U.S. patent application number 09/746375 was filed with the patent office on 2003-09-11 for novel cytokine zcyto18.
Invention is credited to Kindsvogel, Wayne, Presnell, Scott R..
Application Number | 20030170823 09/746375 |
Document ID | / |
Family ID | 29554130 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170823 |
Kind Code |
A1 |
Presnell, Scott R. ; et
al. |
September 11, 2003 |
Novel cytokine ZCYTO18
Abstract
The present invention relates to ZCYTO18 polynucleotide and
polypeptide molecules. The ZCYTO18 is a novel cytokine. The
polypeptides may be used within methods for stimulating the
proliferation and/or development of hematopoietic cells in vitro
and in vivo. The present invention also includes methods for
producing the protein, uses therefor and antibodies thereto.
Inventors: |
Presnell, Scott R.; (Tacoma,
WA) ; Kindsvogel, Wayne; (Seattle, WA) |
Correspondence
Address: |
Jennifer K. Johnson, J.D.
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Family ID: |
29554130 |
Appl. No.: |
09/746375 |
Filed: |
December 22, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60172105 |
Dec 23, 1999 |
|
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60250841 |
Dec 1, 2000 |
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Current U.S.
Class: |
435/69.5 ;
435/325; 435/4; 530/351; 536/23.5 |
Current CPC
Class: |
A61P 1/04 20180101; C12N
2799/026 20130101; A61P 1/18 20180101; C07K 16/24 20130101; A61P
11/06 20180101; C07K 14/54 20130101; A61P 19/02 20180101; A61K
38/00 20130101; A61P 29/00 20180101; C07K 2319/00 20130101; A61K
39/00 20130101 |
Class at
Publication: |
435/69.5 ;
435/325; 435/4; 530/351; 536/23.5 |
International
Class: |
C12Q 001/00; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/52 |
Claims
What is claimed is:
1. An isolated polynucleotide that encodes a cytokine polypeptide
comprising a sequence of amino acid residues that is at least 90%
identical to an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 3
from amino acid number 23 (Pro), to amino acid number 167 (Ile);
(b) the amino acid sequence as shown in SEQ ID NO: 3 from amino
acid number 1 (Met), to amino acid number 167 (Ile); and (c) the
amino acid sequence as shown in SEQ ID NO: 2 from amino acid number
1 (Met), to amino acid number 179 (Ile); and wherein the
polypeptide produced by the cell induces proliferation of cells
expressing a receptor for the polypeptide comprising zcytor11 (SEQ
ID NO: 19) or induces cytotoxicity in K562 cells.
2. An isolated polynucleotide according to claim 1, wherein the
polynucleotide is selected from the group consisting of: (a) a
polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide
123 to nucleotide 557; (b) a polynucleotide sequence as shown in
SEQ ID NO: 1 from nucleotide 57 to nucleotide 557; (c) a
polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 21
to nucleotide 557; and (d) a polynucleotide sequence complementary
to (a), (b) or (c).
3. An isolated polynucleotide sequence according to claim 1,
wherein the polynucleotide comprises nucleotide 1 to nucleotide 501
of SEQ ID NO: 4.
4. An isolated polynucleotide according to claim 1, wherein the
cytokine polypeptide comprises a sequence of amino acid residues
selected from the group consisting of: (a) the amino acid sequence
as shown in SEQ ID NO: 3 from amino acid number 23 (Pro), to amino
acid number 167 (Ile); (b) the amino acid sequence as shown in SEQ
ID NO: 3 from amino acid number 1 (Met), to amino acid number 167
(Ile); and (c) the amino acid sequence as shown in SEQ ID NO: 2
from amino acid number 1 (Met), to amino acid number 179 (Ile).
5. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment encoding a
cytokine polypeptide as shown in SEQ ID NO: 3 from amino acid
number 23 (Pro), to amino acid number 167 (Ile); and a
transcription terminator, wherein the promoter is operably linked
to the DNA segment, and the DNA segment is operably linked to the
transcription terminator.
6. An expression vector according to claim 5, further comprising a
secretory signal sequence operably linked to the DNA segment.
7. A cultured cell comprising an expression vector according to
claim 5, wherein the cell expresses a polypeptide encoded by the
DNA segment.
8. A DNA construct encoding a fusion protein, the DNA construct
comprising: a first DNA segment encoding a polypeptide comprising a
sequence of amino acid residues selected from the group consisting
of: (a) the amino acid sequence as shown in SEQ ID NO: 3 from amino
acid number 1 (Met), to amino acid number 21 (Ala); (b) the amino
acid sequence as shown in SEQ ID NO: 3 from amino acid number 41
(Thr), to amino acid number 53 (Leu); (c) the amino acid sequence
as shown in SEQ ID NO: 3 from amino acid number 80 (Met), to amino
acid number 91 (Val); (d) the amino acid sequence as shown in SEQ
ID NO: 3 from amino acid number 103 (Gln), to amino acid number 116
(Arg); (e) the amino acid sequence as shown in SEQ ID NO: 3 from
amino acid number 149 (Ile), to amino acid number 162 (Leu); and
(f) the amino acid sequence as shown in SEQ ID NO: 3 from amino
acid number 23 (Pro), to amino acid number 167 (Ile); and at least
one other DNA segment encoding an additional polypeptide, wherein
the first and other DNA segments are connected in-frame; and
wherein the first and other DNA segments encode the fusion
protein.
9. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA construct encoding a
fusion protein according to claim 8; and a transcription
terminator, wherein the promoter is operably linked to the DNA
construct, and the DNA construct is operably linked to the
transcription terminator.
10. A cultured cell comprising an expression vector according to
claim 9, wherein the cell expresses a polypeptide encoded by the
DNA construct.
11. A method of producing a fusion protein comprising: culturing a
cell according to claim 10; and isolating the polypeptide produced
by the cell.
12. An isolated cytokine polypeptide comprising a sequence of amino
acid residues that is at least 90% identical to an amino acid
sequence selected from the group consisting of: (a) the amino acid
sequence as shown in SEQ ID NO: 3 from amino acid number 23 (Pro),
to amino acid number 167 (Ile); (b) the amino acid sequence as
shown in SEQ ID NO: 3 from amino acid number 1 (Met), to amino acid
number 167 (Ile); and (c) the amino acid sequence as shown in SEQ
ID NO: 2 from amino acid number 1 (Met), to amino acid number 179
(Ile); and wherein the polypeptide produced by the cell induces
proliferation of cells expressing a receptor for the polypeptide
comprising zcytor11 (SEQ ID NO: 19) or induces cytotoxicity in K562
cells.
13. An isolated polypeptide according to claim 12, wherein the
polypeptide comprises a sequence of amino acid residues selected
from the group consisting of: (a) the amino acid sequence as shown
in SEQ ID NO: 3 from amino acid number 23 (Pro), to amino acid
number 167 (Ile); (b) the amino acid sequence as shown in SEQ ID
NO: 3 from amino acid number 1 (Met), to amino acid number 167
(Ile); and (c) the amino acid sequence as shown in SEQ ID NO: 2
from amino acid number 1 (Met), to amino acid number 179 (Ile).
14. A method of producing a cytokine polypeptide comprising:
culturing a cell according to claim 7; and isolating the cytokine
polypeptide produced by the cell.
15. A method of producing an antibody to a polypeptide comprising:
inoculating an animal with a polypeptide selected from the group
consisting of: (a) a polypeptide consisting of 30 to 144 amino
acids, wherein the polypeptide is identical to a contiguous
sequence of amino acids in SEQ ID NO: 3 from amino acid number 23
(Gly) to amino acid number 779 (Thr); (b) a polypeptide according
to claim 13; (c) a polypeptide consisting of the amino acid
sequence of SEQ ID NO: 3 from amino acid number 29 (Arg) to amino
acid number 34 (Asn); (d) a polypeptide consisting of the amino
acid sequence of SEQ ID NO: 3 from amino acid number 121 (His) to
amino acid number 126 (Asp); (e) a polypeptide consisting of the
amino acid sequence of SEQ ID NO: 3 from amino acid number 134
(Gln) to amino acid number 139 (Thr); (f) a polypeptide consisting
of the amino acid sequence of SEQ ID NO: 3 from amino acid number
137 (Lys) to amino acid number 142 (Lys); (g) a polypeptide
consisting of the amino acid sequence of SEQ ID NO: 3 from amino
acid number 145 (Glu) to amino acid number 150 (Lys); (h) a
polypeptide consisting of the amino acid sequence of SEQ ID NO: 3
from amino acid number 41 (Thr), to amino acid number 53 (Leu); (i)
a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3
from amino acid number 80 (Met) to amino acid number 91 (Val); (j)
polypeptide consisting of the amino acid sequence of SEQ ID NO: 3
from amino acid number 103 (Met) to amino acid number 116 (Arg);
(k) a polypeptide consisting of the amino acid sequence of SEQ ID
NO: 3 from amino acid number 149 (Ile) to amino acid number 162
(Leu); and wherein the polypeptide elicits an immune response in
the animal to produce the antibody; and isolating the antibody from
the animal.
16. An antibody produced by the method of claim 15, which binds to
a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3.
17. The antibody of claim 16, wherein the antibody is a monoclonal
antibody.
18. An antibody that specifically binds to a polypeptide of claim
13.
19. A method of detecting, in a test sample, the presence of an
antagonist of ZCYTO18 protein activity, comprising: culturing a
cell that is responsive to a ZCYTO18-stimulated cellular pathway;
and producing a polypeptide by the method of claim 14; and exposing
the polypeptide to the cell, in the presence and absence of a test
sample; and comparing levels of response to the polypeptide, in the
presence and absence of the test sample, by a biological or
biochemical assay; and determining from the comparison, the
presence of the antagonist of ZCYTO18 activity in the test
sample.
20. A method of detecting, in a test sample, the presence of an
agonist of ZCYTO18 protein activity, comprising: culturing a cell
that is responsive to a ZCYTO18-stimulated cellular pathway; and
adding a test sample; and comparing levels of response in the
presence and absence of the test sample, by a biological or
biochemical assay; and determining from the comparison, the
presence of the agonist of ZCYTO18 activity in the test sample.
21. A method for detecting a genetic abnormality in a patient,
comprising: obtaining a genetic sample from a patient; producing a
first reaction product by incubating the genetic sample with a
polynucleotide comprising at least 14 contiguous nucleotides of SEQ
ID NO: 1 or the complement of SEQ ID NO: 1, under conditions
wherein said polynucleotide will hybridize to complementary
polynucleotide sequence; visualizing the first reaction product;
and comparing said first reaction product to a control reaction
product from a wild type patient, wherein a difference between said
first reaction product and said control reaction product is
indicative of a genetic abnormality in the patient.
22. A method for detecting a cancer in a patient, comprising:
obtaining a tissue or biological sample from a patient; incubating
the tissue or biological sample with an antibody of claim 18 under
conditions wherein the antibody binds to its complementary
polypeptide in the tissue or biological sample; visualizing the
antibody bound in the tissue or biological sample; and comparing
levels of antibody bound in the tissue or biological sample from
the patient to a normal control tissue or biological sample,
wherein an increase or decrease in the level of antibody bound to
the patient tissue or biological sample relative to the normal
control tissue or biological sample is indicative of a cancer in
the patient.
23. A method for detecting a cancer in a patient, comprising:
obtaining a tissue or biological sample from a patient; labeling a
polynucleotide comprising at least 14 contiguous nucleotides of SEQ
ID NO: 1 or the complement of SEQ ID NO: 1; incubating the tissue
or biological sample with under conditions wherein the
polynucleotide will hybridize to complementary polynucleotide
sequence; visualizing the labeled polynucleotide in the tissue or
biological sample; and comparing the level of labeled
polynucleotide hybridization in the tissue or biological sample
from the patient to a normal control tissue or biological sample,
wherein an increase or decrease in the labeled polynucleotide
hybridization to the patient tissue or biological sample relative
to the normal control tissue or biological sample is indicative of
a cancer in the patient.
24. A method of killing cancer cells comprising, obtaining ex vivo
a tissue or biological sample containing cancer cells from a
patient, or identifying cancer cells in vivo; producing a
polypeptide by the method of claim 14; formulating the polypeptide
in a pharmaceutically acceptable vehicle; and administering to the
patient or exposing the cancer cells to the polypeptide; wherein
the polypeptide kills the cells.
25. A method of killing cancer cells of claim 24, wherein the
polypeptide is further conjugated to a toxin.
26. A method of increasing platelets in a patient or injured
tissue, producing a polypeptide by the method of claim 14;
administering the polypeptide to the patient or injured tissue in a
pharmaceutically acceptable vehicle, wherein the polypeptide
increases the level pf platelets in the patient or injured
tissue.
27. A method for detecting inflammation in a patient, comprising:
obtaining a tissue or biological sample from a patient; incubating
the tissue or biological sample with an antibody of claim 18 under
conditions wherein the antibody binds to its complementary
polypeptide in the tissue or biological sample; visualizing the
antibody bound in the tissue or biological sample; and comparing
levels of antibody bound in the tissue or biological sample from
the patient to a normal control tissue or biological sample,
wherein an increase in the level of antibody bound to the patient
tissue or biological sample relative to the normal control tissue
or biological sample is indicative of inflammation in the
patient.
28. A method for detecting inflammation in a patient, comprising:
obtaining a tissue or biological sample from a patient; labeling a
polynucleotide comprising at least 14 contiguous nucleotides of SEQ
ID NO: 1 or the complement of SEQ ID NO: 1; incubating the tissue
or biological sample with under conditions wherein the
polynucleotide will hybridize to complementary polynucleotide
sequence; visualizing the labeled polynucleotide in the tissue or
biological sample; and comparing the level of labeled
polynucleotide hybridization in the tissue or biological sample
from the patient to a normal control tissue or biological sample,
wherein an increase in the labeled polynucleotide hybridization to
the patient tissue or biological sample relative to the normal
control tissue or biological sample is indicative of inflammation
in the patient.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to Provisional Application
60/172,105, filed on Dec. 23, 1999. This application is also
related to Provisional Application 60/###,###, filed on Dec. 1,
2000. Under 35 U.S.C. .sctn. 119(e)(1), this application claims
benefit of said Provisional Applications.
BACKGROUND OF THE INVENTION
[0002] Hormones and polypeptide growth factors control
proliferation and differentiation of cells of multicellular
organisms. These diffusable molecules allow cells to communicate
with each other and act in concert to form cells 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 nuclear receptors or 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 that 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 is the T cell, which produce many cytokines and adaptive
immunity to antigens. Cytokines produced by the T cell 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] Natural killer (NK) cells have a common progenitor cell with
T cells and B cells, and play a role in immune surveillance. NK
cells, which comprise up 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.
[0009] The demonstrated in vivo activities of the cytokine family
illustrates the enormous clinical potential of, and need for, other
cytokines, cytokine agonists, and cytokine antagonists. The present
invention addresses these needs by providing a new cytokine that
stimulates multiple cell types including hematopoietic cells, and
participates in the inflammatory response and tumor cell growth, as
well as related compositions and methods.
BRIEF DESCRIPTION OF THE DRAWING
[0010] FIG. 1 is a multiple alignment of the human ZCYTO18
polypeptide (hZCYTO18) (SEQ ID NO: 3), and the mouse ZCYTO18
polypeptide (mZCYTO18) (SEQ ID NO: 38) of the present invention.
The ":" in the figure indicates amino acids that are identical
between the mouse and human sequences, and the "." in the figure
indicates amino acids that are conserved substitutions. There is a
78.4% identity between the human and mouse sequences over the
entire sequence (167 amino acid overlap).
DESCRIPTION OF THE INVENTION
[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.
[0012] Within one aspect, the present invention provides an
isolated polynucleotide that encodes a cytokine polypeptide
comprising a sequence of amino acid residues that is at least 90%
identical to an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 3
from amino acid number 23 (Pro), to amino acid number 167 (Ile);
(b) the amino acid sequence as shown in SEQ ID NO: 3 from amino
acid number 1 (Met), to amino acid number 167 (Ile); and (c) the
amino acid sequence as shown in SEQ ID NO: 2 from amino acid number
1 (Met), to amino acid number 179 (Ile); and wherein the
polypeptide produced by the cell induces proliferation of cells
expressing a receptor for the polypeptide comprising zcytor11 (SEQ
ID NO: 19) or induces cytotoxicity in K562 cells. In one
embodiment, the isolated polynucleotide disclosed above is selected
from the group consisting of: (a) a polynucleotide sequence as
shown in SEQ ID NO: 1 from nucleotide 123 to nucleotide 557; (b) a
polynucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 57
to nucleotide 557; and (c) a polynucleotide sequence as shown in
SEQ ID NO: 1 from nucleotide 21 to nucleotide 557; and (d) a
polynucleotide sequence complementary to (a), (b) or (c). In
another embodiment, the isolated polynucleotide disclosed above
comprises nucleotide 1 to nucleotide 501 of SEQ ID NO: 4. In
another embodiment, the isolated polynucleotide disclosed above
encodes a cytokine polypeptide that comprises a sequence of amino
acid residues selected from the group consisting of: (a) the amino
acid sequence as shown in SEQ ID NO: 3 from amino acid number 23
(Pro), to amino acid number 167 (Ile); (b) the amino acid sequence
as shown in SEQ ID NO: 3 from amino acid number 1 (Met), to amino
acid number 167 (He); and (c) the amino acid sequence as shown in
SEQ ID NO: 2 from amino acid number 1 (Met), to amino acid number
179 (Ile).
[0013] Within a second aspect, the present invention provides an
expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment encoding a
cytokine polypeptide as shown in SEQ ID NO: 3 from amino acid
number 23 (Pro), to amino acid number 167 (Ile); and a
transcription terminator, wherein the promoter is operably linked
to the DNA segment, and the DNA segment is operably linked to the
transcription terminator. In one embodiment, the expression vector
disclosed above further comprises a secretory signal sequence
operably linked to the DNA segment.
[0014] Within a third aspect, the present invention provides a
cultured cell comprising an expression vector according as
disclosed above, wherein the cell expresses a polypeptide encoded
by the DNA segment.
[0015] Within a fourth aspect, the present invention provides a DNA
construct encoding a fusion protein, the DNA construct comprising:
a first DNA segment encoding a polypeptide comprising a sequence of
amino acid residues selected from the group consisting of: (a) the
amino acid sequence as shown in SEQ ID NO: 3 from amino acid number
1 (Met), to amino acid number 21 (Ala); (b) the amino acid sequence
as shown in SEQ ID NO: 3 from amino acid number 41 (Thr), to amino
acid number 53 (Leu); (c) the amino acid sequence as shown in SEQ
ID NO: 3 from amino acid number 80 (Met), to amino acid number 91
(Val); (d) the amino acid sequence as shown in SEQ ID NO: 3 from
amino acid number 103 (Gln), to amino acid number 116 (Arg); (e)
the amino acid sequence as shown in SEQ ID NO: 3 from amino acid
number 149 (Ile), to amino acid number 162 (Leu); and (f) the amino
acid sequence as shown in SEQ ID NO: 3 from amino acid number 23
(Pro), to amino acid number 167 (Ile); and at least one other DNA
segment encoding an additional polypeptide, wherein the first and
other DNA segments are connected in-frame; and wherein the first
and other DNA segments encode the fusion protein.
[0016] Within another aspect, the present invention provides an
expression vector comprising the following operably linked
elements: a transcription promoter; a DNA construct encoding a
fusion protein as disclosed above; and a transcription terminator,
wherein the promoter is operably linked to the DNA construct, and
the DNA construct is operably linked to the transcription
terminator.
[0017] Within another aspect, the present invention provides a
cultured cell comprising an expression vector as disclosed above,
wherein the cell expresses a polypeptide encoded by the DNA
construct.
[0018] Within another aspect, the present invention provides a
method of producing a fusion protein comprising: culturing a cell
according as disclosed above; and isolating the polypeptide
produced by the cell.
[0019] Within another aspect, the present invention provides an
isolated cytokine polypeptide comprising a sequence of amino acid
residues that is at least 90% identical to an amino acid sequence
selected from the group consisting of: (a) the amino acid sequence
as shown in SEQ ID NO: 3 from amino acid number 23 (Pro), to amino
acid number 167 (Ile); (b) the amino acid sequence as shown in SEQ
ID NO: 3 from amino acid number 1 (Met), to amino acid number 167
(Ile); and (c) the amino acid sequence as shown in SEQ ID NO: 2
from amino acid number 1 (Met), to amino acid number 179 (Ile); and
wherein the polypeptide produced by the cell induces proliferation
of cells expressing a receptor for the polypeptide comprising
zcytor11 (SEQ ID NO: 19) or induces cytotoxicity in K562 cells. In
one embodiment, the isolated polypeptide disclosed above comprises
a sequence of amino acid residues selected from the group
consisting of: (a) the amino acid sequence as shown in SEQ ID NO: 3
from amino acid number 23 (Pro), to amino acid number 167 (Ile);
(b) the amino acid sequence as shown in SEQ ID NO: 3 from amino
acid number 1 (Met), to amino acid number 167 (Ile); and (c) the
amino acid sequence as shown in SEQ ID NO: 2 from amino acid number
1 (Met), to amino acid number 179 (Ile).
[0020] Within another aspect, the present invention provides a
method of producing a cytokine polypeptide comprising: culturing a
cell as disclosed above; and isolating the cytokine polypeptide
produced by the cell.
[0021] Within another aspect, the present invention provides a
method of producing an antibody to a polypeptide comprising:
inoculating an animal with a polypeptide selected from the group
consisting of: (a) a polypeptide consisting of 30 to 144 amino
acids, wherein the polypeptide is identical to a contiguous
sequence of amino acids in SEQ ID NO: 3 from amino acid number 23
(Gly) to amino acid number 779 (Thr); (b) a polypeptide as
disclosed above; (c) a polypeptide consisting of the amino acid
sequence of SEQ ID NO: 3 from amino acid number 29 (Arg) to amino
acid number 34 (Asn); (d) a polypeptide consisting of the amino
acid sequence of SEQ ID NO: 3 from amino acid number 121 (His) to
amino acid number 126 (Asp); (e) a polypeptide consisting of the
amino acid sequence of SEQ ID NO: 3 from amino acid number 134
(Gln) to amino acid number 139 (Thr); (f) a polypeptide consisting
of the amino acid sequence of SEQ ID NO: 3 from amino acid number
137 (Lys) to amino acid number 142 (Lys); (g) a polypeptide
consisting of the amino acid sequence of SEQ ID NO: 3 from amino
acid number 145 (Glu) to amino acid number 150 (Lys); (h) a
polypeptide consisting of the amino acid sequence of SEQ ID NO: 3
from amino acid number 41 (Thr), to amino acid number 53 (Leu); (i)
a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3
from amino acid number 80 (Met) to amino acid number 91 (Val); (j)
a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3
from amino acid number 103 (Met) to amino acid number 116 (Arg);
(k) a polypeptide consisting of the amino acid sequence of SEQ ID
NO: 3 from amino acid number 149 (Ile) to amino acid number 162
(Leu); and wherein the polypeptide elicits 25 an immune response in
the animal to produce the antibody; and isolating the antibody from
the animal.
[0022] Within another aspect, the present invention provides an
antibody produced by the method as disclosed above, which binds to
a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment,
the antibody disclosed above is a monoclonal antibody. Within
another aspect, the present invention provides an antibody which
specifically binds to a polypeptide as disclosed above.
[0023] Within another aspect, the present invention provides a
method of detecting, in a test sample, the presence of an
antagonist of ZCYTO18 protein activity, comprising: culturing a
cell that is responsive to a ZCYTO18-stimulated cellular pathway;
and producing a polypeptide by the method as disclosed above; and
exposing the polypeptide to the cell, in the presence and absence
of a test sample; and comparing levels of response to the
polypeptide, in the presence and absence of the test sample, by a
biological or biochemical assay; and determining from the
comparison, the presence of the antagonist of ZCYTO 18 activity in
the test sample.
[0024] Within another aspect, the present invention provides a
method of detecting, in a test sample, the presence of an agonist
of ZCYTO18 protein activity, comprising: culturing a cell that is
responsive to a ZCYTO18-stimulated cellular pathway; and adding a
test sample; and comparing levels of response in the presence and
absence of the test sample, by a biological or biochemical assay;
and determining from the comparison, the presence of the agonist of
ZCYTO18 activity in the test sample.
[0025] Within another aspect, the present invention provides a
method for detecting a genetic abnormality in a patient,
comprising: obtaining a genetic sample from a patient; producing a
first reaction product by incubating the genetic sample with a
polynucleotide comprising at least 14 contiguous nucleotides of SEQ
ID NO: 1 or the complement of SEQ ID NO: 1, under conditions
wherein said polynucleotide will hybridize to complementary
polynucleotide sequence; visualizing the first reaction product;
and comparing said first reaction product to a control reaction
product from a wild type patient, wherein a difference between said
first reaction product and said control reaction product is
indicative of a genetic abnormality in the patient.
[0026] Within another aspect, the present invention provides a
method for detecting a cancer in a patient, comprising: obtaining a
tissue or biological sample from a patient; incubating the tissue
or biological sample with an antibody as disclosed above under
conditions wherein the antibody binds to its complementary
polypeptide in the tissue or biological sample; visualizing the
antibody bound in the tissue or biological sample; and comparing
levels of antibody bound in the tissue or biological sample from
the patient to a normal control tissue or biological sample,
wherein an increase or decrease in the level of antibody bound to
the patient tissue or biological sample relative to the normal
control tissue or biological sample is indicative of a cancer in
the patient.
