life Technologies) supplemented with 1% Fetal Bovine Serum
(Hyclone). Human recombinant zcytor17lig (test reagent) was added
at various concentrations (from 0.4 to 10 ng/mL) (see Table 21
below), to test for the effect of the protein on immune
cell-endothelial cell interactions resulting in adhesion. Some
wells received 0.3 ng/ml Tumor Necrosis Factor (TNFalpha R&D
Systems), a known pro-inflammatory cytokine, in addition to
zcytor17lig, to test an effect of the protein on endothelial cells
under inflammatory conditions. TNFalpha at 0.3 ng/ml alone was used
as positive control and the concentration used represents
approximately 70% of the maximal TNFalpha effect in this system,
i.e., it does not induce maximal adherence of U937 cells (a human
monocyte-like cell line) to the endothelium. For this reason, this
setup can detect both upregulation and downregulation of the
TNFalpha effects. Basal levels of adhesion both with and without
TNFalpha were used as baseline to assess effect of test
reagents.
[0578] After overnight incubation of the endothelial cells with the
test reagents (zcytor17ligand.+-.TNFalpha), U937 cells, stained
with 5 .mu.M Calcein-AM fluorescent marker (Molecular Probes), the
cells were suspended in RPMI 1640 (no phenol-red) supplemented with
1% FBS and plated at 100,000 cells/well on the rinsed TRBMEC
monolayer. Fluorescence levels at excitation/emission wavelengths
of 485/538 nm (Molecular Devices micro-plate reader, CytoFluor
application) were measured 30 minutes later, before and after
rinsing the well three times with warm RPMI 1640 (no phenol-red),
to remove non-adherent U937. Pre-rinse (total) and post-rinse
(adherence-specific) fluorescence levels were used to determine
percent adherence (net adherent/net total.times.100=%
adherence).
[0579] As can be seen in Table 21 below, zcytor17lig when added
alone affected the basal adherence of U937 cells to the endothelial
monolayers at the concentration range used (less than 2-fold
increases, p<0.01 by ANOVA test). By itself, the positive
control, 0.3 ng/mL TNFalpha, increased the adherence of U937 cells
from a basal 5.8% to 35% (6-fold). In the presence of TNFalpha,
zcytor17lig synergized with TNFalpha and further enhanced U937
adhesion in a concentration-dependent manner between 0.4 and 10
ng/mL (p<0.01 by ANOVA test). At 10 ng/mL, zcytor17lig enhanced
the effect of TNFalpha by 62%. These results indicate that
zcytor17lig may by itself be a pro-inflammatory agent. Zcytor17lig
was able to synergize with sub-maximal concentrations of TNFalpha
to increase monocyte adherence to endothelial cells. These results
also show that endothelial cells, especially when exposed to
pro-inflammatory cytokines such as TNFalpha, are a likely target
tissue of zcytor17lig action. The consequence of zcytor17ligand on
endothelial cells may be to heighten monocyte or macrophage
adhesion to a site of proinflammatory activity. Activated monocytes
and macrophages are important in many inflammatory diseases.
Therefore inhibition of monocyte/macrophage adhesions may provide a
therapeutic rationale for zcytor17ligand antagonists. This data
would support the use of zcytor17 ligand antagonists for treatment
lung diseases, vascular diseases, autoimmunity, tumor metastasis,
disease involving allergic reactions, wound healing and diseases of
the skin including contact, allergic or non-allergic dermatistic or
psoriasis and inflammatory bowel disease. Table 21 shows the
effects of zcytor17lig on U937 monocyte adhesion to TRBMEC
endothelial monolayers. Results are expressed in percent adhesion
and numbers are the mean.+-.st.dev of triplicate wells.
TABLE-US-00021 TABLE 21 Basal 0.3 ng/mL TNFalpha Basal 5.8 .+-. 1.2
35 .+-. 5.5 zcytor17lig 0.4 ng/mL 9 .+-. 0.7 44.7 .+-. 2.5
zcytor17lig 1.1 ng/mL 10.4 .+-. 0.8 45.2 .+-. 0.6 zcytor17lig 3.3
ng/mL 7.9 .+-. 1.7 51.1 .+-. 4 zcytor17lig 10 ng/mL 9.5 .+-. 0.5
56.6 .+-. 3.9
[0580] 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.
* * * * *