[0027] Within another aspect, the present invention provides a
method for detecting a cancer in a patient, comprising: obtaining a
tissue or biological sample from a patient; labeling a
polynucleotide comprising at least 14 contiguous nucleotides of SEQ
ID NO: 1 or the complement of SEQ ID NO: 1;incubating the tissue or
biological sample with under conditions wherein the polynucleotide
will hybridize to complementary polynucleotide sequence;
visualizing the labeled polynucleotide in the tissue or biological
sample; and comparing the level of labeled polynucleotide
hybridization in the tissue or biological sample from the patient
to a normal control tissue or biological sample, wherein an
increase or decrease in the labeled polynucleotide hybridization to
the patient tissue or biological sample relative to the normal
control tissue or biological sample is indicative of a cancer in
the patient.
[0028] Within another aspect, the present invention provides a
method of killing cancer cells comprising, obtaining ex vivo a
tissue or biological sample containing cancer cells from a patient,
or identifying cancer cells in vivo; producing a polypeptide by the
method as disclosed above; formulating the polypeptide in a
pharmaceutically acceptable vehicle; and administering to the
patient or exposing the cancer cells to the polypeptide; wherein
the polypeptide kills the cells. In one embodiment, the method of
killing cancer cells is as disclosed above, wherein the polypeptide
is further conjugated to a toxin.
[0029] Within another aspect, the present invention provides a
method of increasing platelets in a patient or injured tissue,
producing a polypeptide by the method as disclosed above;
administering the polypeptide to the patient or injured tissue in a
pharmaceutically acceptable vehicle, wherein the polypeptide
increases the level pf platelets in the patient or injured
tissue.
[0030] Within another aspect, the present invention provides a
method for detecting inflammation in a patient, comprising:
obtaining a tissue or biological sample from a patient; incubating
the tissue or biological sample with an antibody as disclosed above
under conditions wherein the antibody binds to its complementary
polypeptide in the tissue or biological sample; visualizing the
antibody bound in the tissue or biological sample; and comparing
levels of antibody bound in the tissue or biological sample from
the patient to a normal control tissue or biological sample,
wherein an increase in the level of antibody bound to the patient
tissue or biological sample relative to the normal control tissue
or biological sample is indicative of inflammation in the
patient.
[0031] Within another aspect, the present invention provides a
method for detecting inflammation in a patient, comprising:
obtaining a tissue or biological sample from a patient; labeling a
polynucleotide comprising at least 14 contiguous nucleotides of SEQ
ID NO: 1 or the complement of SEQ ID NO: 1; incubating the tissue
or biological sample with under conditions wherein the
polynucleotide will hybridize to complementary polynucleotide
sequence; visualizing the labeled polynucleotide in the tissue or
biological sample; and comparing the level of labeled
polynucleotide hybridization in the tissue or biological sample
from the patient to a normal control tissue or biological sample,
wherein an increase in the labeled polynucleotide hybridization to
the patient tissue or biological sample relative to the normal
control tissue or biological sample is indicative of inflammation
in the patient.
[0032] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention.
[0033] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0034] 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.).
[0035] 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.
[0036] 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.
[0037] 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-complem- ent pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0038] 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'.
[0039] 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.
[0040] 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).
[0041] 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.
[0042] 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).
[0043] 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.
[0044] 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.
[0045] 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.
[0046] "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.
[0047] 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.
[0048] 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".
[0049] "Probes and/or primers" as used herein can be RNA or DNA.
DNA can be either cDNA or genomic DNA. Polynucleotide probes and
primers are single or double-stranded DNA or RNA, generally
synthetic oligonucleotides, but may be generated from cloned cDNA
or genomic sequences or its complements. Analytical probes will
generally be at least 20 nucleotides in length, although somewhat
shorter probes (14-17 nucleotides) can be used. PCR primers are at
least 5 nucleotides in length, preferably 15 or more nt, more
preferably 20-30 nt. Short polynucleotides can be used when a small
region of the gene is targeted for analysis. For gross analysis of
genes, a polynucleotide probe may comprise an entire exon or more.
Probes can be labeled to provide a detectable signal, such as with
an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,
paramagnetic particle and the like, which are commercially
available from many sources, such as Molecular Probes, Inc.,
Eugene, Oreg., and Amersham Corp., Arlington Heights, Ill., using
techniques that are well known in the art.
[0050] 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.
[0051] 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.
[0052] 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, L-3 receptor, GM-CSF
receptor, G-CSF receptor, erythropoietin receptor and IL-6
receptor).
[0053] 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.
[0054] 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.
[0055] 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 .+-.10%.
[0056] All references cited herein are incorporated by reference in
their entirety.
[0057] The present invention is based in part upon the discovery of
a novel DNA sequence that encodes a protein having the structure of
a four-helical-bundle cytokine. Through processes of cloning, and
expression studies described herein, a polynucleotide sequence
encoding a novel ligand polypeptide has been identified. This
polypeptide ligand, designated ZCYTO18, was isolated from T-cell
cDNA library and mixed lymphocyte reaction (MLR) cDNA and is
expressed in 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. Based on Northern and
RT-PCR analysis, ZCYTO18 polynucleotides are expressed in T-cells,
activated T- and B- cells, and lymphoid tissue. The human ZCYTO18
nucleotide sequence is represented in SEQ ID NO: 1.
[0058] Analysis of SEQ ID NO: 1 reveals that there are two possible
initiation Methionine residues for a ZCYTO18 cytokine polypeptide
translated therefrom. The two deduced ZCYTO18 polypeptide amino
acid sequences are shown in SEQ ID NO: 2 (a 179 amino acid
polypeptide having the initiating Met at nucleotide 21 in SEQ ID
NO: 1) and SEQ ID NO: 3 (a 167 amino acid polypeptide having the
initiating Met at nucleotide 57 in SEQ ID NO: 1). Although both of
these sequences encode a ZCYTO18 polypeptide, based on similarity
of the ZCYTO18 sequence to IL-10 and other cytokines, and the
presence of a strong signal sequence, SEQ ID NO: 3 encodes a fully
functional secreted cytokine polypeptide.
[0059] Sequence analysis of the deduced amino acid sequence as
represented in SEQ ID NO: 3 indicates a 167 amino acid polypeptide
containing a 22 amino acid residue secretory signal sequence (amino
acid residues 1 (Met) to 21 (Ala) of SEQ ID NO: 3), and a mature
polypeptide of 146 amino acids (amino acid residues 22 (Ala) to 167
(Ile) of SEQ ID NO: 3). N-terminal sequence shows that the mature
start at residue 22 (Ala) of SEQ ID NO: 3 or 34 (Ala) of SEQ ID NO:
2.
[0060] In general, cytokines are predicted to have a four-alpha
helix structure, with the 1.sup.st and 4.sup.th helices being most
important in ligand-receptor interactions. The 1.sup.st and
4.sup.th helices are more highly conserved among members of the
family. Referring to the human ZCYTO18 amino acid sequence shown in
SEQ ID NO: 3, alignment of human ZCYTO18, human IL-10, human
zcyto10 (WO US98/25228), and human Human MDA7 (Genbank Accession
No. Q13007) amino acid sequences suggests that ZCYTO18 helix A is
defined by amino acid residues 41 (Thr) to 53 (leu) of SEQ ID NO:
3; helix B by amino acid residues 80 (Met) to 91 (Val) of SEQ ID
NO: 3; helix C by amino acid residues 103 (Met) to 116 (Arg) of SEQ
ID NO: 3; and helix D by amino acid residues 149 (Ile) to 162 Leu)
of SEQ ID NO: 3. 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. Four
cysteine residues are conserved between IL-10 and ZCYTO18
corresponding to amino acid residues 8, 28, 77 and 120 of SEQ ID
NO: 3. Consistent cysteine placement is further confirmation of the
four-helical-bundle structure.
[0061] The corresponding polynucleotides encoding the ZCYTO18
polypeptide regions, domains, motifs, residues and sequences
described herein are as shown in SEQ ID NO: 1. Moreover, the
corresponding ZCYTO18 polypeptide regions, domains, motifs,
residues and sequences described herein are also as shown in SEQ ID
NO: 2.
[0062] 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. Zcyto18 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 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). Using similar methods,
putative regions conferring receptor binding specificity in ZCYTO18
comprise the regions of amino acid residues of SEQ ID NO: 3 that
include: residues 53-60, residues 85-91, and residues 121-140.
These regions will be useful for preparing chimeric molecules,
particularly with other short-helix form cytokines to determine and
modulate receptor binding specificity.
[0063] Subsequent to filing, ZCYTO18 was annotated in the
literature as IL-TIF. Oreover, receptors for ZCYTO18 were
identified comprising zcytor16 (SEQ ID NO: 32, and SEQ ID NO: 33)
((commonly owned PCT International Application No. [#######])),
zcytor11 (SEQ ID NO: 18, and SEQ ID NO: 19) (Commonly owned U.S.
Pat. No. 5,965,704), and CRF2-4 (Genbank Accession No. Z17227).
Moreover several ZCYTO18 responsive cell lines have been identified
(Dumontier et al., J. Immunol. 164:1814-1819, 2000; Dumoutier, L.
et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; Xie MH et
al., J. Biol. Chem. 275: 31335-31339, 2000; Kotenko SV et al., JBC
in press), as well as those that express the ZCYTO18 receptor
subunit zcytor11. Moreover, commonly owned zcytor16 receptor was
shown to bind ZCYTO18 and antagonize its activity (SEQ ID NO: 3)
(commonly owned PCT International Application No. [#######]); the
mouse IL-TIF (ZCYTO18) sequence is shown in Dumontier et al., J.
Immunol. 164:1814-1819, 2000), and was independently cloned,
designated, mouse ZCYTO18 herein, and is shown in SEQ ID NO: 37 and
corresponding plypeptide sequence shown in SEQ ID NO: 38. Moreover,
commonly owned zcytor11 (U.S. Pat. No. 5,965,704) and CRF2-4
receptor also bind ZCYTO18 (See, WIPO publication WO 00/24758;
Dumontier et al., J. Immunol. 164:1814-1819, 2000; Spencer, S D et
al., J. Exp. Med. 187:571-578, 1998; Gibbs, V C and Pennica Gene
186:97-101, 1997 (CRF2-4 cDNA); Xie, M H et al., J. Biol. Chem.
275: 31335-31339, 2000; and Kotenko, S V et al., J. Biol. Chem.
manuscript in press M007837200). Moreover, IL-10.beta. receptor may
be involved as a receptor for ZCYTO18, and it is believed to be
synonymous with CRF2-4 (Dumoutier, L. et al., Proc. Nat'l. Acad.
Sci. 15 97:10144-10149, 2000; Liu Y et al, J Immunol. 152;
1821-1829, 1994 (IL-10R cDNA). These receptors are discussed herein
in relation to the uses of ZCTYTO18.
[0064] The present invention provides polynucleotide molecules,
including DNA and RNA molecules, that encode the ZCYTO18
polypeptides disclosed herein. Those skilled in the art will
readily recognize that, in view of the degeneracy of the genetic
code, considerable sequence variation is possible among these
polynucleotide molecules. SEQ ID NO: 4 is a degenerate DNA sequence
that encompasses all DNAs that encode the ZCYTO18 polypeptide of
SEQ ID NO: 3. Those skilled in the art will recognize that the
degenerate sequence of SEQ ID NO: 4 also provides all RNA sequences
encoding SEQ ID NO: 3 by substituting U for T. Thus, ZCYTO18
polypeptide-encoding polynucleotides comprising nucleotide 1 or 66
to nucleotide 501 of SEQ ID NO: 4 and their RNA equivalents are
contemplated by the present invention. Table 1 sets forth the
one-letter codes used within SEQ ID NO: 4 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, with A being
complementary to T, and G being complementary to C.
1TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G
G G G C C T T A A R A.vertline.G Y C.vertline.T Y C.vertline.T R
A.vertline.G M A.vertline.C K G.vertline.T K G.vertline.T M
A.vertline.C S G.vertline.G S G.vertline.G W A.vertline.T W AlT H
A.vertline.C.vertline.T D A.vertline.G.vertline.T B
C.vertline.G.vertline.T V A.vertline.C.vertline.G V
A.vertline.C.vertline.G B C.vertline.G.vertline.T D
A.vertline.G.vertline.T H A.vertline.C.vertline.T N
A.vertline.C.vertline.G.vertlin- e.T N
A.vertline.C.vertline.G.vertline.T
[0065] The degenerate codons used in SEQ ID NO: 4, encompassing all
possible codons for a given armino acid, are set forth in Table
2.
2TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C
TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT
ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA
GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG
GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG
CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L
CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT
TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter TAA TAG TGA TRR
Asn.vertline.Asp B RAY Glu.vertline.Gln Z SAR Any X NNN
[0066] 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 sequence of SEQ ID NO: 3.
Variant sequences can be readily tested for functionality as
described herein.
[0067] 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 2). For
example, the amino acid Threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequence
disclosed in SEQ ID NO: 4 serves as a template for optimizing
expression of polynucleotides in various cell types and species
commonly used in the art and disclosed herein. Sequences containing
preferential codons can be tested and optimized for expression in
various species, and tested for functionality as disclosed
herein.
[0068] 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 ZCYTO18 RNA. Such
tissues and cells are identified by Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA 77:5201, 1980), reverse transcriptase
PCR (RT-PCR) or by screening conditioned medium from various cell
types for activity on target cells or tissue. Once the activity or
RNA producing cell or tissue is identified, 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 ZCYTO18
polypeptides are then identified and isolated by, for example,
hybridization or PCR.
[0069] A full-length clone encoding ZCYTO18 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 ZCYTO18 fragments, or other specific binding
partners.
[0070] Zcyto18 polynucleotide sequences disclosed herein can also
be used as probes or primers to clone 5' non-coding regions of a
ZCYTO18 gene. In view of the tissue-specific expression observed
for ZCYTO18 by Northern blotting and RT PCR (See, Examples 2 and
3), this gene region is expected to provide for hematopoietic- and
lymphoid-specific expression. Promoter elements from a ZCYTO18 gene
could thus be used to direct the tissue-specific expression of
heterologous genes in, for example, transgenic animals or patients
treated with gene therapy. Cloning of 5' flanking sequences also
facilitates production of ZCYTO18 proteins by "gene activation" as
disclosed in U.S. Pat. No. 5,641,670. Briefly, expression of an
endogenous ZCYTO18 gene in a cell is altered by introducing into
the ZCYTO18 locus a DNA construct comprising at least a targeting
sequence, a regulatory sequence, an exon, and an unpaired splice
donor site. The targeting sequence is a ZCYTO18 5' non-coding
sequence that permits homologous recombination of the construct
with the endogenous ZCYTO18 locus, whereby the sequences within the
construct become operably linked with the endogenous ZCYTO18 coding
sequence. In this way, an endogenous ZCYTO18 promoter can be
replaced or supplemented with other regulatory sequences to provide
enhanced, tissue-specific, or otherwise regulated expression.
[0071] 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 ZCYTO18
polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human ZCYTO18 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 ZCYTO18 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
ZCYTO18-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 the polymerase chain
reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primers
designed from the representative human ZCYTO18 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 ZCYTO18 polypeptide,
binding studies or activity assays. Similar techniques can also be
applied to the isolation of genomic clones. Example 5 shows that a
ZCYTO18 ortholog is present in mouse genomic DNA.
[0072] A polynucleotide sequence for the mouse ortholog of human
ZCYTO18 has been identified and is shown in SEQ ID NO: 37 and the
corresponding amino acid sequence shown in SEQ ID NO: 38. Analysis
of the mouse ZCYTO18 polypeptide encoded by the DNA sequence of SEQ
ID NO: 37 revealed an open reading frame encoding 179 amino acids
(SEQ ID NO: 38) comprising a predicted secretory signal peptide of
33 amino acid residues (residue 1 (Met) to residue 33 (Ala) of SEQ
ID NO: 38), and a mature polypeptide of 146 amino acids (residue 34
(Leu) to residue 179 (Val) of SEQ ID NO: 38). ZCYTO18 helix A is
defined by amino acid residues 53 to 65 of SEQ ID NO: 38; helix B
by amino acid residues 92 to 103 of SEQ ID NO: 38; helix C by amino
acid residues 115 to 124 of SEQ ID NO: 38; and helix D by amino
acid residues 161 to 174 of SEQ ID NO: 38. Four conserved cysteine
residues in mouse ZCYTO18 are conserved with the human sequence
corresponding to amino acid residues 20, 40, 89; and 132 of SEQ ID
NO: 38. Moreover, in the mouse sequence altenative starting
Methionine residues exist at postitions 8 and 13 as shown in SEQ ID
NO: 38, but the signal peptide cleavage after residue 33 (Ala)
would still result in the 146 amino acid mature sequence as
described above. The mature sequence for the mouse ZCYTO18 begins
at Leu.sub.34 (as shown in SEQ ID NO: 38), which corresponds to
Ala.sub.22 (as shown in SEQ ID NO: 3) in the human sequence. There
is about 78% identity between the mouse and human sequences over
the entire amino acid sequence corresponding to SEQ ID NO: 3 and
SEQ ID NO: 38. The above percent identities were determined using a
FASTA program with ktup=1, gap opening penalty=12, gap extension
penalty=2, and substitution matrix=BLOSUM62, with other FASTA
parameters set as default. The corresponding polynucleotides
encoding the mouse ZCYTO18 polypeptide regions, domains, motifs,
residues and sequences described above are as shown in SEQ ID NO:
37.
[0073] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO: 1 represents a single allele of human
ZCYTO18 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: 1, 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: 3. cDNAs
generated from alternatively spliced mRNAs, which retain the
properties of the ZCYTO18 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.
[0074] Moreover, the genomic structure of ZCYTO18 is readily
determined by one of skill in the art by comparing the cDNA
sequence of SEQ ID NO: 1 and the translated amino acid of SEQ ID
NO: 3 or SEQ ID NO: 2 with the genomic DNA in which the gene is
contained (e.g, Genbank Accession No. AC007458). For example, such
analysis can be readily done using FASTA as described herein. As
such, the intron and exon junctions in this region of genomic DNA
can be determined for the ZCYTO18 gene. Thus, the present invention
includes the ZCYTO18 gene as located in human genomic DNA. Based on
annotation of a fragment of human genomic DNA containing a part of
ZCYTO18 genomic DNA (Genbank Accession No. AC007458), ZCYTO18 is
located at the 12q15 region of chromosome 12.
[0075] Within preferred embodiments of the invention, isolated
ZCYTO18-encoding nucleic acid molecules can hybridize under
stringent conditions to nucleic acid molecules having the
nucleotide sequence of SEQ ID NO: 1, to nucleic acid molecules
having the nucleotide sequence of nucleotides 87 to 587 of SEQ ID
NO: 1, or to nucleic acid molecules having a nucleotide sequence
complementary to SEQ ID NO: 1. In general, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH.
[0076] The T.sub.m is the temperature (under defined ionic strength
and pH) at which 50% of the target sequence hybridizes to a
perfectly matched probe.
[0077] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Stringent hybridization conditions encompass
temperatures of about 5-25.degree. C. below the T.sub.m of the
hybrid and a hybridization buffer having up to 1 M Na.sup.+. Higher
degrees of stringency at lower temperatures can be achieved with
the addition of formamide which reduces the T.sub.m of the hybrid
about 1.degree. C. for each 1% formamide in the buffer solution.
Generally, such stringent conditions include temperatures of
20-70.degree. C. and a hybridization buffer containing up to
6.times.SSC and 0-50% formamide. A higher degree of stringency can
be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having up to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 1.times.SSC and 0-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0078] The above conditions are meant to serve as a guide, and it
is well within the abilities of one skilled in the art to adapt
these conditions for use with a particular polynucleotide hybrid.
The T.sub.m for a specific target sequence is the temperature
(under defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those conditions
which influence the T.sub.m include, the size and base pair content
of the polynucleotide probe, the ionic strength of the
hybridization solution, and the presence of destabilizing agents in
the hybridization solution. Numerous equations for calculating
T.sub.m are known in the art, and are specific for DNA, RNA and
DNA-RNA hybrids and polynucleotide probe sequences of varying
length (see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989);
Ausubel et al., (eds.), Current Protocols in Molecular Biology
(John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide
to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and
Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence
analysis software such as OLIGO 6.0 (LSR; Long Lake, Minn.) and
Primer Premier 4.0 (Premier Biosoft International; Palo Alto,
Calif.), as well as sites on the Internet, are available tools for
analyzing a given sequence and calculating T.sub.m based on user
defined criteria. Such programs can also analyze a given sequence
under defined conditions and identify suitable probe sequences.
Typically, hybridization of longer polynucleotide sequences, >50
base pairs, is performed at temperatures of about 20-25.degree. C.
below the calculated T.sub.m. For smaller probes, <50 base
pairs, hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below the calculated T.sub.m. This allows for the
maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.
[0079] The length of the polynucleotide sequence influences the
rate and stability of hybrid formation. Smaller probe sequences,
<50 base pairs, reach equilibrium with complementary sequences
rapidly, but may form less stable hybrids. Incubation times of
anywhere from minutes to hours can be used to achieve hybrid
formation. Longer probe sequences come to equilibrium more slowly,
but form more stable complexes, even at lower temperatures. In such
cases, incubations are allowed to proceed overnight or longer.
Generally, incubations are carried out for a period equal to three
times the calculated Cot time. Cot time, the time it takes for the
polynucleotide sequences to reassociate, can be calculated for a
particular sequence by methods known in the art.
[0080] The base pair composition of a polynucleotide sequence will
affect the thermal stability of its hybrid complex, thereby
influencing the choice of hybridization temperature and the ionic
strength of the hybridization buffer. A-T pairs are less stable
than G-C pairs in aqueous solutions containing sodium chloride.
Therefore, the higher the G-C content, the more stable the hybrid.
Even distribution of G and C residues within the sequence also
contributes positively to hybrid stability. In addition, the base
pair composition can be manipulated to alter the T.sub.m of a given
sequence. For example, 5-methyldeoxycytidine can be substituted for
deoxycytidine and 5-bromodeoxuridine can be substituted for
thymidine to increase the Tm, whereas 7-deazzo-2'-deoxyguanosine
can be substituted for guanosine to reduce dependence on
T.sub.m.
[0081] The ionic concentration of the hybridization buffer also
affects the stability of the hybrid. Hybridization buffers
generally contain blocking agents such as Denhardt's solution
(Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA,
tRNA, milk powders (BLOTTO), heparin or SDS, and a Na.sup.+ source,
such as SSC (1.times.SSC: 0.15 M sodium chloride, 15 mM sodium
citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). By decreasing the ionic
concentration of the buffer, the stability of the hybrid is
increased. Typically, hybridization buffers contain from between 10
mM -1 M Na.sup.+. The addition of destabilizing or denaturing
agents such as formamide, tetralkylammonium salts, guanidinium
cations or thiocyanate cations to the hybridization solution will
alter the T.sub.m of a hybrid. Typically, formamide is used at a
concentration of up to 50% to allow incubations to be carried out
at more convenient and lower temperatures. Formamide also acts to
reduce non-specific background when using RNA probes.
[0082] As an illustration, a nucleic acid molecule encoding a
variant ZCYTO18 polypeptide can be hybridized with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO: 1 (or its
complement) at 42.degree. C. overnight in a solution comprising 50%
formamide, 5.times.SSC (1.times.SSC: 0.15 M sodium chloride and 15
mM sodium citrate), 50 mM sodium phosphate (pH 7.6),
5.times.Denhardt's solution (100.times.Denhardt's solution: 2%
(w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v)
bovine serum albumin), 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA. One of skill in the art can
devise variations of these hybridization conditions. For example,
the hybridization mixture can be incubated at a higher temperature,
such as about 65.degree. C., in a solution that does not contain
formamide. Moreover, premixed hybridization solutions are available
(e.g., EXPRESSHYB Hybridization Solution from CLONTECH
Laboratories, Inc.), and hybridization can be performed according
to the manufacturer's instructions.
[0083] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. That is, nucleic acid molecules encoding a variant
ZCYTO18 polypeptide hybridize with a nucleic acid molecule having
the nucleotide sequence of SEQ ID NO: 1 (or its complement) under
stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree.
C., including 0.5.times.SSC with 0.1% SDS at 55.degree. C., or
2.times.SSC with 0.1% SDS at 65.degree. C. One of skill in the art
can readily devise equivalent conditions, for example, by
substituting SSPE for SSC in the wash solution.
[0084] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. In other words, nucleic acid
molecules encoding a variant ZCYTO18 polypeptide hybridize with a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:
1 (or its complement) under highly stringent washing conditions, in
which the wash stringency is equivalent to 0.1.times.-0.2.times.SSC
with 0.1% SDS at 50-65.degree. C., including 0.1.times.SSC with
0.1% SDS at 50.degree. C., or 0.2.times.SSC with 0.1% SDS at
65.degree. C.
[0085] The present invention also provides isolated ZCYTO18
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO: 3, or their orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides comprising at least 70%, at least 80%, at least 90%,
at least 95%, or greater than 95% sequence identity to the
sequences shown in SEQ ID NO: 3, or their orthologs. The present
invention also includes polypeptides that comprise an amino acid
sequence having at least 70%, at least 80%, at least 90%, at least
95% or greater than 95% sequence identity to the sequence of amino
acid residues 1 to 167, or 23 to 167 of SEQ ID NO: 3; or amino acid
residues 1 to 179, or 35 to 179 of SEQ ID NO: 2. The present
invention further includes nucleic acid molecules that encode such
polypeptides. Methods for determining percent identity are
described below.
[0086] The present invention also contemplates variant ZCYTO18
nucleic acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NO: 3, and/or a
hybridization assay, as described above. Such ZCYTO18 variants
include nucleic acid molecules: (1) that hybridize with a nucleic
acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or
its complement) under stringent washing conditions, in which the
wash stringency is equivalent to 0.5.times.-2.times.SSC with 0.1%
SDS at 55-65.degree. C; or (2) that encode a polypeptide having at
least 70%, at least 80%, at least 90%, at least 95% or greater than
95% sequence identity to the amino acid sequence of 30 SEQ ID NO:
3. Alternatively, ZCYTO18 variants can be characterized as nucleic
acid molecules: (1) that hybridize with a nucleic acid molecule
having the nucleotide sequence of SEQ ID NO: 1 (or its complement)
under highly stringent washing conditions, in which the wash
stringency is equivalent to 0.1.times.-0.2.times.SSC with 0.1% SDS
at 50-65.degree. C.; and (2) that encode a polypeptide having at
least 70%, at least 80%, at least 90%, at least 95% or greater than
95% sequence identity to the amino acid sequence of SEQ ID NO:
3.
[0087] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). 1 Total number of
identical matches [ length of the longer sequence plus the number
of gaps introduced into the longer sequence in order to align the
two sequences ] .times. 100
3 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3
-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1
-1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3
-2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3
-3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0088] 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 ZCYTO18. 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).
[0089] Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID NO:
3) 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=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0090] 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.
[0091] Variant ZCYTO18 polypeptides or polypeptides with
substantially similar sequence identity 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 4) 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 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 of from about 110 to 180 amino acid
residues that comprise a sequence that is at least 70%, preferably
at least 90%, and more preferably 95% or more identical to the
corresponding region of SEQ ID NO: 3. Polypeptides comprising
affinity tags can further comprise a proteolytic cleavage site
between the ZCYTO18 polypeptide and the affinity tag. Preferred
such sites include thrombin cleavage sites and factor Xa cleavage
sites.
4TABLE 4 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
[0092] Determination of amino acid residues that comprise 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, 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.
[0093] Amino acid sequence changes are made in ZCYTO18 polypeptides
so as to minimize disruption of higher order structure essential to
biological activity. For example, when the ZCYTO18 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, an active site, or 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 cysteine 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 structurally similarities between proteins and
polypeptides (Schaanan et al., Science 257:961-964, 1992).
[0094] A Hopp/Woods hydrophilicity profile of the ZCYTO18 protein
sequence as shown in SEQ ID NO: 3 can be generated (Hopp et al.,
Proc. Natl. Acad. Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth.
88:1-18, 1986 and Triquier et al., Protein Engineering 11:153-169,
1998). The profile is based on a sliding six-residue window. Buried
G, S, and T residues and exposed H, Y, and W residues were ignored.
For example, in ZCYTO18, hydrophilic regions include: (1) amino
acid number 29 (Arg) to amino acid number 34 (Asn) of SEQ ID NO: 3;
(2) amino acid number 121 (His) to amino acid number 126 (Asp) of
SEQ ID NO: 3; (3) amino acid number 134 (Gln) to amino acid number
139 (Thr) of SEQ ID NO: 3; (4) amino acid number 137 (Lys) to amino
acid number 142 (Lys) of SEQ ID NO: 3; and (5) amino acid number
145 (Glu) to amino acid number 150 (Lys) of SEQ ID NO: 2.
[0095] 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 ZCYTO18 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: 3. Cysteine residues at positions 8, 27, 77 and
120 of SEQ ID NO: 3, will be relatively intolerant of
substitution.
[0096] The identities of essential amino acids can also be inferred
from analysis of sequence similarity between IL-10, zcyto10, and
MDA7 with ZCYTO18. 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
ZCYTO18 polynucleotide on the basis of structure is to determine
whether a nucleic acid molecule encoding a potential variant
ZCYTO18 gene can hybridize to a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO: 1, as discussed above.
[0097] 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).
[0098] The present invention also includes functional fragments of
ZCYTO18 polypeptides and nucleic acid molecules encoding such
functional fragments. A "functional" ZCYTO18 or fragment thereof as
defined herein is characterized by its proliferative or
differentiating activity, by its ability to induce or inhibit
specialized cell functions, or by its ability to bind specifically
to an anti-ZCYTO18 antibody, cell, or ZCYTO18 receptor (either
soluble or immobilized). As previously described herein, ZCYTO18 is
characterized by a four-helical-bundle structure comprising helix A
(amino acid residues 41-53), helix B (amino acid residues 80-91),
helix C (amino acid residues 103-116) and helix D (amino acid
residues 149-162), as shown in SEQ ID NO: 3. Thus, the present
invention further provides fusion proteins encompassing: (a)
polypeptide molecules comprising one or more of the helices
described above; and (b) functional fragments comprising one or
more of these helices. The other polypeptide portion of the fusion
protein may be contributed by another four-helical-bundle cytokine,
such as IL-10, zcyto10, MDA7, IL-15, IL-2, IL-4 and GM-CSF, or by a
non-native and/or an unrelated secretory signal peptide that
facilitates secretion of the fusion protein.
[0099] Routine deletion analyses of nucleic acid molecules can be
performed to obtain functional fragments of a nucleic acid molecule
that encodes a ZCYTO18 polypeptide. As an illustration, DNA
molecules having the nucleotide sequence of SEQ ID NO: 1 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 ZCYTO18 activity, or for
the ability to bind anti-ZCYTO18 antibodies or ZCYTO18 receptor.
One alternative to exonuclease digestion is to use
oligonucleotide-directed mutagenesis to introduce deletions or stop
codons to specify production of a desired ZCYTO18 fragment.
Alternatively, particular fragments of a ZCYTO18 gene can be
synthesized using the polymerase chain reaction.
[0100] 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
(Nijhoff 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.,
[0101] Biochem. Pharmacol. 50:1295 (1995); and Meisel et al., Plant
Molec. Biol. 30:1 (1996).
[0102] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204), and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)).
[0103] Variants of the disclosed ZCYTO18 nucleotide and polypeptide
sequences can also be generated through DNA shuffling as disclosed
by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Natl Acad. Sci.
USA 91:10747 (1994), and international publication No. WO 97/20078.
Briefly, variant DNA molecules are generated by in vitro homologous
recombination by random fragmentation of a parent DNA followed by
reassembly using PCR, resulting in randomly introduced point
mutations. This technique can be modified by using a family of
parent DNA molecules, such as allelic variants or DNA molecules
from different species, to introduce additional variability into
the process. Selection or screening for the desired activity,
followed by additional iterations of mutagenesis and assay provides
for rapid "evolution" of sequences by selecting for desirable
mutations while simultaneously selecting against detrimental
changes.
[0104] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-ZCYTO18 antibodies or soluble
ZCYTO18 receptor, 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.
[0105] In addition, the proteins of the present invention (or
polypeptide fragments thereof) can be joined to other bioactive
molecules, particularly other cytokines, to provide
multi-functional molecules. For example, one or more helices from
ZCYTO18 can be joined to other cytokines to enhance their
biological properties or efficiency of production.
[0106] The present invention thus provides a series of novel,
hybrid molecules in which a segment comprising one or more of the
helices of ZCYTO18 is fused to another polypeptide. 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 hybrid molecules may further comprise additional amino acid
residues (e.g. a polypeptide linker) between the component proteins
or polypeptides.
[0107] Non-naturally occurring amino acids include, without
limitation, trans-3-methylproline, 2,4-methanoproline,
cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,
allo-threonine, methylthreonine, hydroxyethylcysteine,
hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic
acid, thiazolidine carboxylic acid, dehydroproline, 3- and
4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline,
2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and
4-fluorophenylalanine. Several methods are known in the art for
incorporating non-naturally occurring amino acid residues into
proteins. For example, an in vitro system can be employed wherein
nonsense mutations are suppressed using chemically aminoacylated
suppressor tRNAs. Methods for synthesizing amino acids and
aminoacylating tRNA are known in the art. Transcription and
translation of plasmids containing nonsense mutations is typically
carried out in a cell-free system comprising an E. coli S30 extract
and commercially available enzymes and other reagents. Proteins are
purified by chromatography. See, for example, Robertson et al., J.
Am. Chem. Soc. 113:2722 (1991), Ellman et al., Methods Enzymol.
202:301 (1991), Chung et al., Science 259:806 (1993), and Chung et
al., Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0108] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993)).
[0109] 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 ZCYTO18 amino acid residues.
[0110] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a ZCYTO18
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0111] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein. Hopp[Woods hydrophilicity
profiles can be used to determine regions that have the most
antigenic potential (Hopp et al., 1981, ibid. and Hopp, 1986,
ibid.). In ZCYTO18 these regions include: (1) amino acid number 29
(Arg) to amino acid number 34 (Asn) of SEQ ID NO: 3; (2) amino acid
number 121 (His) to amino acid number 126 (Asp) of SEQ ID NO: 3;
(3) amino acid number 134 (Gln) to amino acid number 139 (Thr) of
SEQ ID NO: 3; (4) amino acid number 137 (Lys) to amino acid number
142 (Lys) of SEQ ID NO: 3; and (5) amino acid number 145 (Glu) to
amino acid number 150 (Lys) of SEQ ID NO: 2. Moreover, ZCYTO18
antigenic epitopes as predicted by a Jameson-Wolf plot, e.g., using
DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.) serve as
preferred antigens, and are readily determined by one of skill in
the art.
[0112] Antigenic epitope-bearing peptides and polypeptides
preferably contain at least four to ten amino acids, at least ten
to fifteen amino acids, or about 15 to about 30 amino acids of SEQ
ID NO: 3. Such epitope-bearing peptides and polypeptides can be
produced by fragmenting a ZCYTO18 polypeptide, or by chemical
peptide synthesis, as described herein. Moreover, epitopes can be
selected by phage display of random peptide libraries (see, for
example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993); and
Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)). Standard
methods for identifying epitopes and producing antibodies from
small peptides that comprise an epitope are described, for example,
by Mole, "Epitope Mapping," in Methods in Molecular Biology, Vol.
10, Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992);
Price, "Production and Characterization of Synthetic
Peptide-Derived Antibodies," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 60-84 (Cambridge University Press 1995), and Coligan et al.
(eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and
pages 9.4.1-9.4.11 (John Wiley & Sons 1997).
[0113] Regardless of the particular nucleotide sequence of a
variant ZCYTO18 polynucleotide, the polynucleotide encodes a
polypeptide that is characterized by its proliferative or
differentiating activity, its ability to induce or inhibit
specialized cell functions, or by the ability to bind specifically
to an anti-ZCYTO18 antibody or ZCYTO18 receptor. More specifically,
variant ZCYTO18 polynucleotides will encode polypeptides which
exhibit at least 50% and preferably, greater than 70%, 80% or 90%,
of the activity of the polypeptide as shown in SEQ ID NO: 3.
[0114] For any ZCYTO18 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above.
[0115] The present invention further provides a variety of other
polypeptide fusions (and related multimeric proteins comprising one
or more polypeptide fusions). For example, a ZCYTO18 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- ZCYTO18 polypeptide fusions can be
expressed in genetically engineered cells (to produce a variety of
multimeric ZCYTO18 analogs). Auxiliary domains can be fused to
ZCYTO18 polypeptides to target them to specific cells, tissues, or
macromolecules. For example, a ZCYTO18 polypeptide or protein could
be targeted to a predetermined cell type by fusing a ZCYTO18
polypeptide to a ligand that specifically binds to a receptor on
the surface of that target cell. In this way, polypeptides and
proteins can be targeted for therapeutic or diagnostic purposes. A
ZCYTO18 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
Research34:1-9, 1996.
[0116] Using the methods discussed herein, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptides that
have substantially similar sequence identity to amino acid residues
1-167 or 23-167 of SEQ ID NO: 3, or functional fragments and
fusions thereof, wherein such polypeptides or fragments or fusions
retain the properties of the wild-type protein such as the ability
to stimulate proliferation, differentiation, induce specialized
cell function or bind the ZCYTO18 receptor or ZCYTO18
antibodies.
[0117] The ZCYTO18 polypeptides of the present invention, including
full-length polypeptides, functional 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.
[0118] In general, a DNA sequence encoding a ZCYTO18 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.
[0119] To direct a ZCYTO18 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
ZCYTO18, 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 ZCYTO18 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).
[0120] 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 comprising amino
acid residue 1 (Met) to 21 (Ala) of SEQ ID NO: 3 is be operably
linked to a DNA sequence encoding 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.
[0121] 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.
1:841-5, 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. 2:714-6, 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. Virol. 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, Manassas, Va. In general, strong transcription
promoters are preferred, such as promoters from SV-40 or
cytomegalovirus. See, e.g., U.S. Pat. No. 10 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.
[0122] 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.
[0123] 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. The second method of making
recombinant baculovirus utilizes a transposon-based system
described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79,
1993). This system is sold in the Bac-to-Bac 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 ZCYTO18 polypeptide into a
baculovirus genome maintained in E. coli as a large plasmid called
a "bacmid." The pFastBac1.TM. transfer vector utilizes the AcNPV
polyhedrin promoter to drive the expression of the gene of
interest, in this case ZCYTO18. However, pFastBac1.TM. can be
modified to a considerable degree. The polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins. See, 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 such transfer
vector constructs, a short or long version of the basic protein
promoter can be used. Moreover, transfer vectors can be constructed
which replace the native ZCYTO18 secretory signal sequences with
secretory signal sequences derived from insect proteins. For
example, a secretory signal sequence from Ecdysteroid
Glucosyltransferase (EGT), honey bee Melittin (Invitrogen,
Carlsbad, Calif.), or baculovirus gp67 (PharMingen, San Diego,
Calif.) can be used in constructs to replace the native ZCYTO18
secretory signal sequence. 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 ZCYTO18 polypeptide, for example,
a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad.
Sci. 82:7952-4, 1985). Using techniques known in the art, a
transfer vector containing ZCYTO18 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 ZCYTO18 is subsequently
produced. Recombinant viral stocks are made by methods commonly
used the art.
[0124] 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. The
cells are grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3. 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 ZCYTO18
polypeptide from the supernatant can be achieved using methods
described herein.
[0125] 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
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459-65, 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.
[0126] 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-aminoirnidazole 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.
[0127] 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 ZCYTO18 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.
[0128] 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. methanolica 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.
methanolica is YEPD (2% D-glucose, 2% Bacto.TM. Peptone (Difco
Laboratories, Detroit, Mich.), 1% Bactom yeast extract (Difco
Laboratories), 0.004% adenine and 0.006% L-leucine).
[0129] It is preferred to purify the polypeptides of the present
invention to .gtoreq.80% purity, more preferably to .gtoreq.90%
purity, even more preferably .gtoreq.95% purity, and particularly
preferred is a pharmaceutically pure state, that is greater than
99.9% pure with respect to contaminating macromolecules,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents. Preferably, a purified polypeptide
is substantially free of other polypeptides, particularly other
polypeptides of animal origin.
[0130] Expressed recombinant ZCYTO18 polypeptides (or chimeric
ZCYTO18 polypeptides) can be purified using fractionation and/or
conventional purification methods and media. Ammonium sulfate
precipitation and acid or chaotrope extraction may be used for
fractionation of samples. Exemplary purification steps may include
hydroxyapatite, size exclusion, FPLC and reverse-phase high
performance liquid chromatography. Suitable chromatographic media
include derivatized dextrans, agarose, cellulose, polyacrylamide,
specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives
are preferred. Exemplary chromatographic media include those media
derivatized with phenyl, butyl, or octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the
like. Suitable solid supports include glass beads, silica-based
resins, cellulosic resins, agarose beads, cross-linked agarose
beads, polystyrene beads, cross-linked polyacrylamide resins and
the like that are insoluble under the conditions in which they are
to be used. These supports may be modified with reactive groups
that allow attachment of proteins by amino groups, carboxyl groups,
sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfflydryl activation, hydrazide activation, and carboxyl and
amino derivatives for carbodiimide coupling chemistries. These and
other solid media are well known and widely used in the art, and
are available from commercial suppliers. Methods for binding
receptor polypeptides to support media are well known in the art.
Selection of a particular method is a matter of routine design and
is determined in part by the properties of the chosen support. See,
for example, Affinity Chromatography: Principles & Methods,
Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
[0131] The polypeptides of the present invention can be isolated by
exploitation of their physical properties. For example, immobilized
metal ion adsorption (IMAC) chromatography can be used to purify
histidine-rich proteins, including those comprising polyhistidine
tags. Briefly, a gel is first charged with divalent metal ions to
form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985).
Histidine-rich proteins will be adsorbed to this matrix with
differing affinities, depending upon the metal ion used, and will
be eluted by competitive elution, lowering the pH, or use of strong
chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (Methods in
Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher,
(ed.), Acad. Press, San Diego, 1990, pp.529-39) and use of the
soluble ZCYTO18 receptor. Within additional embodiments of the
invention, a fusion of the polypeptide of interest and an affinity
tag (e.g., maltose-binding protein, an immunoglobulin domain) may
be constructed to facilitate purification.
[0132] Moreover, using methods described in the art, polypeptide
fusions, or hybrid ZCYTO18 proteins, are constructed using regions
or domains of the inventive ZCYTO18 in combination with those of
other human cytokine family proteins (e.g. interleukins or GM-CSF),
or heterologous proteins (Sambrook et al., ibid., Altschul et al.,
ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994, and references
therein). These methods allow the determination of the biological
importance of larger domains or regions in a polypeptide of
interest. Such hybrids may alter reaction kinetics, binding, alter
cell proliferative activity, constrict or expand the substrate
specificity, or alter tissue and cellular localization of a
polypeptide, and can be applied to polypeptides of unknown
structure.
[0133] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. For example, part or
all of a helix conferring a biological function may be swapped
between ZCYTO18 of the present invention with the functionally
equivalent helices from another family member, such as IL-10,
zcyto10, MDA7, IL-15, L-2, IL-4 and GM-CSF. Such components
include, but are not limited to, the secretory signal sequence,
helices A, B, C, D and four-helical-bundle cytokines. Such fusion
proteins would be expected to have a biological functional profile
that is the same or similar to polypeptides of the present
invention or other known four-helical-bundle cytokine family
proteins, depending on the fusion constructed. Moreover, such
fusion proteins may exhibit other properties as disclosed
herein.
[0134] Standard molecular biological and cloning techniques can be
used to swap the equivalent domtains between the ZCYTO18
polypeptide and those polypeptides to which they are fused.
Generally, a DNA segment that encodes a domain of interest, e.g.,
ZCYTO18 helices A through D, or other domain described herein, is
operably linked in frame to at least one other DNA segment encoding
an additional polypeptide (for instance a domain or region from
another cytokine, such as IL-10, or zcytolo, MDA7 or the like), and
inserted into an appropriate expression vector, as described
herein. Generally DNA constructs are made such that the several DNA
segments that encode the corresponding regions of a polypeptide are
operably linked in frame to make a single construct that encodes
the entire fusion protein, or a functional portion thereof. For
example, a DNA construct would encode from N-terminus to C-terminus
a fusion protein comprising a signal polypeptide followed by a
mature four helical bundle cytokine fusion protein containing helix
A, followed by helix B, followed by helix C, followed by helix D.
or for example, any of the above as interchanged with equivalent
regions from another four helical bundle cytokine family protein.
Such fusion proteins can be expressed, isolated, and assayed for
activity as described herein. Moreover, such fusion proteins can be
used to express and secrete fragments of the ZCYTO18 polypeptide,
to be used, for example to inoculate an animal to generate
anti-ZCYTO18 antibodies as described herein. For example a
secretory signal sequence can be operably linked to helix A, B, C
or D, or a combination thereof (e.g., operably linked polypeptides
comprising helices A-B, B-C, C-D, A-C, A-D, B-D, or ZCYTO18
polypeptide fragments described herein), to secrete a fragment of
ZCYTO18 polypeptide that can be purified as described herein and
serve as an antigen to be inoculated into an animal to produce
anti-ZCYTO18 antibodies, as described herein.
[0135] Zcyto18 polypeptides or fragments thereof may also be
prepared through chemical synthesis. ZCYTO18 polypeptides may be
monomers or multimers; glycosylated or non-glycosylated; pegylated
or non-pegylated; and may or may not include an initial methionine
amino acid residue. For example, the polypeptides can be prepared
by solid phase peptide synthesis, for example as described by
Merrifield, J. Am. Chem. Soc. 85:2149,1963.
[0136] The activity of molecules of the present invention can be
measured using a variety of assays that measure proliferation of
and/or binding to cells expressing the ZCYTO18 receptor. Of
particular interest are changes in ZCYTO18-dependent cells.
Suitable cell lines to be engineered to be ZCYTO18-dependent
include the IL-3-dependent BaF3 cell line (Palacios and Steinmetz,
Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:
4133-4135, 1986), FDC-P1 (Hapel et al., Blood 64: 786-790, 1984),
and MO7e (Kiss et al., Leukemia 7: 235-240, 1993). Growth
factor-dependent cell lines can be established according to
published methods (e.g. Greenberger et al., Leukemia Res. 8:
363-375, 1984; Dexter et al., in Baum et al. Eds., Experimental
Hematology Today, 8th Ann. Mtg. Int. Soc. Exp. Hematol. 1979,
145-156, 1980). For example, Baf3 cells expressing the ZCYTO18
heterodimeric receptor zcytor11/CRF2-4, as described herein, can be
used to assay the activity of ZCYTO18, ZCYTO18 receptor-binding
fragments, and ZCYTO18 variants of the present invention. The BaF3
stable cell line that co-expressing zcytor11 and CRF2-4 (ZCYTO18
receptor) exhibits dose-dependent proliferative response to ZCYTO18
protein in the media without IL-3.
[0137] Proteins of the present invention are 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, ZCYTO18 polypeptides are useful for stimulating
proliferation, activation, differentiation, induction or inhibition
of specialized cell functions of cells of the hematopoetic
lineages, including, but not limited to, T cells, B cells, NK
cells, dendritic cells, monocytes, and macrophages. Proliferation
and/or differentiation of hematopoietic cells can be measured in
vitro using cultured cells or in vivo by administering molecules of
the present invention to the appropriate animal model. Assays
measuring cell proliferation or differentiation are well known in
the art. For example, assays measuring proliferation include such
assays as chemosensitivity to neutral red dye (Cavanaugh et al.,
Investigational New Drugs 8:347-354, 1990, incorporated herein by
reference), incorporation of radiolabelled nucleotides (Cook et
al., Analytical Biochem. 179:1-7, 1989, incorporated herein by
reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the
DNA of proliferating cells (Porstmann et al., J. Immunol. Methods
82:169-179, 1985, incorporated herein by reference), and use of
tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983;
Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth
Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833,
1988; all incorporated herein by reference). Assays measuring
differentiation include, for example, measuring cell-surface
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; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171,
1989; all incorporated herein by reference).
[0138] IL-10 is a cytokine that inhibits production of other
cytokines, induces proliferation and differentiation of activated B
lymphocytes, inhibits HIV-1 replication and exhibits antagonistic
effects on gamma interferon. IL-10 appears to exist as a dimer
formed from two alpha-helical polypeptide regions related by a
180.degree. rotation. See, for example, Zdanov et al., Structure:
3(6): 591-601 (1996). IL-10 has been reported to be a product of
activated Th2 T-cells, B-cells, keratinocytes and
monocytes/macrophages that is capable of modulating a Th1 T-cell
response. Such modulation may be accomplished by inhibiting
cytokine synthesis by Th1 T-cells. See, for example, Hus et al.,
Int. Immunol. 4: 563 (1992) and D'Andrea et al., J. Exp. Med. 178:
1042 (1992). IL-10 has also been reported to inhibit cytokine
synthesis by natural killer cells and monocytes/macrophages. See,
for example, Hus et al. cited above and Fiorentino et al., J.
Immunol. 146: 3444 (1991). In addition, IL-10 has been found to
have a protective effect with respect to insulin dependent diabetes
mellitus. Similarly, as a cytokine sharing polypeptide structure
and some sequence similarity to IL-10, ZCYTO18 can have these above
disclosed activities, and the assays used to assess IL-10 activity
can be applied to assay ZCYTO18 activity.
[0139] The molecules of the present invention can be assayed in
vivo using viral delivery systems. Exemplary viruses for this
purpose include adenovirus, herpesvirus, retroviruses, vaccinia
virus, and adeno-associated virus (AAV). Adenovirus, a
double-stranded DNA virus, is currently the best studied gene
transfer vector for delivery of heterologous nucleic acid (for
review, see T. C. Becker et al., Meth. Cell Biol. 43:161-15 89,
1994; and J. T. Douglas and D. T. Curiel, Science & Medicine
4:44-53, 1997). The adenovirus system offers several advantages:
(i) adenovirus can accommodate relatively large DNA inserts; (ii)
can be grown to high-titer; (iii) infect a broad range of mammalian
cell types; and (iv) can be used with many different promoters
including ubiquitous, tissue specific, and regulatable promoters.
Also, because adenoviruses are stable in the bloodstream, they can
be administered by intravenous injection.
[0140] Using adenovirus vectors where portions of the adenovirus
genome are deleted, inserts are incorporated into the viral DNA by
direct ligation or by homologous recombination with a
co-transfected plasmid. In an exemplary system, the essential E1
gene has been deleted from the viral vector, and the virus will not
replicate unless the E1 gene is provided by the host cell (the
human 293 cell line is exemplary). When intravenously administered
to intact animals, adenovirus primarily targets the liver. If the
adenoviral delivery system has an El gene deletion, the virus
cannot replicate in the host cells. However, the host's tissue
(e.g., liver) will express and process (and, if a secretory signal
sequence is present, secrete) the heterologous protein. Secreted
proteins will enter the circulation in the highly vascularized
liver, and effects on the infected animal can be determined.
[0141] Moreover, adenoviral vectors containing various deletions of
viral genes can be used in an attempt to reduce or eliminate immune
responses to the vector. Such adenoviruses are E1 deleted, and in
addition contain deletions of E2A or E4 (Lusky, M. et al., J.
Virol. 72:2022-2032, 1998; Raper, S. E. et al., Human Gene Therapy
9:671-679, 1998). In addition, deletion of E2b is reported to
reduce immune responses (Amalfitano, A. et al., J. Virol.
72:926-933, 1998). Moreover, by deleting the entire adenovirus
genome, very large inserts of heterologous DNA can be accommodated.
Generation of so called "gutless" adenoviruses where all viral
genes are deleted are particularly advantageous for insertion of
large inserts of heterologous DNA. For review, see Yeh, P. and
Perricaudet, M., FASEB J. 11:615-623, 1997.
[0142] The adenovirus system can also be used for protein
production in vitro. By culturing adenovirus-infected cells under
conditions where the cells are not rapidly dividing, the cells can
produce proteins for extended periods of time. For instance, BHK
cells are grown to confluence in cell factories, then exposed to
the adenoviral vector encoding the secreted protein of interest.
The cells are then grown under serum-free conditions, which allows
infected cells to survive for several weeks without significant
cell division. Alternatively, adenovirus vector infected 293 cells
can be grown as adherent cells or in suspension culture at
relatively high cell density to produce significant amounts of
protein (See Garnier et al., Cztotechnol. 15:145-55, 1994). With
either protocol, an expressed, secreted heterologous protein can be
repeatedly isolated from the cell culture supernatant, lysate, or
membrane fractions depending on the disposition of the expressed
protein in the cell. Within the infected 293 cell production
protocol, non-secreted proteins may also be effectively
obtained.
[0143] In view of the tissue distribution observed for ZCYTO18
receptor agonists (including the natural
ligand/substrate/cofactor/etc.) and/or antagonists have enormous
potential in both in vitro and in vivo applications. Compounds
identified as ZCYTO18 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,
ZCYTO18 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, and other cells
of the lymphoid and myeloid lineages in culture.
[0144] 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 ZCYTO18 activity (ZCYTO18 antagonists) include
anti-ZCYTO18 antibodies and soluble ZCYTO18 receptors, as well as
other peptidic and non-peptidic agents (including ribozymes).
[0145] ZCYTO18 can also be used to identify inhibitors
(antagonists) of its activity. Test compounds are added to the
assays disclosed herein to identify compounds that inhibit the
activity of ZCYTO18. In addition to those assays disclosed herein,
samples can be tested for inhibition of ZCYTO18 activity within a
variety of assays designed to measure receptor binding, the
stimulation/inhibition of ZCYTO18-dependent cellular responses or
proliferation of ZCYTO18 receptor-expressing cells.
[0146] A ZCYTO18 polypeptide can be expressed as a fusion with an
immunoglobulin heavy chain constant region, typically an Fc
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 Fc 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 to (e.g., for dimerization, increasing stability and in
vivo half-life, affinity purify ligand, in vitro assay tool,
antagonist). For use in assays, the chimeras are bound to a support
via the Fc region and used in an ELISA format. Fc fusions may
represent preferred therapeutic proteins wth different
pharmacokinetics and altered action.
[0147] Polypeptides containing the receptor-binding region of the
ligand can be used for purification of receptor. The ligand
polypeptide 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, sulfflydryl
activation, and hydrazide activation. The resulting media will
generally be configured in the form of a column, and fluids
containing receptors are passed through the column one or more
times to allow receptor to bind to the ligand polypeptide. The
receptor is then eluted using changes in salt concentration,
chaotropic agents (MnCl.sub.2), or pH to disrupt ligand-receptor
binding.
[0148] ZCYTO18 polypeptides or ZCYTO18 fusion proteins are used,
for example, to identify the ZCYTO18 receptor. Using labeled
ZCYTO18 polypeptides, cells expressing the receptor are identified
by fluorescence immunocytometry or immunohistochemistry. ZCYTO18
polypeptides are useful in determining the distribution of the
receptor on tissues or specific cell lineages, and to provide
insight into receptor/ligand biology. An exemplary method to
identify a ZCYTO18 receptor in vivo or in vitro, e.g., in cell
lines, is to us a ZCYTO18 polypeptide fused to the catalytic domain
of Alkaline phosphatase (AP), as described in Feiner, L. et al.,
Neuron 19:539-545, 1997. Such AP fusions, as well as radiolabeled
ZCYTO18, ZCYTO18 fusions with fluorescent lables, and others
described herein, combined with standard cloning techniques enable
one of skill in the art to visualize, identify and clone the
ZCYTO18 receptor.
[0149] Conversely, a ZCYTO18-binding polypeptide can be used for
purification of ligand. The polypeptide 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 HCl), or pH to
disrupt ligand-receptor binding.
[0150] 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 sulfflydryl
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.).
[0151] 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; Cunningham et
al., Science 245:821-25, 1991).
[0152] Zcyto18 polypeptides can also be used to prepare antibodies
that bind to ZCYTO18 epitopes, peptides or polypeptides. The
ZCYTO18 polypeptide 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 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 ZCYTO18 polypeptide (e.g., SEQ
ID NO: 3). Polypeptides comprising a larger portion of a ZCYTO18
polypeptide, 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 and carriers, as
described herein. Suitable antigens include the ZCYTO18 polypeptide
encoded by SEQ ID NO: 3 from amino acid number 23 to amino acid
number 167, or a contiguous 9 to 144, or 30 to 144 amino acid
fragment thereof. Other suitable antigens include helices of the
four-helical-bundle structure, as described herein. Preferred
peptides to use as antigens are hydrophilic peptides such as those
predicted by one of skill in the art from a hydrophobicity plot, as
described herein. For example suitable hydrophilic peptides
include: (1) amino acid number 29 (Arg) to amino acid number 34
(Asn) of SEQ ID NO: 3; (2) amino acid number 121 (His) to amino
acid number 126 (Asp) of SEQ ID NO: 3; (3) amino acid number 134
(Gln) to amino acid number 139 (Thr) of SEQ ID NO: 3; (4) amino
acid number 137 (Lys) to amino acid number 142 (Lys) of SEQ ID NO:
3; and (5) amino acid number 145 (Glu) to amino acid number 150
(Lys) of SEQ ID NO: 2. Moreover, ZCYTO18 antigenic epitopes as
predicted by a Jameson-Wolf plot, e.g., using DNASTAR Protean
program (DNASTAR, Inc., Madison, Wis.) serve as preferred antigens,
and are readily determined by one of skill in the art.
[0153] Antibodies from an immune response generated by inoculation
of an animal with these antigens (or immunogens) 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.
[0154] 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 ZCYTO18 polypeptide or a
fragment thereof. The immunogenicity of a ZCYTO18 polypeptide may
be increased through the use of an adjuvant, such as alum (aluminum
hydroxide) or Freund's complete or incomplete adjuvant.
Polypeptides useful for immunization also include fusion
polypeptides, such as fusions of ZCYTO18 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.
[0155] 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.
[0156] 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-ZCYTO18 antibodies
herein bind to a ZCYTO18 polypeptide, peptide or epitope with an
affinity at least 10-fold greater than the binding affinity to
control (non-ZCYTO18) polypeptide. 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).
[0157] Whether anti-ZCYTO18 antibodies do not significantly
cross-react with related polypeptide molecules is shown, for
example, by the antibody detecting ZCYTO18 polypeptide 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 ZCYTO18, and ZCYTO18 mutant
polypeptides. Moreover, antibodies can be "screened against" known
related polypeptides, to isolate a population that specifically
binds to the ZCYTO18 polypeptides. For example, antibodies raised
to ZCYTO18 are adsorbed to related polypeptides adhered to
insoluble matrix; antibodies specific to ZCYTO18 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-ZCYTO18 antibodies can be detected
by a number of methods in the art, and disclosed below.
[0158] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which bind to ZCYTO18 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
ZCYTO18 protein or polypeptide.
[0159] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to ZCYTO18 protein or peptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled ZCYTO18 protein or peptide). Genes encoding
polypeptides having potential ZCYTO18 polypeptide binding domains
can be obtained by screening random peptide libraries displayed on
phage (phage display) or on bacteria, such as E. coli. Nucleotide
sequences encoding the polypeptides can be obtained in a number of
ways, such as through random mutagenesis and random polynucleotide
synthesis. These random peptide display libraries can be used to
screen for peptides which interact with a known target which can be
a protein or polypeptide, such as a ligand or receptor, a
biological or synthetic macromolecule, or organic or inorganic
substances. Techniques for creating and screening such random
peptide display libraries are known in the art (Ladner et al., U.S.
Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner
et al., U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No.
5,571,698) and random peptide display libraries and kits for
screening such libraries are available commercially, for instance
from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego,
Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia
LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the ZCYTO18 sequences disclosed
herein to identify proteins which bind to ZCYTO18. These "binding
polypeptides" which interact with ZCYTO18 polypeptides can be used
for tagging cells; for isolating homolog polypeptides by affinity
purification; they can be directly or indirectly conjugated to
drugs, toxins, radionuclides and the like. These binding
polypeptides can also be used in analytical methods such as for
screening expression libraries and neutralizing activity, e.g., for
blocking interaction between ligand and receptor, or viral binding
to a receptor. The binding polypeptides can also be used for
diagnostic assays for determining circulating levels of ZCYTO18
polypeptides; for detecting or quantitating soluble ZCYTO18
polypeptides as marker of underlying pathology or disease. These
binding polypeptides can also act as ZCYTO18 "antagonists" to block
ZCYTO18 binding and signal transduction in vitro and in vivo. These
anti-ZCYTO18 binding polypeptides would be useful for inhibiting
ZCYTO18 activity or protein-binding.
[0160] Antibodies to ZCYTO18 may be used for tagging cells that
express ZCYTO18; for isolating ZCYTO18 by affinity purification;
for diagnostic assays for determining circulating levels of ZCYTO18
polypeptides; for detecting or quantitating soluble ZCYTO18 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 ZCYTO18 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 ZCYTO18 or fragments thereof may be used in vitro to
detect denatured ZCYTO18 or fragments thereof in assays, for
example, Western Blots or other assays known in the art.
[0161] Antibodies or polypeptides herein can also be directly or
indirectly conjugated to drugs, toxins, radionuclides and the like,
and these conjugates used for in vivo diagnostic or therapeutic
applications. For instance, polypeptides or antibodies of the
present invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary molecule
(receptor or antigen, respectively, for instance). More
specifically, ZCYTO18 polypeptides or anti-ZCYTO18 antibodies, or
bioactive fragments or portions thereof, can be coupled to
detectable or cytotoxic molecules and delivered to a mammal having
cells, tissues or organs that express the anti-complementary
molecule.
[0162] Suitable detectable molecules may be directly or indirectly
attached to the polypeptide 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, 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). Polypeptides or antibodies
may also be conjugated to cytotoxic drugs, such as adriamycin. For
indirect attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule can be conjugated with a member of
a complementary/anticomplementary pair, where the other member is
bound to the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/anticomplementary
pair.
[0163] In another embodiment, 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. Such
cytokine toxin fusion proteins can be used for in vivo killing of
target tissues.
[0164] In another embodiment, ZCYTO18 cytokine fusion proteins or
antibody-cytokine fusion proteins can be used for in vivo killing
of target tissues (for example, leukemia, lymphoma, lung cancer,
colon cancer, melanoma, pancreatic cancer, ovanian cancer, blood
and bone marrow cancers, or other cancers wherein ZCYTO18 receptors
ar expressed) (See, generally, Hornick et al., Blood 89:4437-47,
1997). The described fusion proteins enable targeting of a cytokine
to a desired site of action, thereby providing an elevated local
concentration of cytokine. Suitable ZCYTO18 polypeptides or
anti-ZCYTO18 antibodies target an undesirable cell or tissue (i.e.,
a tumor or a leukemia), and the fused cytokine mediated improved
target cell lysis by effector cells. Suitable cytokines for this
purpose include interleukin 2 and granulocyte-macrophage
colony-stimulating factor (GM-CSF), for instance.
[0165] In yet another embodiment, if the ZCYTO18 polypeptide or
anti-ZCYTO18 antibody targets vascular cells or tissues, such
polypeptide or antibody may be conjugated with a radionuclide, and
particularly with a beta-emitting radionuclide, to reduce
restenosis. Such therapeutic approaches pose less danger to
clinicians who administer the radioactive therapy. For instance,
iridium-192 impregnated ribbons placed into stented vessels of
patients until the required radiation dose was delivered showed
decreased tissue growth in the vessel and greater luminal diameter
than the control group, which received placebo ribbons. Further,
revascularisation and stent thrombosis were significantly lower in
the treatment group. Similar results are predicted with targeting
of a bioactive conjugate containing a radionuclide, as described
herein.
[0166] The bioactive polypeptide or antibody conjugates described
herein can be delivered intravenously, intraarterially or
intraductally, or may be introduced locally at the intended site of
action.
[0167] Zcyto18 was isolated from tissue known to have important
immunological function and which contain cells which play a role in
the immune system. ZCYTO18 ligand is expressed in CD3+ selected,
activated peripheral blood cells. This suggests that ZCYTO18
expression may be regulated and increase after T cell activation.
Moreover, polypeptides of the present invention may have an effect
on the growth/expansion and/or differentiated state of T- or
B-Cells, T- or B-cell progenitors, NK cells or NK progenitors.
Moreover, ZCYTO18 can effect proliferation and/or differentiation
of T cells and B cells in vivo. Factor that both stimulate
proliferation of hematopoietic progenitors and activate mature
cells are generally known. NK cells are responsive to IL-2 alone,
but proliferation and activation generally 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+IL-2 in combination with IL-7 and Steel Factor
was more effective (Mrzek 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). A composition comprising
ZCYTO18 and IL-15 may stimulate NK progenitors and NK cells, as a
composition that is more potent than previously described factors
and combinations of factors. Similarly, such combinations of
factors that include ZCYTO18 may also affect other hematopoietic
and lymphoid cell types, such as T-cells, B-cells, macrophages,
dendritic cells, and the like.
[0168] Most four-helix bundle cytokines as well as other proteins
produced by activated lymphocytes play an important biological role
in cell differentiation, activation, recruitment and homeostasis of
cells throughout the body. Therapeutic utility includes treatment
of diseases which require immune regulation including autoimmune
diseases, such as, rheumatoid arthritis, multiple sclerosis,
myasthenia gravis, systemic lupus erythomatosis (SLE) and diabetes.
Zcyto18 may be important in the regulation of inflammation, and
therefore would be useful in treating rheumatoid arthritis, asthma,
ulcerative colitis, inflammatory bowel disease, Crohn's disease,
pancreatitis, and sepsis. There may be a role of ZCYTO18 in
mediating tumor cell killing and therefore would be useful in the
treatment of cancer such as ovarian cancer, lung cancer, melanoma,
and colon cancer. Zcyto18 may be a potential therapeutic in
suppressing the immune system which would be important for reducing
graft rejection. Zcyto18 may have usefulness in prevention of
graft-vs-host disease.
[0169] The proteins of the present invention can also be used ex
vivo, such as in autologous marrow culture. Briefly, bone marrow is
removed from a patient prior to chemotherapy or organ transplant
and treated with ZCYTO18, optionally in combination with one or
more other cytokines. The treated marrow is then returned to the
patient after chemotherapy to speed the recovery of the marrow or
after transplant to suppress graft vs. Host disease. In addition,
the proteins of the present invention can also be used for the ex
vivo expansion of marrow or peripheral blood progenitor (PBPC)
cells. Prior to treatment, marrow can be stimulated with stem cell
factor (SCF) to release early progenitor cells into peripheral
circulation. These progenitors can be collected and concentrated
from peripheral blood and then treated in culture with ZCYTO18,
optionally in combination with one or more other cytokines,
including but not limited to L-10, zcyto10, MDA7, SCF, IL-2, EL-4,
IL-7 or L-15, to differentiate and proliferate into high-density
lymphoid cultures, which can then be returned to the patient
following chemotherapy or transplantation.
[0170] Alternatively, ZCYTO18 may activate the immune system which
would be important in boosting immunity to infectious diseases,
treating immunocompromised patients, such as HIV+ patients, or in
improving vaccines. In particular, ZCYTO18 stimulation or expansion
of T-cells, B-cells, NK cells, and the like, or their progenitors,
would provide therapeutic value in treatment of viral infection,
and as an anti- neoplastic factor. 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).
[0171] Further analysis of mice injected with ZCYTO18 adenovirus
reveals that albumin levels are reduced relative to control
adenovirus injected animals, and glucose levels are depressed
significantly. However liver enzymes (ALT and AST) are at Asimilar
levels to those seen for mice injected with control adenovirus.
ZCYTO18 may specifically inhibit or alter liver cell functions.
Alternatively excess ZCYTO18 may synergize with viral infection
leading to adverse effects on the liver. Thus antagonists
(antibodies, muteins, soluble receptors) may be useful to treat
viral disease. Especially viral diseases that target the liver such
as: Hepatitis B, Hepatitis C and Adenovirus. Viral disease in other
tissues may be treated with antagonists to ZCYTO18, for example
viral meningitis, and HIV-related disease.
[0172] Mice injected with ZCYTO18 adenovirus display weight-loss,
loss of mobility and a failure to groom, and a reduction in
circulating lymphocytes. These changes are typical of those seen
during septic shock and other inflammatory conditions. These
effects may be caused directly by ZCYTO18 or indirectly by
induction of elevated levels of proinflammatory cytokines such as
IL-1, TNF.alpha., and IL-6. Antagonists to ZCYTO18 may be useful to
treat septic shock, adult respiratory distress syndrome,
endotoxemia, and meningitis. Other diseases that may benefit from
ZCYTO18 antagonists include: Hemorrhagic shock, disseminated
intravascular coagulopathy, myocardial ischemia, stroke, rejection
of transplanted organs, pulmonary fibrosis, inflammatory
hyperalgesia and cachexia.
[0173] Mice injected with ZCYTO18 adenovirus display reduced
numbers of peripheral blood lymphocytes. This is likely to be a
direct inhibitory effect of ZCYTO18 on peripheral blood
lymphocytes. Antagonizing ZCYTO18 may promote lymphocyte
maintenance and growth especially when they are needed to eradicate
bacterial, viral or parasitic pathogens. Thus antagonizing ZCYTO18
may benefit patients with: Tuberculosis, cryptogenic fibrosing
alveolitis, pneumonia, meningococal disease, AIDS, HIV-related lung
disease, hepatitis, viral meningitis, malaria, and dysentery
(Shigella dysenteriae).
[0174] The lymphocyte inhibitory effects of ZCYTO18 may be used to
reduce autoimmunity and to inhibit the growth of lymphoma tumors,
especially non-Hodgkin's lymphoma and lymphoid leukemias. ZCYTO18
may also be used to inhibit lymphocytes and promote graft
acceptance for organ transplant patients. Kidney and bone marrow
grafts would be suitable indications.
[0175] Mice injected with ZCYTO18 adenovirus display significantly
increased numbers of platelets. Mild bleeding disorders (MBDs)
associated with platelet dysfunctions are relatively common
(Bachmann, Seminars in Hematology 17: 292-305, 1980), as are a
number of congenital disorders of platelet function, including
Bernard-Soulier syndrome (deficiency in platelet GPIb), Glanzmann's
thrombasthenia (deficiency of GPIIb and GPIIIa), congenital
afibrinogenemia (diminished or absent levels of fibrinogen in
plasma and platelets), and gray platelet syndrome (absence of
a-granules). In addition there are a number of disorders associated
with platelet secretion, storage pool deficiency, abnormalities in
platelet arachidonic acid pathway, deficiencies of platelet
cyclooxygenase and thromboxane synthetase and defects in platelet
activation (reviewed by Rao and Holmsen, Seminars in Hematology 23:
102-118, 1986).
[0176] The proteins of the present invention were shown to increase
platelet and neutrophils in vivo in animals, and can be used
therapeutically wherever it is desirable to increase the level of
platelets and neutrophils, such as in the treatment of cytopenia,
such as that induced by aplastic anemia, myelodisplastic syndromes,
chemotherapy or congenital cytopenias. The proteins are also useful
for increasing platelet production, such as in the treatment of
thrombocytopenia. Thrombocytopenia is associated with a diverse
group of diseases and clinical situations that may act alone or in
concert to produce the condition. Lowered platelet counts can
result from, for example, defects in platelet production, abnormal
platelet distribution, dilutional losses due to massive
transfusions, or abnormal destruction of platelets. For example,
chemotherapeutic drugs used in cancer therapy may suppress
development of platelet progenitor cells in the bone marrow, and
the resulting thrombocytopenia limits the chemotherapy and may
necessitate transfusions. In addition, certain malignancies can
impair platelet production and platelet distribution. Radiation
therapy used to kill malignant cells also kills platelet progenitor
cells. Thrombocytopenia may also arise from various platelet
autoimmune disorders induced by drugs, neonatal alloimmunity or
platelet transfusion alloimmunity. The proteins of the present
invention can reduce or eliminate the need for transfusions,
thereby reducing the incidence of platelet alloimmunity. Abnormal
destruction of platelets can result from: (1) increased platelet
consumption in vascular grafts or traumatized tissue; or (2) immune
mechanisms associated with, for example, drug-induced
thrombocytopenia, idiopathic thrombocytopenic purpura (ITP),
autoinumune diseases, hematologic disorders such as leukemia and
lymphoma or metastatic cancers involving bone marrow. Other
indications for the proteins of the present invention include
aplastic anemia and drug-induced marrow suppression resulting from,
for example, chemotherapy or treatment of HIV infection with
AZT.
[0177] Thrombocytopenia is manifested as increased bleeding, such
as mucosal bleedings from the nasal-oral area or the
gastrointestinal tract, as well as oozing from wounds, ulcers or
injection sites.
[0178] For pharmaceutical use, the proteins of the present
invention are formulated for parenteral, particularly intravenous
or subcutaneous, delivery according to conventional methods.
Intravenous administration will be by bolus injection, controlled
release, e.g, using mini-pumps or other appropriate technology, or
by infusion over a typical period of one to several hours. In
general, pharmaceutical formulations will include a hematopoietic
protein in combination with a pharmaceutically acceptable vehicle,
such as saline, buffered saline, 5% dextrose in water or the like.
Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to provent
protein loss on vial surfaces, etc. In addition, the hematopoietic
proteins of the present invention may be combined with other
cytokines, particularly early-acting cytokines such as stem cell
factor, IL-3, IL-6, IL-11 or GM-CSF. When utilizing such a
combination therapy, the cytokines may be combined in a single
formulation or may be administered in separate formulations.
Methods of formulation are well known in the art and are disclosed,
for example, in Remington's Pharmaceutical Sciences, Gennaro, ed.,
Mack Publishing Co., Easton PA, 1990, which is incorporated herein
by reference. Therapeutic doses will generally be in the range of
0.1 to 100 mg/kg of patient weight per day, preferably 0.5-20 mg/kg
per day, with the exact dose determined by the clinician according
to accepted standards, taking into account the nature and severity
of the condition to be treated, patient traits, etc. Determination
of dose is within the level of ordinary skill in the art. The
proteins will commonly be administered over a period of up to 28
days following chemotherapy or bone-marrow transplant or until a
platelet count of >20,000/mm.sup.3, preferably
>50,000/mm.sup.3, is achieved. More commonly, the proteins will
be administered over one week or less, often over a period of one
to three days. In general, a therapeutically effective amount of
ZCYTO18 is an amount sufficient to produce a clinically significant
increase in the proliferation and/or differentiation of lymphoid or
myeloid progenitor cells, which will be manifested as an increase
in circulating levels of mature cells (e.g. platelets or
neutrophils). Treatment of platelet disorders will thus be
continued until a platelet count of at least 20,000/mm.sup.3,
preferably 50,000/mm.sup.3, is reached. The proteins of the present
invention can also be administered in combination with other
cytokines such as L-3,-6 and -11; stem cell factor; erythropoietin;
G-CSF and GM-CSF. Within regimens of combination therapy, daily
doses of other cytokines will in general be: EPO, 150 U/kg; GM-CSF,
5-15 lg/kg; IL-3, 1-5 lg/kg; and G-CSF, 1-25 lg/kg. Combination
therapy with EPO, for example, is indicated in anemic patients with
low EPO levels.
[0179] The proteins of the present invention can also be used ex
vivo, such as in autologous marrow culture or liver cultures. For
example, briefly, bone marrow is removed from a patient prior to
chemotherapy and treated with ZCYTO18, optionally in combination
with one or more other cytokines. The treated marrow is then
returned to the patient after chemotherapy to speed the recovery of
the marrow. In addition, the proteins of the present invention can
also be used for the ex vivo expansion of marrow or peripheral
blood progenitor (PBPC) cells. Prior to chemotherapy treatment,
marrow can be stimulated with stem cell factor (SCF) or G-CSF to
release early progenitor cells into peripheral circulation. These
progenitors can be collected and concentrated from peripheral blood
and then treated in culture with ZCYTO18, optionally in combination
with one or more other cytokines, including but not limited to SCF,
G-CSF, IL-3, GM-CSF, IL-6 or IL-11, to differentiate and
proliferate into high-density megakaryocyte cultures, which can
then be returned to the patient following high-dose chemotherapy.
Such ex vivo uses are especially desirable in the instance that
systemic administration is not tolerated by a patient. Thus the
present invention to provide methods for stimulating the production
of platelets and neutrophils in mammals including humans. The
invention provides methods for stimulating platelet and neutrophil
production in a mammal, ex vivo tissue sample, or cell cultures.
The methods comprise administering to a mammal, ex vivo tissue
sample, or cell culture a therapeutically effective amount of a
hematopoietic protein selected from the group consisting of (a)
proteins comprising the sequence of amino acids of SEQ ID NO: 3
from amino acid residue 22 to amino acid residue 167; (b) allelic
variants of (a); and (d) species homologs of (a) or (b), wherein
the protein stimulates proliferation or differentiation of myeloid
or lymphoid precursors, or the production of platelets, in
combination with a pharmaceutically acceptable vehicle.
[0180] Moreover, the increase of platelets and neutrophils is
desirable at a wound site not only in patients with blood diseases
or undergoing chemotherapy as described above, but under normal
conditions. A polypeptide such as ZCYTO18, that increases platelet
levels in vivo, can be used in topological formulations including
gels, meshes, poultices, liquids, and the like to aid in the
healing of common cuts, burns, lacerations, abrasions, and the
like. Moreover, such applications can be applied in any instance
where the healing of skin, muscle, or the like is desired, even
internally, such as after surgery.
[0181] The proteins of the present invention are also valuable
tools for the in vitro study of the differentiation and development
of hematopoietic cells, such as for elucidating the mechanisms of
cell differentiation and for determining the lineages of mature
cells, and may also find utility as proliferative agents in cell
culture.
[0182] 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. Myocytes, osteoblasts, adipocytes,
chrondrocytes, fibroblasts and reticular cells are believed to
originate from a common mesenchymal stem cell (Owen et al., Ciba
Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stem cells
have not been well identified (Owen et al., J. of Cell Sci.
87:731-738, 1987), so identification is usually made at the
progenitor and mature cell stages. The novel polypeptides of the
present invention may be useful for studies to isolate mesenchymal
stem cells and myocyte or other progenitor cells, both in vivo and
ex vivo.
[0183] 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. Thus, the present invention
includes stimulating or inhibiting the proliferation of myocytes,
smooth muscle cells, osteoblasts, adipocytes, chrondrocytes,
neuronal and endothelial cells. Molecules of the present invention
for example, may while stimulating proliferation or differentiation
of cardiac myocytes, inhibit proliferation or differentiation of
adipocytes, by virtue of the affect on their common precursor/stem
cells. Thus molecules of the present invention may have use in
inhibiting chondrosarcomas, atherosclerosis, restenosis and
obesity.
[0184] 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; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses,
161-171, 1989; all incorporated herein by reference).
Alternatively, ZCYTO18 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 ZCYTO18 polypeptide, or its loss of expression in a tissue as it
differentiates, can serve as a marker for differentiation of
tissues.
[0185] Similarly, direct measurement of ZCYTO18 polypeptide, or its
loss of expression in a tissue can be determined in a tissue or
cells as they undergo tumor progression. Increases in invasiveness
and motility of cells, or the gain or loss of expression of ZCYTO18
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 ZCYTO18
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, ZCYTO18 gain or loss of expression may serve as a
diagnostic for prostate and other cancers.
[0186] Moreover, the activity and effect of ZCYTO18 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 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
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 ZCYTO18, 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., ZCYTO18, 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 ZCYTO18. Use of stable
ZCYTO18 transfectants as well as use of induceable promoters to
activate ZCYTO18 expression in vivo are known in the art and can be
used in this system to assess ZCYTO18 induction of metastasis.
Moreover, purified ZCYTO18 or ZCYTO18 conditioned media can be
directly injected in to this mouse model, and hence be used in this
system. For general reference see, O'Reilly MS, et al. Cell
79:315-328, 1994; and Rusciano D, et al. Murine Models of Liver
Metastasis. Invasion Metastasis 14:349-361, 1995.
[0187] Polynucleotides encoding ZCYTO18 polypeptides are useful
within gene therapy applications where it is desired to increase or
inhibit ZCYTO18 activity. If a mammal has a mutated or absent
ZCYTO18 gene, the ZCYTO18 gene can be introduced into the cells of
the mammal. In one embodiment, a gene encoding a ZCYTO18
polypeptide 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; Samulski et
al., J. Virol. 63:3822-8, 1989).
[0188] In another embodiment, a ZCYTO18 gene 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 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.
[0189] 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;
Wu et al., J. Biol. Chem. 263:14621-4, 1988.
[0190] Antisense methodology can be used to inhibit ZCYTO18 gene
transcription, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a
ZCYTO18-encoding polynucleotide (e.g., a polynucleotide as set
froth in SEQ ID NO: 1) are designed to bind to ZCYTO18-encoding
mRNA and to inhibit translation of such mRNA. Such antisense
polynucleotides are used to inhibit expression of ZCYTO18
polypeptide-encoding genes in cell culture or in a subject.
[0191] The present invention also provides reagents which will find
use in diagnostic applications. For example, the ZCYTO18 gene, a
probe comprising ZCYTO18 DNA or RNA or a subsequence thereof can be
used to determine if the ZCYTO18 gene is present on a human
chromosome, such as chromosome 12, or if a mutation has occurred.
Based on annotation of a fragment of human genomic DNA containing a
part of ZCYTO18 genomic DNA (Genbank Accession No. AC007458),
ZCYTO18 is located at the 12q15 region of chromosome 12. Detectable
chromosomal aberrations at the ZCYTO18 gene locus include, but are
not limited to, aneuploidy, gene copy number changes, loss of
heterogeneity (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).
[0192] 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.
[0193] ZCYTO18 is located at the 12q15 region of chromosome 12.
Another T-cell expressed cytokine, interferon-gamma (IFN-.gamma.)
maps near this locus (12q14), suggesting that the 12q14-15 locus is
an important region for T-cell expressed cytokines. Moreover,
mutations in IFN-.gamma. are associated with immunodeficiency (See,
e.g., Tzoneva, M. et al., Clin. Genet. 33:454-456, 1988). Mutations
in ZCYTO18, are likely to cause human disease as well, such as
immunodeficiency, autoimmune disease, lymphoid cell cancers, or
other immune dysfunction. Moreover, there are several genes that
map to the ZCYTO18 locus that are associated with human disease
states, such as cancer. 12q13-q15 region is involved in a variety
of malignant and benign solid tumors (including salivary adenomas
and uterine leiomyomas), with 12q15 as a common break point.
Moreover, the high mobility group protein isoform I-C (HMGIC) maps
to 12q15 and is involved in benign lipomas and other tumors. As
ZCYTO18 maps to 12q15 as well, there can be an association between
loss of ZCYTO18 function and tumor formation or progression.
Moreover, translocations in 12q13-15 are prevalent in soft tissue
tumors, multiple lipomatosis and malignant muixoid liposarcoma.
ZCYTO18 polynucleotide probes can be used to detect abnormalities
or genotypes associated with these cancer susceptibility markers.
Because there is abundant evidence for cancer resulting from
mutations in the 12q15 region, and ZCYTO18 also maps to this
chromosomal locus, mutations in ZCYTO18 may also be directly
involved in or associated with cancers, such as lymphoid cell
cancers or other tumors.
[0194] 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-ZCYTO18 antibodies,
polynucleotides, and polypeptides can be used for the detection of
ZCYTO18 polypeptide, mRNA or anti-ZCYTO18 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, ZCYTO18 polynucleotide probes can be
used to detect abnormalities or genotypes associated with
chromosome 12q15 deletions and translocations associated with human
diseases, such as multiple lipomatosis and malignant mixoid
liposarcoma (above), or other translocations involved with
malignant progression of tumors or other 12q15 mutations, which are
expected to be involved in chromosome rearrangements in malignancy;
or in other cancers. Similarly, ZCYTO18 polynucleotide probes can
be used to detect abnormalities or genotypes associated with
chromosome 12q15 trisomy and chromosome loss associated with human
diseases or spontaneous abortion. Moreover, amongst other genetic
loci, those for Scapuloperoneal spinal muscular atrophy
(12q13.3-q15), mucopolysaccaridosis (12q14), pseudo-vitamin D
deficiency Rickets as a result of mutation in Cytochrome CYP27B1
(12q14) and others, all manifest themselves in human disease states
as well as map to this region of the human genome. See the Online
Mendellian Inheritance of Man (OMIM) gene map, and references
therein, for this region of chromosome 3 on a publicly available
WWW server (http://www3.ncbi.nlm.nih.gov/htbin-po-
st/Omim/getmap?chromosome=12q15). All of these serve as possible
candidate genes for an inheritable disease which show linkage to
the same chromosomal region as the ZCYTO18 gene. Thus, ZCYTO18
polynucleotide probes can be used to detect abnormalities or
genotypes associated with these defects.
[0195] As discussed above, defects in the ZCYTO18 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 ZCYTO18 genetic defect. In addition, ZCYTO18 polynucleotide
probes can be used to detect allelic differences between diseased
or non-diseased individuals at the ZCYTO18 chromosomal locus. As
such, the ZCYTO18 sequences can be used as diagnostics in forensic
DNA profiling.
[0196] 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 ZCYTO18 polynucleotide probe may
comprise an entire exon or more. Exons are readily determined by
one of skill in the art by comparing ZCYTO18 sequences (SEQ ID NO:
1) with the human genomic DNA for ZCYTO18 (Genbank Accession No.
AC007458). 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 ZCYTO18
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 method such as visualizing the first reaction
product with a ZCYTO18 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. 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 ZCYTO18 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 form any
tissue or other biological sample from a patient, such as but 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
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)).
[0197] Aberrations associated with the ZCYTO18 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 al. (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 ZCYTO18 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 al.
(eds.), Current Protocols in Human Genetics, at pages 7.1.6 to
7.1.7 (John Wiley & Sons 1998)).
[0198] Mice engineered to express the ZCYTO18 gene, referred to as
"transgenic mice," and mice that exhibit a complete absence of
ZCYTO18 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 ZCYTO18, 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 ZCYTO18
polypeptide, polypeptide fragment or a mutant thereof may alter
normal cellular processes, resulting in a phenotype that identifies
a tissue in which ZCYTO18 expression is functionally relevant and
may indicate a therapeutic target for the ZCYTO18, its agonists or
antagonists. For example, a preferred transgenic mouse to engineer
is one that over-expresses the mature ZCYTO18 polypeptide (amino
acid residues 23 (Pro) to 167 (Ile) of SEQ ID NO: 3). Moreover,
such over-expression may result in a phenotype that shows
similarity with human diseases. Similarly, knockout ZCYTO18 mice
can be used to determine where ZCYTO18 is absolutely required in
vivo. The phenotype of knockout mice is predictive of the in vivo
effects of that a ZCYTO18 antagonist, such as those described
herein, may have. The human or mouse ZCYTO18 cDNA can be used to
generate knockout mice. These mice may be employed to study the
ZCYTO18 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 ZCYTO18 antisense
polynucleotides or ribozymes directed against ZCYTO18, described
herein, can be used analogously to transgenic mice described above.
Studies may be carried out by administration of purified ZCYTO18
protein, as well.
[0199] For pharmaceutical use, the proteins of the present
invention are formulated for parenteral, particularly intravenous
or subcutaneous, delivery according to conventional methods.
Intravenous administration will be by bolus injection or infusion
over a typical period of one to several hours. In general,
pharmaceutical formulations will include a ZCYTO18 protein in
combination with a pharmaceutically acceptable vehicle, such as
saline, buffered saline, 5% dextrose in water or the like.
Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to prevent
protein loss on vial surfaces, etc. Methods of formulation are well
known in the art and are disclosed, for example, in Remington: The
Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing
Co., Easton, Pa., 19th ed., 1995. Therapeutic doses will generally
be in the range of 0.1 to 100 .mu.g/kg of patient weight per day,
preferably 0.5-20 mg/kg per day, with the exact dose determined by
the clinician according to accepted standards, taking into account
the nature and severity of the condition to be treated, patient
traits, etc. Determination of dose is within the level of ordinary
skill in the art. The proteins may be administered for acute
treatment, over one week or less, often over a period of one to
three days or may be used in chronic treatment, over several months
or years. In general, a therapeutically effective amount of ZCYTO18
is an amount sufficient to produce a clinically significant change
in hematopoietic or immune function.
[0200] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Using an EST Sequence to Identify and Clone ZCYTO18
[0201] Novel ZCYTO18 encoding polynucleotides and polypeptides of
the present invention were initially identified by querying an EST
database for sequences homologous to conserved motifs within the
cytokine family. A primary expressed sequence tag (EST) from a
human T-lymphocyte cDNA library was identified.
[0202] An initial partial sequence was obtained from the sequencing
of the EST (INC4345486). Additional 5' sequence was obtained from
sequencing the cDNA fragment obtained by PCR from the Northern
Analysis (Example 2, below) and by further PCR using
oligonucleotides ZC25,840 (SEQ ID NO: 5) and ZC25,841 (SEQ ID NO:
6) in a PCR using human mixed lymphocyte reaction (MLR) cDNA.
Thermocycler conditions were as described in Example 2 below. The
resulting 1082 bp full length sequence is disclosed in SEQ ID NO: 1
and the corresponding amino acid sequence is shown in SEQ ID NO: 2
and SEQ ID NO: 3. The full length novel cytokine was designated
ZCYTO18.
Example 2
Zcyto18 Tissue Distribution
[0203] Northerns were performed using Human Multiple Tissue Blots
(MTN1, MTN2 and MTN3) from Clontech (Palo Alto, Calif.) to
determine the tissue distribution of human ZCYTO18. A 237 bp cDNA
probe was obtained using the PCR. Oligonucleotides ZC25,838 (SEQ ID
NO: 7) and ZC25,839 (SEQ ID NO: 8) were used as primers. Marathon
cDNA, synthesized in-house using Marathon cDNA Kit (Clontech) and
protocol, was used as a template. The following human tissue
specific cDNAs were also used: lymph node, bone marrow, CD4+, CD8+,
spleen, and MLR, along with human genomic DNA (Clontech).
Thermocycler conditions were as follows: one cycle at 94.degree. C.
for 2 min.; 35 cycles of 94.degree. C. for 15 sec., 62.degree. C.
for 20 sec., and 72.degree. C. for 30 sec.; one cycle at 72.degree.
C. for 7 min.; followed by a 4.degree. C. hold. The correct
predicted band size (237 bp) was observed on a 4% agarose gel in
CD4+ and MLR reactions, along with the genomic DNA reaction. A band
was excised and purified using a Gel Extraction Kit (Qiagen,
Chatsworth, Calif.) according to manufacturer's instructions. The
cDNA was radioactively labeled using a Rediprime II DNA labeling
kit (Amersham, Arlington Heights, Ill.) according to the
manufacturer's specifications. The probe was purified using a
NUCTRAP push column (Stratagene Cloning Systems, La Jolla, Calif.).
EXPRESSHYB (Clontech, Palo Alto, Calif.) solution was used for
prehybridization and as a hybridizing solution. Hybridization took
place overnight at 55.degree. C., using 2.times.10.sup.6 cpm/ml
labeled probe. The blots were then washed in 2.times.SSC and 0.1%
SDS at room temperature, then with 2.times.SSC and 0.1% SDS at
65.degree. C., followed by a wash in 0.1.times.SSC and 0.1% SDS at
65.degree. C. The blots were exposed 5 days to Biomax MS film
(Kodak, Rochester, N.Y.). No transcript signals were observed on
the MTN blots after development.
[0204] A RNA Master Dot Blot (Clontech) that contained RNAs from
various tissues that were normalized to 8 housekeeping genes was
also probed and hybridized as described above. A signal was
observed in genomic DNA. While a faint signal in lymph node and
very faint signals in fetal liver, skeletal muscle, and placenta
were observed it was inconclusive whether these signals were
significantly above background.
Example 3
Identification of Cells Expressing ZCYTO18 Using RT-PCR
[0205] Specific human cell types were isolated and screened for
ZCYTO18 expression by RT-PCR. B-cells were isolated from fresh
human tonsils by mechanical disruption through 100 .mu.m nylon cell
strainers (Becton Dickinson Biosciences, Franklin Lakes, N.J.). The
B-cell suspensions were enriched for CD19+ B-cells by positive
selection with VarioMACS VS+ magnetic column and CD19 microbeads
(Miltenyi Biotec, Auburn, Calif.) as per manufacturer's
instructions. T-cells were isolated from human apheresed blood
samples. CD3+ T-cells were purified by CD3 microbead to VarioMACS
positive selection and monocytes were purified by VarioMACS
negative iselection columns (Miltenyi) as per manufacturer's
instructions. Samples from each population were stained and
analyzed by fluorescent antibody cell sorting (FACS) (Bectin
Dickinson, San Jose, Calif.) analysis to determine the percent
enrichment and resulting yields. CD19+ B-cells were approximately
96% purified, CD3+ T-cells were approximately 95% purified, and
monocytes were approximately 96% purified.
[0206] RNA was prepared, using a standard method in the art, from
all three cell types that were either resting or activated. RNA was
isolated from resting cells directly from the column preparations
above. The CD19+ and CD3+ cells were activated by culturing at
500,000 cells/ml in RPMI+10% FBS containing PMA 5 ng/ml
(Calbiochem, La Jolla, Calif.) and lonomycin 0.5 ug/ml (Calbiochem)
for 4 and 24 hours. The monocytes were activated by culturing in
RPMI+10% FBS containing LPS 10 ng/ml (Sigma St. Louis Mo.) and
rhIFN-g 10 ng/ml (R&D, Minneapolis, Minn.) for 24 hours. Cells
were harvested and washed in PBS. RNA was prepared from the cell
pellets using RNeasy Midiprep.TM. Kit (Qiagen, Valencia, Calif.) as
per manufacturer's instructions and first strand cDNA synthesis was
generated with Superscript II.TM. Kit (GIBCO BRL, Grand Island,
N.Y.) as per manufacturers protocol.
[0207] Oligos ZC25,838 (SEQ ID NO: 7) and ZC25,840 (SEQ ID NO: 5)
were used in a PCR reaction to screen the above described samples
for a 473 bp fragment corresponding to ZCYTO18 message. PCR
amplification was performed with Taq Polymerase (BRL Grand Island
N.Y.), and reaction conditions as follows: 35 cycles of 94.degree.
C. for 15 sec., 62.degree. C. for 20 sec., 72.degree. C. for 30
sec.; 1 cycle at 72.degree. C. for 7 min.; and 4.degree. C. soak. 5
ul of each 50 .mu.l reaction volume was run on a 0.9% agarose
0.5XTBE gel to identify resultant products. Table 5 below describes
the results. PCR products were scored as (-) for no product, (+)
for expected PCR product visible, (++) increased presence of PCR
product and (+++) being the strongest signal.
5TABLE 5 Cells expressing ZCYTO18 using RT-PCR cDNA Source
Activation PCR Product CD3 + cells 0 hr resting + 4-hr activated
+++ CD19 + cells 4 hr activated ++ 24 hr activated + Monocytes 24
hr activated -
[0208] These results indicated that ZCYTO18 message is present in
resting CD3+ T-cells and increases with mitogenic activation. It
also appears to be expressed by 4-hr activated human CD19+ B-cells
and reduced in expression in 24 hr activated B-cells. There was no
apparent message in activated monocytes.
Example 4
Identification of hZCYTO18 Message in an Activated T-Cell
Library
[0209] A. The vector for CD3+ Selected Library Construction
[0210] The vector for CD3+ selected library construction was
pZP7NX. The pZP7NX vector was previously 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: 9) and
ZC13,945 (SEQ ID NO: 10), and pZeoSV2(+) as a template. There are
additional PstI and BclI restriction sites in primer ZC13,946 (SEQ
ID NO: 9), and additional NcoI and SfuI sites in primer ZC13,945
(SEQ ID NO: 10). 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 with the same
restriction enzymes (BclI and SfuI).
[0211] 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 Xho1 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 EcoR1 (Life Technologies
Gaithersberg, Md.) and 20 units of Xho1 (Boehringer Mannheim
Indianapolis, Ind.) 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 1XTAE 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.
[0212] B. Preparation of Primary Human Activated CD3+ Selected Cell
cDNA Library
[0213] 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. 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.
[0214] 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: 11)
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.SUPERSCRIPT.RTM. 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. at 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.lwash with 70% ethanol. The
cDNA was resuspended in 98 .mu.l water for use in second strand
synthesis.
[0215] 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 MgCl2, 50 mM (NH4)2SO4), 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.
[0216] 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 tO l of 10.times. mung bean nuclease
buffer (Life technologies), 5 .mu.l of mung bean nuclease
(Pharmacia Biotech Corp.) diluted to 1U/.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.
[0217] 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 MgCl2), 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.
[0218] Eco RI 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.
[0219] 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 primer. Restriction enzyme
digestion was carried out in a reaction mixture containing 35 .mu.l
of the ligation mix described above, 6 .rho.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.
[0220] 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.
[0221] 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 10 mM
ATP, 1 .mu.l pZP7NX digested with EcoR1-Xho1, 11 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 11. 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 controllera (Biorad) set to 2.5 KV, 251F, 200
ohms. These cells were immediately resuspended in 1 ml. SOC broth
(Manniatis, et al. su ra.) followed by 50011 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.
[0222] C. PCR Identification of ZCYTO18 Message in Activated T-Cell
Library
[0223] PCR was performed using oligos ZC25,838 (SEQ ID NO: 7) and
ZC25,840 (SEQ ID NO: 5) to screen the above library for presence of
a 473 bp product corresponding to ZCYTO18 clones. PCR amplification
was performed with Taq Polymerase (BRL Grand Island N.Y.), and
conditions as follows: 30 cycles of 94.degree. C. for 15 sec.,
62.degree. C. 20 sec., 72.degree. C. 30 sec.; 1 cycle at 72.degree.
C. for 7 min.; and a 4.degree. C. soak. 5 .mu.l of each 50 .mu.l
reaction volume was run on a 0.9% agarose 0.5.times. TBE gel to
identify resultant products. Table 6 below describes the results.
PCR products were scored as (-) for no product, (+) for expected
PCR product visible, (++) increased presence of PCR product and
(+++) being the strongest signal.
6TABLE 6 Identification of ZCYTO18 message in activated T-Cell
Library Template PCR Product 1 ng Activated Library + 10 ng
Activated Library ++ 100 ng Activated Library +++ 100 ng Vector
Control - No Template Control -
[0224] These results indicate the presence of a ZCYTO18 cDNA clone
and therefore message in activated CD3+ T-cells.
Example 5
Southern Blot Analysis
[0225] Southern blots were performed using EVO Mammalian
Group/EcoRI Southern Blots (Quantum Biotechnologies, Inc.,
Montreal, Canada) to determine the presence of orthologous ZCYTO18
sequences. A ZCYTO18 probe was generated by labeling 25 ng of
ZCYTO18 fragment, as described in Example 2, using Prime-It II
Random Primer labeling kit (Stratagene, La Jolla, Calif.).
Hybridization was performed using Expresshyb (Clontech) with
5.times.10.sup.5 cpm/ml probe and conditions of 65.degree. C.
overnight. Stringency washes were performed with 0.2.times.SSC,
0.1% SDS at 45.degree. C. The blot was exposed overnight at
-80.degree. C. to X-ray film and analyzed.
[0226] Results showed a strong approximately 1 kb band in the human
genomic DNA sample with weaker bands present at approximately 7 and
20 kb for murine genomic DNA demonstrating the presence of a
putative murine homolog for ZCYTO18.
[0227] The mouse cDNA sequence was cloned using standard methods
and is shown in SEQ ID NO: 37, and corresponding polypeptides
sequence shown in SEQ ID NO: 38.
Example 6
Chromosomal Assignment and Placement of Zcyto18
[0228] Zcyto18 was mapped to 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
(http:H/shgc-www.stanford.edu) allows chromosomal localization of
markers and genes.
[0229] For the mapping of Zcyto18 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
85 PCR reactions consisted of 2 .mu.l 10.times. KlenTaq PCR
reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.),
1.6 .mu.l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,
Calif.), 1 .mu.l sense primer, ZC 26,414 (SEQ ID NO: 12), 1 .mu.l
antisense primer, ZC 26,415 (SEQ ID NO: 13), 2 .mu.l "RediLoad"
(Research Genetics, Inc., Huntsville, Ala.), 0.4 .mu.l 50.times.
Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 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 5 minute
denaturation at 94.degree. C., 35 cycles of a 45 seconds
denaturation at 94.degree. C., 45 seconds annealing at 66.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.
[0230] The results showed linkage of Zcyto18 to the chromosome 12
marker SHGC-17533 with a LOD score of >12 and at a distance of 0
cR.sub.--10000 from the marker. The use of surrounding genes and
markers positions Zcyto18 in the 12q14-q24.3 chromosomal
region.
Example 7
Construct for Generating CEE-tagged ZCYTO18
[0231] Oligonucleotides were designed to generate a PCR fragment
containing the Kozak sequence and the coding region for ZCYTO18,
without its stop codon. These oligonucleotides were designed with a
KpnI site at the 5' end and a BamHI site at the 3' end to
facilitate cloning into pHZ200-CEE, our standard vector for
mammalian expression of C-terminal Glu-Glu tagged (SEQ ID NO: 14)
proteins. The pHZ200 vector contains an MT-1 promoter.
[0232] PCR reactions were carried out using Turbo Pfu polymerase
(Stratagene) to amplify a ZCYTO18 cDNA fragment. About 20 ng human
ZCYTO18 polynucleotide template (SEQ ID NO: 1), and
oligonucleotides ZC28590 (SEQ ID NO: 16) and ZC28580 (SEQ ID NO:
17) were used in the PCR reaction. PCR reaction conditions were as
follows: 95.degree. C. for 5 minutes,; 30 cycles of 95.degree. C.
for 60 seconds, 55.degree. C. for 60 seconds, and 72.degree. C. for
60 seconds; and 72.degree. C. for 10 minutes; followed by a
4.degree. C. hold. PCR products were separated by agarose gel
electrophoresis and purified using a QiaQuick.TM. (Qiagen) gel
extraction kit. The isolated, approximately 600 bp, DNA fragment
was digested with KpnI and BanmHI (Boerhinger-Mannheim), gel
purified as above and ligated into pHZ200-CEE that was previously
digested with KpnI and BamHI.
[0233] About one microliter of the ligation reaction was
electroporated into DH10B ElectroMax.TM. competent cells (GIBCO
BRL, Gaithersburg, Md.) according to manufacturer's direction and
plated onto LB plates containing 100 .mu.g/ml ampicillin, and
incubated overnight. Colonies were picked and screened by PCR using
oligonucleotides ZC28,590 (SEQ ID NO: 16) and ZC28,580 (SEQ ID NO:
17), with PCR conditions as described above. Clones containing
inserts were then sequenced to confirm error-free ZCYTO18 inserts.
Maxipreps of the correct pHZ200-ZCYTO18-CEE construct, as verified
by sequence analysis, were performed.
Example 8
Transfection And Expression Of ZCYTO18-CEE Polypeptides
[0234] BHK 570 cells (ATCC No. CRL-10314), were plated at about
1.times.10.sup.6 cells/100 mm culture dish in 6.4 ml of serum free
(SF) DMEM media (DMEM, Gibco/BRL High Glucose) (Gibco BRL,
Gaithersburg, Md.). The cells were transfected with an expression
plasmid containing ZCYTO18-CEE described above (Example 7), using
Lipofectin.TM. (Gibco BRL), in serum free (SF) DMEM according to
manufacturer's instructions.
[0235] The cells were incubated at 37.degree. C. for approximately
five hours, then 10 ml of DMEM/10% fetal bovine serum (FBS)
(Hyclone, Logan, Utah) was added. The plates were incubated at
37.degree. C., 5% CO.sub.2, overnight and the DMEM/10% FBS media
was replaced with selection media (5% FBS/DMEM with 1 .mu.M
methotrexate (MTX)) the next day.
[0236] Approximately 7-10 days post-transfection, pools of cells or
colonies were mechanically picked to 12-well plates in one ml of 5%
FCS/DMEM with 5 .mu.M MTX, then grown to confluence. Cells were
then incubated in 5% FCS/DMEM with 10 .mu.M MTX for at least 14
days. Conditioned media samples from positive expressing clonal
colonies and pools were then tested for expression levels via
SDS-PAGE and Western analysis. A high-expressing clones or pools
were picked and expanded for ample generation of conditioned media
for purification of the ZCYTO18-CEE expressed by the cells (Example
9).
Example 9
Purification of ZCYTO18-CEE From BHK 570 Cells
[0237] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying ZCYTO18
polypeptide containing C-terminal GluGlu (EE) tags (SEQ ID NO: 14).
A Protease inhibitor solution was added to the concentrated
conditioned media containing ZCYTO18-CEE (Example 8) to final
concentrations of 2.5 mM ethylenediamlnetetraacetic acid (EDTA,
Sigma Chemical Co. St. Louis, Mo.), 0.003 mM leupeptin
(Boehringer-Mannheim, Indianapolis, Ind.), 0.001 mM pepstatin
(Boehringer-Mannheim) and 0.4 mM Pefabloc
(Boehringer-Mannheim).
[0238] About 100 ml column of anti-EE G-Sepharose (prepared as
described below) was poured in a Waters AP-5, 5 cm.times.10 cm
glass column. The column was flow packed and equilibrated on a
BioCad Sprint (PerSeptive BioSystems, Framingham, Mass.) with
phosphate buffered saline (PBS) pH 7.4. The concentrated
conditioned media was 0.2 micron sterile filtered, pH adjusted to
7.4, then loaded on the column overnight with about 1 ml/minute
flow rate. The column was washed with 10 column volumes (CVs) of
phosphate buffered saline (PBS, pH 7.4), then plug eluted with 200
ml of PBS (pH 6.0) containing 0.1 mg/ml EE peptide (Anaspec, San
Jose, Calif.) at 5 ml/minute. The EE peptide used has the sequence
EYMPME (SEQ ID NO: 14). Five ml fractions were collected over the
entire elution chromatography and absorbance at 280 and 215 nM were
monitored; the pass through and wash pools were also saved and
analyzed. The EE-polypeptide elution peak fractions were analyzed
for the target protein via SDS-PAGE Silver staining and Western
Blotting with the anti-EE HRP conjugated antibody. The polypeptide
elution fractions of interest were pooled and concentrated from 60
ml to 5.0 ml using a 10,000 Dalton molecular weight cutoff membrane
spin concentrator (Millipore, Bedford, Mass.) according to the
manufacturer's instructions.
[0239] To separate ZCYTO18-CEE polypeptide from free EE peptide and
any contaminating co-purifying proteins, the pooled concentrated
fractions were subjected to size exclusion chromatography on a
1.5.times.90 cm Sephadex S200 (Pharmacia, Piscataway, N.J.) column
equilibrated and loaded in PBS at a flow rate of 1.0 ml/min using a
BioCad Sprint. 1.5 ml fractions were collected across the entire
chromatography and the absorbance at 280 and 215 nM were monitored.
The peak fractions were characterized via SDS-PAGE Silver staining,
and only the most pure fractions were pooled. This material
represented purified ZCYTO18-CEE polypeptide.
[0240] This purified material was finally subjected to a 4 ml
ActiClean Etox (Sterogene) column to remove any remaining
endotoxins. The sample was passed over the PBS equilibrated gravity
column four times then the column was washed with a single 3 ml
volume of PBS, which was pooled with the "cleaned" sample. The
material was then 0.2 micron sterile filtered and stored at
-80.degree. C. until it was aliquoted.
[0241] On Western blotted, Coomassie Blue and Silver stained
SDS-PAGE gels, the ZCYTO18-CEE polypeptide was two major bands and
two mionor bands. 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. In a Western blot analysis, all bands were
immunoreactive with a rabbit anti-ZCYTO18-peptide antibody (Example
16). The 4 bands likely represent different glycosylated forms of
the ZCYTO18 polypeptide.
[0242] To prepare anti-EE Sepharose, a 100 ml bed volume of protein
G-Sepharose (Pharmacia, Piscataway, N.J.) was washed 3 times with
100 ml of PBS containing 0.02% sodium azide using a 500 ml Nalgene
0.45 micron filter unit. The gel was washed with 6.0 volumes of 200
mM triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.), and an
equal volume of EE antibody solution containing 900 mg of antibody
was added. After an overnight incubation at 4.degree. C., unbound
antibody was removed by washing the resin with 5 volumes of 200 mM
TEA as described above. The resin was resuspended in 2 volumes of
TEA, transferred to a suitable container, and
dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in
TEA, was added to a final concentration of 36 mg/ml of protein
G-Sepharose gel. The gel was rocked at room temperature for 45 min
and the liquid was removed using the filter unit as described
above. Nonspecific sites on the gel were then blocked by incubating
for 10 min. at room temperature with 5 volumes of 20 mM
ethanolamine in 200 mM TEA. The gel was then washed with 5 volumes
of PBS containing 0.02% sodium azide and stored in this solution at
4.degree. C.
Example 10
Generation of Non-tagged ZCYTO18 Recombinant Adenovirus
[0243] The protein coding region of human ZCYTO18 (SEQ ID NO: 1;
SEQ ID NO: 2) was amplified by PCR using primers that added FseI
and AscI restriction sties at the 5' and 3' termini respectively.
PCR primers ZC26665 (SEQ ID NO: 20) and ZC26666 (SEQ ID NO: 21)
were used with pINCY template plasmid containing the full-length
ZCYTO18 cDNA in a PCR reaction as follows: one cycle at 95.degree.
C. for 5 minutes; followed by 18 cycles at 95.degree. C. for 0.5
min., 58.degree. C. for 0.5 min., and 72.degree. C. for 0.5 min.;
followed by 72.degree. C. for 7 min.; followed by a 4.degree. C.
soak. The PCR reaction product was loaded onto a 1.2% (low melt)
SeaPlaque GTG (FMC, Rockland, Me.) gel in TAE buffer. The ZCYTO18
PCR product was excised from the gel and the gel slice melted at
70.mu..degree. C., extracted twice with an equal volume of Tris
buffered phenol, and EtOH precipitated.
[0244] The 540 bp ZCYTO18 PCR product was digested with FseI and
AscI enzymes. The cDNA was isolated on a 1% low melt SeaPlaque
GTG.TM. (FMC, Rockland, Me.) gel and was then excised from the gel
and the gel slice melted at 70.degree. C., extracted twice with an
equal volume of Tris buffered phenol, and EtOH precipitated. The
DNA was resuspended in 10 .mu.l H.sub.2O.
[0245] The ZCYTO18 cDNA was cloned into the FseI-AscI sites of a
modified pAdTrack CMV (He, T-C. et al., PNAS 95:2509-2514, 1998).
This construct contains the GFP marker gene. The CMV promoter
driving GFP expression was replaced with the SV40 promoter and the
SV40 polyadenylation signal was replaced with the human growth
hormone polyadenylation signal. In addition, the native polylinker
was replaced with Fsel, EcoRV, and AscI sites. This modified form
of pAdTrack CMV was named pZyTrack. Ligation was performed using
the Fast-Link.TM. DNA ligation and screening kit (Epicentre
Technologies, Madison, Wis.). Clones containing the ZCYTO18 insert
were identified by standard mini prep analysis. The cloned ZCYTO18
cDNA was sequenced to verify no errors were introduced during PCR.
In order to linearize the plasmid, approximately 5 .mu.g of the
pZyTrack ZCYTO18 plasmid was digested with PmeI. Approximately 1
.mu.g of the linearized plasmid was cotransformed with 200 ng of
supercoiled pAdEasy (He et al., supra.) into BJ5183 cells. The
co-transformation was done using a Bio-Rad Gene Pulser at 2.5 kV,
200 ohms and 25 mFa. The entire co-transformation was plated on 4
LB plates containing 25 .mu.g/ml kanamycin. The smallest colonies
were picked and expanded in LB/kanamycin and recombinant adenovirus
DNA identified by standard DNA miniprep procedures. Digestion of
the recombinant adenovirus DNA with FseI-AscI confirmed the
presence of ZCYTO18. The recombinant adenovirus miniprep DNA was
transformed into DH10B competent cells and DNA prepared using a
Qiagen maxi prep kit as per kit instructions.
[0246] Transfection of 293a Cells with Recombinant DNA
[0247] Approximately 5 .mu.g of recombinant adenoviral DNA was
digested with Pacd enzyme (New England Biolabs) for 3 hours at
37.degree. C. in a reaction volume of 100 .mu.l containing 20-30U
of Pacd. The digested DNA was extracted twice with an equal volume
of phenol/chloroform and precipitated with ethanol. The DNA pellet
was resuspended in 5 .mu.l distilled water. A T25 flask of QBI-293A
cells (Quantum Biotechnologies, Inc. Montreal, Qc. Canada),
inoculated the day before and grown to 60-70% confluence, were
transfected with the Pacd digested DNA. The PacI-digested DNA was
diluted up to a total volume of 50 .mu.l with sterile HBS (150 mM
NaCl, 20 mM HEPES). In a separate tube, 25 .mu.l DOTAP (Boehringer
Mannheim, 1 mg/ml) was diluted to a total volume of 100 .mu.l with
HBS. The DNA was added to the DOTAP, mixed gently by pipeting up
and down, and left at room temperature for 15 minutes. The media
was removed from the 293A cells and washed with 5 ml serum-free
MEMalpha (Gibco BRL) containing 1 mM Sodium Pyruvate (GibcoBRL),
0.1 mM MEM non-essential amino acids (GibcoBRL) and 25 mM HEPES
buffer (GibcoBRL). 5 ml of serum-free MEM was added to the 293A
cells and held at 37.degree. C. The DNA/lipid mixture was added
drop-wise to the T25 flask of 293A cells, mixed gently and
incubated at 37.degree. C. for 4 hours. After 4 hours the media
containing the DNA/lipid mixture was aspirated off and replaced
with 5 ml complete MEM containing 5% fetal bovine serum. The
transfected cells were monitored for Green Fluorescent Protein
(GFP) expression and formation of foci, i.e., viral plaques.
[0248] Seven days after transfection of 293A cells with the
recombinant adenoviral DNA, the cells expressed the GFP protein and
started to form foci. These foci are viral "plaques" and the crude
viral lysate was collected by using a cell scraper to collect all
of the 293A cells. The lysate was transferred to a 50 ml conical
tube. To release most of the virus particles from the cells, three
freeze/thaw cycles were done in a dry ice/ethanol bath and a
37.degree. C. waterbath.
[0249] Amplification of Recombinant Adenovirus (rAdV)
[0250] The crude lysate was amplified (Primary (1.degree.)
amplification) to obtain a working "stock" of zsig45 rAdV lysate.
Ten 10 cm plates of nearly confluent (80-90% ) 293A cells were set
up 20 hours previously, 200 ml of crude rAdV lysate added to each
10 cm plate and monitored for 48 to 72 hours looking for CPE under
the white light microscope and expression of GFP under the
fluorescent microscope. When all of the 293A cells showed CPE
(Cytopathic Effect) this 1.degree. stock lysate was collected and
freeze/thaw cycles performed as described under Crude rAdV
Lysate.
[0251] Secondary (2.degree.) Amplification of zsig46 rAdV was
obtained as follows: Twenty 15 cm tissue culture dishes of 293A
cells were prepared so that the cells were 80-90% confluent. All
but 20 mls of 5% MEM media was removed and each dish was inoculated
with 300-500 ml 10 amplified rAdv lysate. After 48 hours the 293A
cells were lysed from virus production and this lysate was
collected into 250 ml polypropylene centrifuge bottles and the rAdV
purified.
[0252] rAdV/cDNA Purification
[0253] NP-40 detergent was added to a final concentration of 0.5%
to the bottles of crude lysate in order to lyse all cells. Bottles
were placed on a rotating platform for 10 min. agitating as fast as
possible without the bottles falling over. The debris was pelleted
by centrifugation at 20,000.times.G for 15 minutes. The supernatant
was transferred to 250 ml polycarbonate centrifuge bottles and 0.5
volumes of 20% PEG8000/2.5M NaCl solution added. The bottles were
shaken overnight on ice. The bottles were centrifuged at
20,000.times.G for 15 minutes and supernatant discarded into a
bleach solution. The white precipitate in two vertical lines along
the wall of the bottle on either side of the spin mark is the
precipitated virus/PEG. Using a sterile cell scraper, the
precipitate from 2 bottles was resuspended in 2.5 ml PBS. The virus
solution was placed in 2 ml microcentrifuge tubes and centrifuged
at 14,000.times.G in the microfuge for 10 minutes to remove any
additional cell debris. The supernatant from the 2 ml
microcentrifuge tubes was transferred into a 15 ml polypropylene
snapcap tube and adjusted to a density of 1.34 g/ml with cesium
chloride (CsCl). The volume of the virus solution was estimated and
0.55 g/ml of CsCl added. The CsCl was dissolved and 1 ml of this
solution weighed 1.34 g. The solution was transferred polycarbonate
thick-walled centrifuge tubes 3.2 ml (Beckman #362305) and spin at
80,000 rpm (348,000.times.G) for 3-4 hours at 25.degree. C. in a
Beckman Optima TLX microultracentrifuge with the TLA-100.4 rotor.
The virus formed a white band. Using wide-bore pipette tips, the
virus band was collected.
[0254] The virus from the gradient has a large amount of CsCl which
must be removed before it can be used on cells. Pharmacia PD-10
columns prepacked with Sephadex G-25M (Pharmacia) were used to
desalt the virus preparation. The column was equilibrated with 20
ml of PBS. The virus was loaded and allow it to run into the
column. 5 ml of PBS was added to the column and fractions of 8-10
drops collected. The optical densities of 1:50 dilutions of each
fraction was determined at 260 nm on a spectrophotometer. A clear
absorbance peak was present between fractions 7-12. These fractions
were pooled and the optical density (OD) of a 1:25 dilution
determined. A formula is used to convert OD into virus
concentration: (OD at 260 nm)(25)(1.1.times.10.sup.12)=virions/ml.
The OD of a 1:25 dilution of the ZCYTO18 rAdV was 0.134, giving a
virus concentration of 3.7.times.10.sup.12 virions/ml.
[0255] To store the virus, glycerol was added to the purified virus
to a final concentration of 15% , mixed gently but effectively, and
stored in aliquots at -80.degree. C.
[0256] Tissue Culture Infectious Dose at 50% CPE (TCID 50) Viral
Titration Assay
[0257] A protocol developed by Quantum Biotechnologies, Inc.
(Montreal, Qc. Canada) was followed to measure recombinant virus
infectivity. Briefly, two 96-well tissue culture plates were seeded
with 1.times.10.sup.4 293A cells per well in MEM containing 2%
fetal bovine serum for each recombinant virus to be assayed. After
24 hours 10-fold dilutions of each virus from 1.times.10.sup.-2 to
1.times.10.sup.-14 were made in MEM containing 2% fetal bovine
serum. 100 .mu.l of each dilution was placed in each of 20 wells.
After 5 days at 37.degree. C., wells were read either positive or
negative for Cytopathic Effect (CPE) and a value for "Plaque
Forming Units/ml" (PFU) is calculated.
[0258] TCID.sub.50 formulation used was as per Quantum
Biotechnologies, Inc., above. The titer (T) is determined from a
plate where virus used is diluted from 10.sup.-2 to 10.sup.-14, and
read 8 days after the infection. At each dilution a ratio (R) of
positive wells for CPE per the total number of wells is
determined.
[0259] To Calculate titer of the undiluted virus sample: the
factor, "F"=1+d(S-0.5); where "S" is the sum of the ratios (R); and
"d" is Log10 of the dilution series, for example, "d" is equal to 1
for a ten-fold dilution series. The titer of the undiluted sample
is T=10.sup.(1+F)=TCID.sub.50/ml. To convert TCID.sub.50/ml to
pfu/ml, 0.7 is subtracted from the exponent in the calculation for
titer (T). The ZCYTO18 adenovirus had a titer of
2.8.times.10.sup.11 pfu/ml.
Example 11
In vivo Affects of ZCYTO18 Polypeptide
[0260] Mice (female, C57B1, 8 weeks old; Charles River Labs,
Kingston, N.Y.) were divided into three groups. On day 0, parental
or ZCYTO18 adenovirus (Example 10) was administered to the first
(n=8) and second (n=8) groups, respectively, via the tail vein,
with each mouse receiving a dose of .about.1.times.10.sup.11
particles in .about.0.1 ml volume. The third group (n=8) received
no treatment. On days 12, mice were weighed and blood was drawn
from the mice. Samples were analyzed for complete blood count (CBC)
and serum chemistry. Statistically significant elevations in
neutrophil and platelet counts were detected in the blood samples
from the ZCYTO18 adenovirus administered group relative to the
parental adenovirus treated group. Also, at day 12 lymphocyte
counts were significantly reduced from the ZCYTO18 adenovirus
administered group relative to the parental adenovirus treated
group, and they rebounded to normal levels by day 21. In addition,
the ZCYTO18 adenovirus treated mice decreased in body weight, while
parental adenovirus treated mice gained weight. The elevated
platelet and neutrophil count, and the loss of body weight are
still significant as compared to the control group. The liver
chemistry test indicated the increased level of globulin and
decreased level of albumin concentration, which is consistant with
the observation of inflammatory response induced by
TNF-.alpha..
[0261] The results suggested that ZCYTO18 affects hematopoiesis,
i.e., blood cell formation in vivo. As such, ZCYTO18 could have
biological activities affecting different blood precursors,
progenitors or stem cells, and a resulting increase or decrease of
certain differentiated blood cells in a specific lineage. For
instance, ZCYTO18 appears to reduce lymphocytes, which is likely
due to inhibition of the committed progenitor cells that give rise
to lymphoid cells. This finding agrees with the inhibitory effects
of ZCYTO18 on the proliferation and/or growth of myeloid stem cells
(Example 23), supporting the notion that ZCYTO18 could play a role
in anemia, infection, inflammation, and/or immune diseases by
influencing blood cells involved in these process. Antagonists
against ZCYTO18, such as antibodies or a soluble receptor
antagonist could be used as therapeutic reagents in these diseases.
It is also possible that ZCYTO18 directly affects the release and
survival of platelets in peripheral blood or other vascularized
tissues such as liver. That is, besides working through a
hematopoisis loop (differentiation, proliferation of stem cells),
zcyto18 might directly affect the release, stablization or
depletion of platelets and neutrophils in peripheral blood or some
target tissue and organs. ZCYTO18 also affects the number of
granulocytes in the peripheral blood. Extramedullary sites of
hematopoiesis (e.g. liver) are also targets for ZCYTO18 action.
[0262] Moreover, these experiments using ZCYTO18 adenovirus in mice
suggest that ZCYTO18 over-expression increases the level of
neutrophils and platelets in vivo. Although increasing neutrophils
and platelets is desirable in certain therapeutic applications
discussed herein, chronic elevation or increased reactivity of
these cells could play a role in cardiovascular disease.
Antagonists against ZCYTO18, such as antibodies or its soluble
receptor, could be used as therapeutic reagents in these diseases.
Although this may appear contradictory to the finding seen in K562
cells (Example 12), it is not uncommon to observe diverse
activities of a particular protein in vitro versus in vivo. It is
conceivable that there are other factors (such as cytokines and
modifier genes) involved in the responses to ZCYTO18 in the whole
animal system. Nevertheless, these data strongly support the
involvement of ZCYTO18 in hematopoiesis. Thus, ZCYTO18 and its
receptors are suitable reagents/targets for the diagnosis and
treatment in variety of disorders, such as inflammation, immune
disorders, infection, anemia, hematopoietic and other cancers, and
the like.
Example 12
The ZCYTO18 Polypeptide Inhibits the Growth of K-562 Cells in A
Cytotoxicity Assay
[0263] The K-562 cell line (CRL-243, ATCC) has attained widespread
use as a highly sensitive in vitro target for cytotoxicity assays.
K-562 blasts are multipotential, hematopoietic malignant cells that
spontaneously differentiate into recognizable progenitors of the
erythrocytic, granulocytic and monocytic series (Lozzio, BB et al.,
Proc. Soc. Exp. Biol. Med. 166: 546-550, 1981).
[0264] K562 cells were plated at 5,000 cells/well in 96-well tissue
culture clusters (Costar) in DMEM phenol-free growth medium (Life
Technologies) supplemented with pyruvate and 10% serum (HyClone).
Next day, human recombinant ZCYTO18 (Example 19), BSA control or
retinoic acid (known to be cytotoxic to K562 cells) were added.
Seventy-two hours later, the vital stain MTT (Sigma, St Louis,
Mo.), a widely used indicator of mitochondrial activity and cell
growth, was added to the cells at a final concentration of 0.5
mg/ml. MTT is converted to a purple formazan derivative by
mitochondrial dehydrogenases. Four hours later, converted MTT was
solubilized by adding an equal volume of acidic isopropanol (0.04N
HCl in absolute isopropanol) to the wells. Absorbance was measured
at 570 nm, with background subtraction at 650 nm. In this
experimental setting, absorbance reflects cell viability. Results
shown in Table 7 are expressed as % cytotoxicity.
7 TABLE 7 Agent Concentration % Cytotoxicity BSA Control 1 ug/ml
1.3 Retinoic acid 100 uM 62 ZCYTO18 100 ng/ml 16.2 ZCYTO18 300
ng/ml 32
[0265] The results indicate that ZCYTO18 may affect myeloid stem
cells. Myeloid stem cells are daughter cells of the universal blood
stem cells. They are progenitors of erythrocytes, monocytes (or
migrated macrophages), neutrophil, basophil, and eosinophils. Since
K-562 blasts differentiate into progenitors of the erythrocytic,
granulocytic and monocytic series, they are considered a model for
myeloid stem cells. Thus, the results demonstrate that ZCYTO18 has
an inhibitory activity on the proliferation and/or growth of a
promyelocytic tumor cell line. Thus ZCYTO18 could play a role in
anemia, infection, inflammation, and/or immune diseases. In
addition, an antagonist against ZCYTO18, such as antibodies or a
soluble receptor antagonist, could be used to block ZCYTO18's
activity on myeloid stem cells, or as therapeutic reagents in
diseases such as anemia, infection, inflammation, and/or immune
diseases. However, as ZCYTO18 exhibits cytotoxicity on tumor cells,
it can be used directly or in combination with other cytokines as
an anti-tumor agent as an anti-tumor agent.
Example 13
Human zcytor16 Tissue Distribution in Tissue Panels Using Northern
Blot and PCR
[0266] A. Human zcytor16 Tissue Distribution using Northern Blot
and Dot Blot
[0267] Commonly owned, human zcytor16 (SEQ ID NO: 32, and SEQ ID
NO: 33) (PCT International Application No. [#####]) is a
naturally-expressed soluble receptor antagonist of ZCYTO18.
Northern blot analysis was performed using Human Multiple Tissue
Northern Blots I, II, III (Clontech) and an in house generated
U-937 northern blot. U-937 is a human promonocytic blast cell line.
The cDNA probe was generated using oligos ZC25,963 (SEQ ID NO: 24)
and ZC28,354 (SEQ ID NO: 25). The PCR conditions were as follows:
94.degree. for 1 minute; 30 cycles of 94.degree., 15 seconds;
60.degree., 30 seconds; 72.degree., 30 seconds and a final
extension for 5 minutes at 72.degree.. The 364 bp product was gel
purified by gel electrophoresis on a 1% TBE gel and the band was
excised with a razor blade. The cDNA was extracted from the agarose
using the QIAquick Gel Extraction Kit (Qiagen). 94 ng of this
fragment was radioactively labeled with .sup.32P-dCTP using
Rediprime II (Amersham), a random prime labeling system, according
to the manufacturer's specifications. Unincorporated radioactivity
was removed using a Nuc-Trap column (Stratagene) according to
manufacturer's instructions. Blots were prehybridized at 65.degree.
for 3 hours in ExpressHyb (Clontech) solution. Blots were
hybridized overnight at 65.degree. in Expresshyb solution
containing 1.0.times.10.sup.6 cpm/ml of labeled probe, 0.1 mg/ml of
salmon sperm DNA and 0.5 .mu.g/ml of human cot-1 DNA. Blots were
washed in 2.times.SSC, 0.1% SDS at room temperature with several
solution changes then washed in 0.1.times.SSC. 0.1% SDS at
55.degree. for 30 minutes twice. Transcripts of approximately 1.6
kb and 3.0 kb size were detected in spleen and placenta, but not
other tissues examined. The same sized transcripts plus an
additional approximate 1.2 kb transcript was detected in U-937 cell
line.
[0268] B. Tissue Distribution in Tissue cDNA Panels Using PCR
[0269] A panel of cDNAs from human tissues was screened for
zcytor16 expression using PCR. The panel was made in-house and
contained 94 marathon cDNA and cDNA samples from various normal and
cancerous human tissues and cell lines are shown in Table 8, below.
The cDNAs came from in-house libraries or marathon cDNAs from
in-house RNA preps, Clontech RNA, or Invitrogen RNA. The marathon
cDNAs were made using the marathon-Ready.TM. kit (Clontech, Palo
Alto, Calif.) and QC tested with clathrin primers ZC21195 (SEQ ID
NO: 26) and ZC21196 (SEQ ID NO: 27) and then diluted based on the
intensity of the clathrin band. To assure quality of the ipanel
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: 28) and 3' alpha tubulin specific oligo primer
ZC17,574 (SEQ ID NO: 29) or 3' G3PDH specific oligo primer ZC17,600
(SEQ ID NO: 30); 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 ZC25,963 (SEQ ID NO: 24) and
ZC27,659 (SEQ ID NO: 25), Advantage 2 DNA Polymerase Mix (Clontech)
and Rediload dye (Research Genetics, Inc., Huntsville, Ala.). The
amplification was carried out as follow: 1 cycle at 94.degree. C.
for 2 minutes, 30 cycles of 94.degree. C. for 20 seconds,
58.degree. C. for 30 seconds and 72.degree. C. for 1 minute,
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 2% agarose gel. The correct predicted DNA
fragment size was not observed in any tissue or cell line.
Subsequent experiments showing expression of zcytor16 indicated
that the negative results from this panel were likely due to the
primers used.
8TABLE 8 # # 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 2 (ATCC # CCL-156)
Colon 1 Testis 4 Fetal brain 1 Thyroid 2 Fetal heart 1 WI38 (ATCC #
CCL-75 2 Fetal kidney 1 ARIP 1 (ATCC # CRL-1674 - rat) 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 1 Ionomycin + PMA stimulated K562 1 HPVS
(ATCC # CRL-2221) - 1 (ATCC # CCL-243) 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
[0270] An additional panel of cDNAs from human tissues was screened
for zcytor16 expression using PCR. This panel was made in-house and
contained 77 marathon cDNA and cDNA samples from various normal and
cancerous human tissues and cell lines are shown in Table 9, below.
Aside from the PCR reaction, the assay was carried out as per
above. The PCR reactions were set up using oligos ZC25,963 (SEQ ID
NO: 24) and ZC25,964 (SEQ ID NO: 31), Advantage 2 DNA Polymerase
Mix (Clontech) and Rediload dye (Research Genetics, Inc.,
Huntsville, Ala.). The amplification was carried out as follow: 1
cycle at 94.degree. C. for 1 minute, 38 cycles of 94.degree. C. for
10 seconds, 60.degree. C. for 30 seconds and 72.degree. C. for
followed by 1 cycle at 72.degree. C. for 5 minutes. The correct
predicted DNA fragment size was observed in bone marrow, fetal
heart, fetal kidney, fetal muscle, fetal skin, heart, mammary
gland, placenta, salivary gland, skeletal muscle, small intestine,
spinal cord, spleen, kidney, fetal brain, esophageal tumor, uterine
tumor, stomach tumor, ovarian tumor, rectal tumor, lung tumor and
RPMI-1788 (a B-lymphocyte cell line). Zcytor16 expression was not
observed in the other tissues and cell lines tested in this panel.
The expression pattern of zcytor16 shows expression in certain
tissue-specific tumors especially, e.g., ovarian cancer, stomach
cancer, uterine cancer, rectal cancer, lung cancer and esophageal
cancer, where zcytor16 is not expressed in normal tissue, but is
expressed in the tumor tissue. One of skill in the art would
recognize that the natural ligand, CYTO18, and receptor binding
fragments of ZCYTO18 of the present invention can be used as a
diagnostic to detect cancer, or cancer tissue in a biopsy, tissue,
or histologic sample, particularly e.g., ovarian cancer, stomach
cancer, uterine cancer, rectal cancer, lung cancer and esophageal
cancer tissue. Such diagnostic uses for the molecules of the
present invention are known in the art and described herein.
[0271] In addition, because the expression pattern of zcytor16, one
of ZCYTO18's receptors, shows expression in certain specific
tissues as well as tissue-specific tumors, binding partners
including the natural ligand, ZCYTO18, can also be used as a
diagnostic to detect specific tissues (normal or abnormal), cancer,
or cancer tissue in a biopsy, tissue, or histologic sample, where
ZCYTO18 receptors are expressed, and particularly e.g., ovarian
cancer, stomach cancer, uterine cancer, rectal cancer, lung cancer
and esophageal cancer tissue. ZCYTO18 can also be used to target
other tissues wherein its receptors, e.g., zcytor16 and zcytor11
(Commonly owned U.S. Pat. No. 5,965,704) are expressed. Moreover,
such binding partners could be conjugated to chemotherapeutic
agents, toxic moieties and the like to target therapy to the site
of a tumor or diseased tissue. Such diagnostic and targeted therapy
uses are known in the art and described herein.
[0272] A commercial 1st strand cDNA panel (Human Blood Fractions
MTC Panel, Clontech, Palo Alto, Calif.) was also assayed as above.
The panel contained the following samples: mononuclear cells,
activated mononuclear cells, resting CD4+ cells, activated CD4+
cells, resting CD8+ cells, activated CD8+ cells, resting CD14+
cells, resting CD19+ cells and activated CD19+ cells. Activated
CD4+ cells and activated CD19+ cells showed zcytor16 expression,
whereas the other cells tested, including resting CD4+ cells and
resting CD19+ cells, did not.
9 TABLE 9 # # Tissue samples Tissue samples adrenal gland 1 bladder
1 bone marrow 3 brain 2 cervix 1 colon 1 fetal brain 3 fetal heart
2 fetal kidney 1 fetal liver 2 fetal lung 1 fetal skin 1 heart 2
fetal muscle 1 kidney 2 liver 1 lung 1 lymph node 1 mammary gland 1
melanoma 1 ovary 1 pancreas 1 pituitary 2 placenta 3 prostate 3
rectum 1 salivary gland 2 skeletal muscle 1 small intestine 1
spinal cord 2 spleen 1 uterus 1 stomach 1 adipocyte library 1
testis 5 islet 1 thymus 1 prostate SMC 1 thyroid 2 RPMI 1788 1
trachea 1 WI38 1 esophageal tumor 1 lung tumor 1 liver tumor 1
ovarian tumor 1 rectal tumor 1 stomach tumor 1 uterine tumor 2 CD3
+ library 1 HaCAT library 1 HPV library 1 HPVS library 1 MG63
library 1 K562 1
[0273] C. Tissue Distribution in Human Tissue and Cell Line RNA
Panels Using RT-PCR
[0274] A panel of RNAs from human cell lines was screened for
zcytor16 expression using RT-PCR. The panels were made in house and
contained 84 RNAs from various normal and cancerous human tissues
and cell lines as shown in Tables 10-13 below. The RNAs were made
from in house or purchased tissues and cell lines using the RNAeasy
Midi or Mini Kit (Qiagen, Valencia, Calif.). The panel was set up
in a 96-well format with 100 ngs of RNA per sample. The RT-PCR
reactions were set up using oligos ZC25,963 (SEQ ID NO: 24) and
ZC25,964 (SEQ ID NO: 31), Rediload dye and SUPERSCRIPT One Step
RT-PCR System (Life Technologies, Gaithersburg, Md.). The
amplification was carried out as follows: one cycle at 550 for 30
minutes followed by 40 cycles of 94.degree., 15 seconds;
59.degree., 30 seconds; 72 seconds; then ended with a final
extension at 72.degree. for 5 minutes. 8 to 10 .mu.ls of the PCR
reaction product was subjected to standard Agarose gel
electrophoresis using a 4% agarose gel. The correct predicted cDNA
fragment size of 184 bps was observed in cell lines U-937, HL-60,
ARPE-19, HaCat#1, HaCat#2, HaCat#3, and HaCat#4; bladder, cancerous
breast, normal breast adjacent to a cancer, bronchus, colon,
ulcerative colitis colon, duodenum, endometrium, esophagus,
gastro-esophageal, heart left ventricle, heart ventricle, ileum,
kidney, lung, lymph node, lymphoma, mammary adenoma, mammary gland,
cancerous ovary, pancreas, parotid and skin, spleen lymphoma and
small bowel. Zcytor16 expression was not observed in the other
tissues and cell lines tested in this panel.
[0275] Zcytor16 is detectably expressed by PCR in normal tissues:
such as, the digestive system, e.g., esophagus, gastro-esophageal,
pancreas, duodenum, ileum, colon, small bowel; the female
reproductive system, e.g., mammary gland, endometrium, breast
(adjacent to cancerous tissues); and others systems, e.g., lymph
nodes, skin, parotid, bladder, bronchus, heart ventricles, and
kidney. Moreover, Zcytor16 is detectably expressed by PCR in
several human tumors: such as tumors associated with female
reproductive tissues e.g., mammary adenoma, ovary cancer, uterine
cancer, other breast cancers; and other tissues such as lymphoma,
stomach tumor, and lung tumor. The expression of zcytor16 is found
in normal tissues of female reproductive organs, and in some tumors
associated with these organs. As such, a ligand for zcytor16, such
as ZCYTO18, or a receptor-binding fragment thereof, can serve as a
marker for these tumors wherein the zcytor16 may be over-expressed.
Several cancers positive for zcytor16 are associated with
ectodermal/epithelial origin (mammary adenoma, and other breast
cancers). Hence, ligand for zcytor16, such as ZCYTO18, or a
receptor-binding fragment thereof, can serve as a marker for
epithelial tissue, such as epithelial tissues in the digestive
system and female reproductive organs (e.g., endometrial tissue,
columnar epithelium), as well as cancers involving epithelial
tissues. Moreover, in a preferred embodiment, ZCYTO18, or a
receptor-binding fragment thereof, can serve as a marker for
certain tissue-specific tumors especially, e.g., ovarian cancer,
stomach cancer, uterine cancer, rectal cancer, lung cancer and
esophageal cancer, where it's receptor zcytor16 is not expressed in
normal tissue, but is expressed in the tumor tissue. Use of
polynucleotides, polypeptides, and antibodies of the present
invention for diagnostic purposes are known in the art, and
disclosed herein.
10TABLE 10 # Tissue samples Tissue #samples adrenal gland 6
duodenum 1 bladder 3 endometrium 5 brain 2 cancerous endometrium 1
brain meningioma 1 gastric cancer 1 breast 1 esophagus 7 cancerous
breast 4 gastro-esophageal 1 normal breast adjacent to 5 heart
aorta 1 cancer bronchus 3 heart left ventricle 4 colon 15 heart
right ventricle 2 cancerous colon 1 heart ventricle 1 normal colon
adjacent to 1 ileum 3 cancer ulcerative colitis colon 1 kidney 15
cancerous kidney 1
[0276]
11TABLE 11 # Tissue/Cell # Tissue/Cell Line samples Line samples
293 1 HBL-100 1 C32 1 Hs-294T 1 HaCat#1 1 Molt4 1 HaCat#2 1 RPMI 1
HaCat#3 1 U-937 1 HaCat#4 1 A-375 1 WI-38 1 HCT-15 1 WI-38 + 2 um
ionomycin#1 1 HT-29 1 WI-38 + 2 um ionomycin#2 1 MRC-5 1 WI-38 + 5
um ionomycin#1 1 RPT-1 1 WI-38 + 5 um ionomycin#2 1 RPT-2 1 Caco-2,
1 WM-115 1 Caco-2,differentiated 1 A-431 1 DLD-1 1 WERI-Rb-1 1 HRE
1 HEL-92.1.7 1 HRCE 1 HuH-7 1 MCF7 1 MV-4-11 1 PC-3 1 U-138 1 TF-1
1 CCRF-CEM 1 5637 1 Y-79 1 143B 1 A-549 1 ME-180 1 EL-4 1 prostate
epithelia 1 HeLa 229 1 U-2 OS 1 HUT 78 1 T-47D 1 NCI-H69 1 Mg-63 1
SaOS2 1 Raji 1 USMC 1 U-373 MG 1 UASMC 2 A-172 1 AoSMC 1 CRL-1964 1
UtSMC 1 CRL-1964 + butryic acid 1 HepG2 1 HUVEC 1 HepG2-IL6 1
SK-Hep-1 1 NHEK#1 1 SK-Lu-1 1 NHEK#2 1 Sk-MEL-2 1 NHEK#3 1 K562 1
NHEK#4 1 BeWo 1 ARPE-19 1 FHS74.Int 1 G-361 1 HL-60 1 HISM 1 Malme
3M 1 3AsubE 1 FHC 1 INT407 1 HREC 1
[0277]
12TABLE 12 Tissue #samples Tissue #samples liver 10 lung 13 lymph
node 1 cancerous lung 2 lymphoma 4 normal lung adjacent to 1 cancer
mammary adenoma 1 muscle 3 mammary gland 3 neuroblastoma 1
melinorioma 1 omentum 2 osteogenic sarcoma 2 ovary 6 pancreas 4
cancerous ovary 2 skin 5 parotid 7 sarcoma 2 salivary gland 4
[0278]
13 TABLE 13 Tissue #samples Tissue #samples small bowel 10 uterus
11 spleen 3 uterine cancer 1 spleen lymphoma 1 thyroid 9 stomach 13
stomach cancer 1
Example 14
Human zcytor11 Tissue Distribution in Tissue Panels Using Northern
Blot and PCR
[0279] A. Human zcytor11 Tissue Distribution in Tissue Panels Using
PCR
[0280] A panel of eDNAs from human tissues was screened for
zcytor11 expression using PCR. Commonly owned, human zcytor11 (SEQ
ID NO: 18, and SEQ ID NO: 19) (U.S. Pat. No. 5,965,704) is a
receptor for ZCYTO18. The panel was made in-house and contained 94
marathon cDNA and cDNA samples from various normal and cancerous
human tissues and cell lines are shown in Table 9 above. Aside from
the PCR reaction, the method used was as shown in Example 13. The
PCR reactions were set up using oligos ZC14,666 (SEQ ID NO: 22) and
ZC14,742 (SEQ ID NO: 23), Advantage 2 cDNA polymerase mix
(Clontech, Palo Alto, Calif.), and Rediload dye (Research Genetics,
Inc., Huntsville, Ala.). The amplification was carried out as
follows: 1 cycle at 94.degree. C. for 2 minutes, 40 cycles of
94.degree. C. for 15 seconds, 51.degree. C. for 30 seconds and
72.degree. C. for 30 seconds, followed by 1 cycle at 72.degree. C.
for 7 minutes. The correct predicted DNA fragment size was observed
in bladder, brain, cervix, colon, fetal brain, fetal heart, fetal
kidney, fetal liver, fetal lung, fetal skin, heart, kidney, liver,
lung, melanoma, ovary, pancreas, placenta, prostate, rectum,
salivary gland, small intestine, testis, thymus, trachea, spinal
cord, thyroid, lung tumor, ovarian tumor, rectal tumor, and stomach
tumor. Zcytor11 expression was not observed in the other tissues
and cell lines tested in this panel.
[0281] A commercial 1st strand cDNA panel (Human Blood Fractions
MTC Panel, Clontech, Palo Alto, Calif.) was also assayed as above.
The panel contained the following samples: mononuclear cells,
activated mononuclear cells, resting CD4+ cells, activated CD4+
cells, resting CD8+ cells, activated CD8+ cells, resting CD14+
cells, resting CD19+ cells and activated CD19+ cells. All samples
except activated CD8+ and Activated CD19+ showed expression of
zcytor11.
[0282] B. Tissue Distribution of Zcytor11 in Human Cell Line and
Tissue Panels Using RT-PCR
[0283] A panel of RNAs from human cell lines was screened for
zcytor11 expression using RT-PCR. The panels were made in house and
contained 84 RNAs from various normal and cancerous human tissues
and cell lines as shown in Tables 10-13 above. The RNAs were made
from in house or purchased tissues and cell lines using the RNAeasy
Midi or Mini Kit (Qiagen, Valencia, Calif.). The panel was set up
in a 96-well format with 100 ngs of RNA per sample. The RT-PCR
reactions were set up using oligos ZC14,666 (SEQ ID NO: 22) and
ZC14,742 (SEQ ID NO: 23), Rediload dye and SUPERSCRIPT One Step
RT-PCR System(Life Technologies, Gaithersburg, Md.). The
amplification was carried out as follows: one cycle at 50.degree.
for 30 minutes followed by 45 cycles of 94.degree., 15 seconds;
52.degree., 30 seconds; 72.degree., 30 seconds; then ended with a
final extension at 72.degree. for 7 minutes. 8 to 10 uls of the PCR
reaction product was subjected to standard Agarose gel
electrophoresis using a 4% agarose gel. The correct predicted cDNA
fragment size was observed in adrenal gland, bladder, breast,
bronchus, normal colon, colon cancer, duodenum, endometrium,
esophagus, gastic cancer, gastro-esophageal cancer, heart
ventricle, ileum, normal kidney, kidney cancer, liver, lung, lymph
node, pancreas, parotid, skin, small bowel, stomach, thyroid, and
uterus. Cell lines showing expression of zcytorl 1 were A-431,
differentiated CaCO2, DLD-1, HBL-100, HCT-15, HepG2, HepG2+IL6,
HuH7, and NHEK #1-4. Zcytor11 expression was not observed in the
other tissues and cell lines tested in this panel.
[0284] In addition, because the expression pattern of zcytorll, one
of ZCYTO18's receptors, shows expression in certain specific
tissues, binding partners including the natural ligand, ZCYTO18,
can also be used as a diagnostic to detect specific tissues (normal
or abnormal), cancer, or cancer tissue in a biopsy, tissue, or
histologic sample, particularly in tissues where ZCYTO18 receptors
are expressed. ZCYTO18 can also be used to target other tissues
wherein its receptors, e.g., zcytor16 and zcytor11 are expressed.
Moreover, such binding partners could be conjugated to
chemotherapeutic agents, toxic moieties and the like to target
therapy to the site of a tumor or diseased tissue. Such diagnostic
and targeted therapy uses are known in the art and described
herein.
[0285] The expression patterns of zcytor11 (above) and zcytor16
(Example 13, and Example 15) indicated target tissues and cell
types for the action of ZCYTO18, and hence ZCYTO18 antagonists. The
zcytorl 1 expression generally overlapped with zcytor16 expression
in three physiologic systems: digestive system, female reproductive
system, and immune system. Moreover, the expression pattern of the
receptor (zcytorl 1) indicated that a ZCYTO18 antagonist such as
zcytor16 would have therapeutic application for human disease in at
least two areas: inflammation (e.g., IBD, Chron's disease,
pancreatitis) and cancer (e.g., ovary, colon). That is, the
polynucleotides, polypeptides and antibodies of the present
invention can be used to antagonize the inflammatory, and other
cytokine-induced effects of ZCYTO18 interaction with the cells
expressing the zcytor11 receptor.
[0286] Moreover, the expression of zcytor11 appeared to be
downregulated or absent in an ulcerative colitis tissue, HepG2
liver cell line induced by IL-6, activated CD8+ T-cells and CD19+
B-cells. However, zcytor16 appeared to be upregulated in activated
CD19+ B-cells (Example 12), while zcytor11 is downregulated in
activated CD19+ cells, as compared to the resting CD19+ cells
(above). The expression of zcytor11 and zcytor16 has a reciprocal
correlation in this case. These RT-PCR experiments demonstrate that
CD19+ peripheral blood cells, B lymphocytes, express receptors for
ZCYTO18, namely zcytorl 1 and zcytor16. Furthermore B cells display
regulated expression of zcytor11 and zcytor16. B-lymphocytes
activated with mitogens decrease expression of zcytor11 and
increase expression of zcytor16. This represents feedback
inhibition that would serve to dampen the activity of ZCYTO18 on B
cells and other cells as well. Soluble zcytor16 would act as an
antagonist to neutralize the effects of ZCYTO18 on B cells. This
would be beneficial in diseases where B cells are the key players:
Autoimmune diseases including systemic lupus erythmatosus (SLE),
myasthenia gravis, immune complex disease, and B-cell cancers that
are exacerbated by ZCYTO18. Also autoimmune diseases where B cells
contribute to the disease pathology would be targets for zcytor16
therapy: Multiple sclerosis, inflammatory bowel disease (IBD) and
rheumatoid arthritis are examples. Zcytor16 therapy would be
beneficial to dampen or inhibit B cells producing IgE in atopic
diseases including asthma, allergy and atopic dermatitis where the
production of IgE contributes to the pathogenesis of disease.
[0287] B cell malignancies may exhibit a loss of the "feedback
inhibition" described above. Administration of zcytor16 would
restore control of ZCYTO18 signaling and inhibit B cell tumor
growth. The administration of zcytor16 following surgical resection
or chemotherapy may be useful to treat minimal residual disease in
patients with B cell malignancies. The loss of regulation may lead
to sustained or increased expression of zcytor11. Thus creating a
target for therapeutic monoclonal antibodies targeting
zcytor11.
Example 15
Identification of Cells Expressing Zcytor16 Using in situ
Hybridization
[0288] Specific human tissues were isolated and screened for
zcytor16 expression by in situ hybridization. Various human tissues
prepared, sectioned and subjected to in situ hybridization included
cartilage, colon, appendix, intestine, fetal liver, lung, lymph
node, lymphoma, ovary, pancreas, placenta, prostate, skin, spleen,
and thymus. The tissues were fixed in 10% buffered formalin and
blocked in paraffin using standard techniques. Tissues were
sectioned at 4 to 8 microns. Tissues were prepared using a standard
protocol ("Development of non-isotopic in situ hybridization" at
The Laboratory of Experimental Pathology (LEP), NIEHS, Research
Triangle Park, NC; web address http://dir.niehs.nih.gov/d-
irlep/ish.html). Briefly, tissue sections were deparaffinized with
HistoClear (National Diagnostics, Atlanta, Ga.) and then dehydrated
with ethanol. Next they were digested with Proteinase K (50
.mu.g/ml) (Boehringer Diagnostics, Indianapolis, Ind.) at
37.degree. C. for 2 to 7 minutes. This step was followed by
acetylation and re-hydration of the tissues.
[0289] One in situ probe was designed against the human zcytor16
sequence (nucleotide 1-693 of SEQ ID NO: 32), and isolated from a
plasmid containing SEQ ID NO: 32 using standard methods. T3 RNA
polymerase was used to generate an antisense probe. The probe was
labeled with digoxigenin (Boehringer) using an In Vitro
transcription System (Promega, Madison, Wis.) as per manufacturer's
instruction.
[0290] In situ hybridization was performed with a
digoxigenin-labeled zcytor16 probe (above). The probe was added to
the slides at a concentration of 1 to 5 pmol/ml for 12 to 16 hours
at 62.5.degree. C. Slides were subsequently washed in 2.times.SSC
and 0.1.times.SSC at 55.degree. C. The signals were amplified using
tyramide signal amplification (TSA) (TSA, in situ indirect kit;
NEN) and visualized with Vector Red substrate kit (Vector Lab) as
per manufacturer's instructions. The slides were then
counter-stained with hematoxylin (Vector Laboratories, Burlingame,
Calif.).
[0291] Signals were observed in several tissues tested: The lymph
node, plasma cells and other mononuclear cells in peripheral
tissues were strongly positive. Most cells in the lymphatic nodule
were negative. In lymphoma samples, positive signals were seen in
the mitotic and multinuclear cells. In spleen, positive signals
were seen in scattered mononuclear cells at the periphery of
follicles were positive. In thymus, positive signals were seen in
scattered mononuclear cells in both cortex and medulla were
positive. In fetal liver, a strong signal was observed in a mixed
population of mononuclear cells in sinusoid spaces. A subset of
hepatocytes might also have been positive. In the inflamed
appendix, mononuclear cells in peyer's patch and infiltration sites
were positive. In intestine, some plasma cells and ganglia nerve
cells were positive. In normal lung, zcytor16 was expressed in
alveolar epithelium and mononuclear cells in interstitial tissue
and circulation. In the lung carcinoma tissue, a strong signal was
observed in mostly plasma cells and some other mononuclear cells in
peripheral of lymphatic aggregates. In ovary carcinoma, epithelium
cells were strongly positive. Some interstitial cells, most likely
the mononuclear cells, were also positive. There was no signal
observed in the normal ovary. In both normal and pancreatitis
pancreas samples, acinar cells and some mononuclear cells in the
mesentery were positive. In the early term (8 weeks) placenta,
signal was observed in trophoblasts. In skin, some mononuclear
cells in the inflamed infiltrates in the superficial dermis were
positive. Keratinocytes were also weakly positive. In prostate
carcinoma, scatted mononuclear cells in interstitial tissues were
positive. In articular cartilage, chondrocytes were positive. Other
tissues tested including normal ovary and a colon adenocarcinoma
were negative.
[0292] In summary, the in situ data was consistent with expression
data described above for the zcytor16. Zcytor16 expression was
observed predominately in mononuclear cells, and a subset of
epithelium was also positive. These results confirmed the presence
of zcytor16 expression in immune cells and point toward a role in
inflammation, autoimmune disease, or other immune function, for
example, in binding pro-inflammatory cytokines, including but not
limited to ZCYTO18. Moreover, detection of zcytor16 expression can
be used for example as an marker for mononuclear cells in
histologic samples.
[0293] Zcytor16 is expressed in mononuclear cells, including normal
tissues (lymph nodes, spleen, thymus, pancreas and fetal liver,
lung), and abnormal tissues (inflamed appendix, lung carcinoma,
ovary carcinoma, pancreatitis, inflamed skin, and prostate
carcinoma). It is notable that plasma cells in the lymph node,
intestine, and lung carcinoma are positive for zcytor16. Plasma
cells are immunologically activated lymphocytes responsible for
antibody synthesis. In addition, ZCYTO18, is expressed in activated
T cells. In addition, the expression of zcytor16 is detected only
in activated (but not in resting) CD4+ and CD19+ cells (Example
13). Thus, zcytor16 can be used as a marker for or as a target in
isolating certain lymphocytes, such as mononuclear leucocytes and
limited type of activated leucocytes, such as activated CD4+ and
CD19+.
[0294] Furthermore, the presence of zcytor16 expression in
activated immune cells such as activated CD4+ and CD19+ cells
showed that zcytor16 may be involved in the body's immune defensive
reactions against foreign invaders: such as microorganisms and cell
debris, and could play a role in immune responses during
inflammation and cancer formation.
[0295] Moreover, as discussed herein, epithelium form several
tissues was positive for zcytor16 expression, such as hepatocytes
(endoderm-derived epithelia), lung alveolar epithelium
(endoderm-derived epithelia), and ovary carcinoma epithelium
(mesoderm-derived epithelium). The epithelium expression of
zcytor16 could be altered in inflammatory responses and/or
cancerous states in liver and lung. Thus, ligand for zcytor16, such
as ZCYTO18, or a receptor-binding fragment thereof, could be used
as marker to monitor changes in these tissues as a result of
inflammation or cancer. Moreover, analysis of zcytor16 in situ
expression showed that normal ovary epithelium is negative for
zcytor16 expression, while it is strongly positive in ovary
carcinoma epithelium providing further evidence that ZCYTO18
polypeptides, or a receptor-binding fragment thereof, can be used
as a diagnostic marker and/or therapeutic target for the diagnosis
and treatment of ovarian cancers, and ovary carcinoma, as described
herein.
[0296] Zcytor16 was also detected in other tissues, such as acinar
cells in pancreas (normal and pancreatitis tissues), trophoblasts
in placenta (ectoderm-derived), chondrocytes in cartilage
(mesoderm-derived), and ganglia cells in intestine
(ectoderm-derived). As such, zcytor16 may be involved in
differentiation and/or normal functions of corresponding cells in
these organs. As such, potential utilities of zcytor16 include
maintenance of normal metabolism and pregnancy, bone
formation/homeostasis, and physiological function of intestine, and
the like.
Example 16
huZCYTO18 Anti-Peptide Antibodies
[0297] Polyclonal anti-peptide antibodies were prepared by
immunizing two female New Zealand white rabbits with the peptide,
huZCYTO18-1 (SEQ ID NO: 34) or huZCYTO18-2 (SEQ ID NO: 35) or
huZCYTO18-3 (SEQ ID NO: 36). The peptides were synthesized using an
Applied Biosystems Model 431A peptide synthesizer (Applied
Biosystems, Inc., Foster City, Calif.) according to manufacturer's
instructions. The peptides huZCYTO18-1, huZCYTO18-2, and
huZCYTO18-3 were then conjugated to the carrier protein
maleimide-activated keyhole limpet hemocyanin (KLH) through
cysteine residues (Pierce, Rockford, Ill.). The rabbits were each
given an initial intraperitoneal (IP) injection of 200 .mu.g of
conjugated peptide in Complete Freund's Adjuvant (Pierce, Rockford,
Ill.) followed by booster IP injections of 100 .mu.g conjugated
peptide in Incomplete Freund's Adjuvant every three weeks. Seven to
ten days after the administration of the third booster injection,
the animals were bled and the serum was collected. The rabbits were
then boosted and bled every three weeks.
[0298] The huZCYTO18 peptide-specific Rabbit seras were
characterized by an ELISA titer check using 1 .mu.g/ml of the
peptide used to make the antibody as an antibody target. The 2
rabbit seras to the huZCYTO18-1 peptide (SEQ ID NO: 34) have titer
to their specific peptide at a dilution of 1:5E6 (1:5,000,000).
[0299] The huZCYTO18-1 peptide-specific antibodies were affinity
purified from the rabbit serum using an EPOXY-SEPHAROSE 6B peptide
column (Pharmacia LKB) that was prepared using 10 mg of the
respective peptides per gram EPOXY-SEPHAROSE 6B, followed by
dialysis in PBS overnight. Peptide-specific huZCYTO18 antibodies
were characterized by an ELISA titer check using 1 .mu.g/ml of the
appropriate peptide as an antibody target. The huZCYTO18-1
peptide-specific antibodies have a lower limit of detection (LLD)
of 500 pg/ml by ELISA on its appropriate antibody target. The
huZCYTO18-1 peptide-specific antibodies recognize full-length
recombinant protein (BV produced) by reducing Western Blot
analysis.
Example 17
Construction of Human ZCYTO18 Transgenic Plasmids
[0300] Approximately 10 .mu.g Zytrack vector containing the
sequence confirmed human ZCYTO18 coding region was digested with
FseI and AscI. The vector was then ethanol precipitated and the
pellet was resuspended in TE. The released 540 bp human ZCYTO18
fragment was isolated by running the digested vector on a 1.2%
SeaPlaque gel and excising the fragment. DNA was purified using the
QiaQuick (Qiagen) gel extraction kit.
[0301] The human ZCYTO18 fragment was then ligated into pTG12-8,
our standard transgenic vector, which was previously digested with
FseI and AscI. The pTG12-8 plasmid, designed for expression of a
gene of interest 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 poly A sequence.
[0302] About one microliter of the ligation reaction was
electroporated into DH10B ElectroMax.RTM. competent cells (GIBCO
BRL, Gaithersburg, Md.) according to manufacturer's direction,
plated onto LB plates containing 100 .mu.g/ml ampicillin, and
incubated overnight at 37.degree. C. Colonies were picked and grown
in LB media containing 100 .mu.g/ml ampicillin. Miniprep DNA was
prepared from the picked clones and screened for the human ZCYTO18
insert by restriction digestion with FseI/AscI, and subsequent
agarose gel electrophoresis. Maxipreps of the correct pTG12-8 human
ZCYTO18 construct were performed.
[0303] A Sall fragment containing 5' and 3' flanking sequences, the
MT promoter, the rat insulin II intron, human ZCYTO18 cDNA and the
human growth hormone poly A sequence was prepared and used for
microinjection into fertilized murine oocytes.
[0304] A second transgenic construct was made by subdloning as
described above, the FseI/AscI fragment containing the human
ZCYTO18 cDNA, into a lymphoid-specific transgenic vector pKFO51.
The pKFO51 transgenic vector is derived from p1026.times. (Iritani,
B. M., et al., EMBO J. 16:7019-31, 1997) and contains the T
cell-specific 1ck proximal promoter, the B/T cell-specific
immunoglobulin E.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).
[0305] Maxi-prep DNA was digested with NotI, and this fragment,
containing the Ick proximal promoter, imrnunoglobulin E.mu.
enhancer, human ZCYTO18 cDNA, and the mutated hGH gene was prepared
to be used for microinjection into fertilized murine oocytes.
[0306] Construction of Mouse ZCYTO18 Transgenic Plasmids
[0307] Transgenic constructs were also made for mouse ZCYTO18.
Oligonucleotides were designed to generate a PCR fragment
containing a consensus Kozak sequence and the exact mouse ZCYTO18
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, a lymphoid-specific transgenic vector
containing the EuLCK promoter to drive expression of ZCYTO18.
[0308] PCR reactions were carried out with 200 ng mouse ZCYTO18
template (SEQ ID NO: 37) and oligonucleotides ZC37,125 (SEQ ID NO:
39) and ZC37,126 (SEQ ID NO: 40). A PCR reaction was performed
using Advantage.TM. cDNA polymerase (Clontech) under the following
conditions: 95.degree. C. for 5 minutes; 15 cycles of 95.degree. C.
for 60 seconds, 60.degree. C. for 60 seconds, and 72.degree. C. for
90 seconds; and 72.degree. C. for 7 minutes. PCR products were
separated by agarose gel electrophoresis and purified using a
QiaQuick (Qiagen) gel extraction kit. The isolated, 540 bp, DNA
fragment was digested with FseI and AscI (Boerhinger-Mannheim),
ethanol precipitated and cloned into pKFO51 as described above. A
correct clone of pKFO51 mouse ZCYTO18 was verified by sequencing,
and a maxiprep of this clone was performed and prepared as above
for injection.
Example 18
Baculovirus Expression of zCyto18-CEE
[0309] An expression vector, zCyto18-CEE/pZBV32L, was prepared to
express zCyto18-CEE polypeptides in insect cells.
zCyto18-CEE/pZBV32L was designed to express a zCyto18 polypeptide
with a C-terminal GLU-GLU tag (SEQ ID NO: 14). This construct can
be used to determine the N-terminal amino acid sequence of zCyto18
after the signal peptide has been cleaved off.
[0310] A. Construction of zCvto18-CEE/pZBV32L
[0311] A 561 bp zCyto18 fragment containing BaniHI and Xbal
restriction sites on the 5' and 3' ends, respectively, was
generated by PCR amplification from a plasmid containing zCyto18
cDNA using primers ZC28,348 (SEQ ID NO: 41) and ZC28,345 (SEQ ID
NO: 42). The PCR reaction conditions were as follows: 1 cycle at
94.degree. C. for 5 minutes; 35 cycles of 94.degree. C. for 90
seconds, 60.degree. C. for 120 seconds, and 72.degree. C. for 180
seconds; 1 cycle at 72.degree. C. for 10 min; followed by 4.degree.
C. soak. The fragment was visualized by gel electrophoresis (1%
agarose). The band was excised and then extracted using a
QIAquick.TM. Gel Extraction Kit (Qiagen, Cat. No. 28704). The cDNA
was digested using BaniHI and XbaI and then was ligated into the
vector pZBV32L. The pZBV32L vector is a modification of the
pFastBac.TM. (Life Technologies) expression vector, where the
polyhedron promoter has been removed and replaced with the late
activating Basic Protein Promoter, and the coding sequence for the
Glu-Glu tag as well as a stop signal was inserted at the 3' end of
the multiple cloning region. Approximately 68 nanograms of the
restriction digested zCyto18 insert and about 100 ng of the
corresponding pZBV32L vector were ligated overnight at 16.degree.
C. The ligation mix was diluted 10 fold in water and 1 fmol of the
diluted ligation mix was transformed into ElectoMAX.TM. DH12S.TM.
cells (Life Technologies, Cat. No. 18312-017) by electroporation at
400 Ohms, 2V and 25 .mu.F in a 2 mm gap electroporation cuvette
(BTX, Model No. 620). The transformed cells were diluted in 450
.mu.l of SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10
ml 1M NaCl, 1.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20
mM glucose) and 100 .mu.l of the dilution were plated onto LB
plates containing 100 .mu.g/ml ampicillin. Clones were analyzed by
PCR and two positive clones were selected to be outgrown and
purified using a QIAprep.RTM. Spin Miniprep Kit (Qiagen, Cat. No.
27106). Two .mu.l of each of the positive clones were transformed
into 20 .mu.l DH1OBac.TM. Max Efficiency.RTM. competent cells
(GIBCO-BRL Cat. No. 10361-012) by heat shock for 45 seconds in a
42.degree. C. heat block. The transformed DH10Bac.TM. cells were
diluted in 980 .mu.l SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast
Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10 mM MgCl.sub.2, 10 mM
MgSO.sub.4 and 20 mM glucose) and 100 .mu.l were plated onto Luria
Agar plates containing 50 .mu.g/ml kanamycin, 7 .mu.g/ml
gentamicin, 10 .mu.g/ml tetracycline, 40 .mu.g/mL IPTG and 200
.mu.g/mL Bluo Gal. The plates were incubated for 48 hours at
37.degree. C. A color selection was used to identify those cells
having transposed viral DNA (referred to as a "bacmid"). Those
colonies, which were white in color, were picked for analysis.
Colonies were analyzed by PCR and positive colonies (containing
desired bacmid) were selected for outgrowth and purified using a
QLAprep.RTM. Spin Miniprep Kit (Qiagen, Cat. No. 27106). Clones
were screened for the correct insert by amplifying DNA using
primers to the transposable element in the bacmid via PCR using
primers ZC447 (SEQ ID NO: 43) and ZC976 (SEQ ID NO: 44). The PCR
reaction conditions were as follows: 1 cycle at 94.degree. C. for 5
minutes; 30 cycles of 94.degree. C. for 60 seconds, 50.degree. C.
for 90 seconds, and 72.degree. C. for 180 seconds; 1 cycle at
72.degree. C. for 10 min; followed by 4.degree. C. soak. The PCR
product was run on a 1% agarose gel to check the insert size. Those
having the correct insert were used to transfect Spodoptera
Frugiperda (Sf9) cells.
[0312] B. Transfection
[0313] Sf9 cells were seeded at 1.times.10.sup.6 cells per well in
a 6-well plate and allowed to attach for 1 hour at 27.degree. C.
Five microliters of bacmid DNA were diluted with 100 .mu.l Sf-900
II SFM (Life Technologies). Twenty .mu.l of Lipofectamine.TM.
Reagent (Life Technologies, Cat. No. 18324-012) were diluted with
100 .mu.l Sf-900 II SFM. The bacmid DNA and lipid solutions were
gently mixed and incubated 30-45 minutes at room temperature. The
media from one well of cells was aspirated, the cells were washed
1.times. with 2 ml fresh Sf-900 II SFM media. Eight hundred
microliters of Sf-900 II SFM was added to the lipid-DNA mixture.
The wash media was aspirated and the DNA-lipid mix added to the
cells. The cells were incubated at 27.degree. C. overnight. The
DNA-lipid mix was aspirated and 2 ml of Sf-900 II media was added
to each plate. The plates were incubated at 27.degree. C., 90%
humidity, for 96 hours after which the virus was harvested.
[0314] C. Amplification
[0315] Sf9 cells were seeded at 1.times.10.sup.6 cells per well in
a 6-well plate. 50 .mu.l of virus from the transfection plate were
placed in the well and the plate was incubated at 27.degree. C.,
90% humidity, for 96 hours after which the virus was harvested.
[0316] Sf9 cells were grown in 50 ml Sf-900 II SFM in a 125 ml
shake flask to an approximate density of 1.times.10.sup.6 cells/ml.
They were then infected with 100 .mu.l of the viral stock from the
above plate and incubated at 27.degree. C. for 3 days after which
time the virus was harvested.
Example 19
Purification of ZCYTO18-CEE from Sf9 Cells
[0317] The following procedure was used for purifying zCyto18
polypeptides containing C-terminal Glu-Glu (EE) tags (SEQ ID NO:
14), that were expressed in baculovirus. Conditioned media from Sf9
cells expressing zCyto18-CEE (Example 18) was filtered using a 0.22
.mu.m Steriflip.TM. filter (Millipore) and one Complete.TM.
protease inhibitor cocktail tablet (Boehringer) was added for every
50 mL of media. Total target protein concentrations of the
concentrated conditioned media were determined via SDS-PAGE and
Western blot analysis using an anti-EE antibody (produced in-house)
followed by a secondary anti-mig HRP conjugated antibody.
[0318] Batch purification was accomplished by adding 250 .mu.l of
Protein G Sepharose.RTM. 4 Fast Flow (Pharmacia) which was treated
with anti-EE antibody (Protein G Sepharose/anti-EE beads), to 40
mLs of Sf9 conditioned media. To capture the ZCYTO18-CEE, the
media-bead mixture was rocked overnight at 4.degree. C. The beads
were spun out of the media at 1000 RPM for 10 minutes in a Beckman
GS6R centrifuge. The beads were washed using the following scheme
(centrifugation and aspiration steps were done after each wash):
1.times. with 1 mL cell lysis buffer (150 mM Sodium Chloride, 50 mM
Tris pH 8.0, and 1% NP-40); 1.times. with 1 mL wash buffer (650mM
Sodium Chloride, 50 mM Tris pH 8.0, and 1% NP-40); 1.times. with 1
mL cell lysis buffer. The beads were then suspended in 500 .mu.l
cell lysis buffer and submitted for N-terminal sequencing.
Example 20
N-terminal Amino Acid Sequence Analysis:
[0319] 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).
[0320] A purified human ZCYTO18-CEE sample was supplied as captured
on Protein G Sepharose/anti-EE beads (Example 19). 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 ZCYTO18 band for N-terminal
protein 5,15 sequencing. The mouse anti-EE IgG HRP conjugated
antibody used was produced in house.
[0321] N-terminal sequence analysis of the secreted ZCYTO18
polypeptide verified the predicted cleavage site of the signal
sequence resulting in a mature start of the ZCYTO18 precursor
sequence at 22 (Ala) as shown in SEQ ID NO: 3.
Example 21
Construction of BaF3 Cells Expressing the CRF2-4 Receptor
(BaF3/CRF2-4 Cells) and BaF3 Cells Expressing the CRF2-4 Receptor
with the Zcytor11 Receptor (BaF3/CRF2-4/zcytor11 Cells)
[0322] BaF3 cells expressing the full-length CFR2-4 receptor were
constructed, using 30 .mu.g of a CFR2-4 expression vector,
described below. The BaF3 cells expressing the CFR2-4 receptor were
designated as BaF3/CFR2-4. These cells were used as a control, and
were further transfected with full-length zcytor11 receptor (SEQ ID
NO: 18 and SEQ ID NO: 19) (U.S. Pat. No. 5,965,704) and used to
construct a screen for ZCYTO18 activity as described below. This
cell assay system can be used to assess ZCYTO18 acitvity and
readily screen for the activity of ZCYTO18 variants.
[0323] A. Construction of BaF3 Cells Expressing the CRF2-4
Receptor
[0324] The full-length cDNA sequence of CRF2-4 (Genbank Accession
No. Z17227) was isolated from a Daudi cell line cDNA library, and
then cloned into an expression vector pZP7P using standard
methods.
[0325] BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell
line derived from murine bone marrow (Palacios and Steinmetz, Cell
41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:
4133-4135, 1986), was maintained in complete media (RPMI medium
(JRH Bioscience Inc., Lenexa, Kans.) supplemented with 10%
heat-inactivated fetal calf serum, 2 ng/ml murine IL-3 (mIL-3) (R
& D, Minneapolis, Minn.), 2 mM L-glutaMax-1.TM. (Gibco BRL), 1
mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics (GIBCO BRL)).
Prior to electroporation, CRF2-4/pZP7P was prepared and purified
using a Qiagen Maxi Prep kit (Qiagen) as per manufacturer's
instructions. For electroporation, BaF3 cells were washed once in
serum-free RPMI media and then resuspended in serum-free RPMI media
at a cell density of 10.sup.7 cells/ml. One ml of resuspended BaF3
cells was mixed with 30 .mu.g of the CRF2-4/pZP7P plasmid DNA and
transferred to separate disposable electroporation chambers (GIBCO
BRL). Following a 15-minute incubation at room temperature the
cells were given two serial shocks (800 1Fad/300 V.; 1180 1Fad/300
V.) delivered by an electroporation apparatus (CELL-PORATORT.TM.;
GIBCO BRL). After a 5-minute recovery time, the electroporated
cells were transferred to 50 ml of complete media and placed in an
incubator for 15-24 hours (37.degree. C., 5% CO.sub.2). The cells
were then spun down and resuspended in 50 ml of complete media
containing 2 .mu.g/ml puromycin in a T-162 flask to isolate the
puromycin-resistant pool. Pools of the transfected BaF3 cells,
hereinafter called BaF3/CRF2-4 cells, were assayed for signaling
capability as described below. Moreover these cells were further
transfected with zcytorl 1 receptor as described below.
[0326] B. Construction of BaF3 Cells Expressing CRF2-4 and Zcytor11
Receptors
[0327] BaF3/CRF2-4 cells expressing the full-length zcytor11
receptor were constructed as per Example 21A above, using 30 .mu.g
of an expression vector containing zcytor11 cDNA (SEQ ID NO: 18).
Following recovery, transfectants were selected using 200 .mu.g/ml
zeocin and 2 .mu.g/ml puromycin. The BaF3/CRF2-4 cells expressing
the zcytor11 receptor were designated as BaF3/CRF2-4/zcytor11
cells. These cells were used to screen for ZCYTO18 activity
(Example 22).
Example 22
Screening for ZCYTO18 Activity Using BaF3/CRF2-4/zcytor11 Cells
Using an Alamar Blue Proliferation Assay
[0328] A. Screening for ZCYTO18 Activity Using BaF3/CRF2-4/zcytor11
Cells Using an Alamar Blue Proliferation Assay
[0329] Purified ZCYTO18-CEE (Example 9) was used to test for the
presence of proliferation activity as described below
[0330] BaF3/CRF2-4/zcytor11 cells were spun down and washed in the
complete media, described in Example 21A 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.
[0331] Proliferation of the BaF3/CRF2-4/zcytor11 cells was assessed
using ZCYTO18-CEE protein diluted with mIL-3 free media to 50, 10,
2, 1, 0.5, 0.25, 0.13, 0.06 ng/ml concentrations. 100 .mu.l of the
diluted protein was added to the BaF3/CRF2-4/zcytor11 cells. The
total assay volume is 200 .mu.l. The assay plates were incubated at
37.degree. C., 5% CO.sub.2 for 3 days at which time Alamar Blue
(Accumed, Chicago, Ill.) was added at 20 .mu.l/well. Plates were
again incubated at 37.degree. C., 5% CO.sub.2 for 24 hours. Alamar
Blue gives a fluourometric readout based on number of live cells,
and is thus a direct measurement of cell proliferation in
comparison to a negative control. Plates were again incubated at
37.degree. C., 5% CO.sub.2 for 24 hours. Plates were read on the
Fmax.TM. plate reader (Molecular Devices Sunnyvale, Calif.) using
the SoftMax.TM. Pro program, at wavelengths 544 (Excitation) and
590 (Emmission). Results confirmed the dose-dependent proliferative
response of the BaF3/CRF2-4/zcytor11 cells to ZCYTO18-CEE. The
response, as measured, was approximately 15-fold over background at
the high end of 50 ng/ml down to a 2-fold induction at the low end
of 0.06 ng/ml. The BaF3 wild type cells, and BaF3/CRF2-4 cells did
not proliferate in response to ZCYTO18-CEE, showing that ZCYTO18 is
specific for the CRF2-4/zcytor11 heterodimeric receptor.
[0332] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
44 1 1116 DNA Homo sapiens CDS (21)...(557) 1 tcgagttaga attgtctgca
atg gcc gcc ctg cag aaa tct gtg agc tct ttc 53 Met Ala Ala Leu Gln
Lys Ser Val Ser Ser Phe 1 5 10 ctt atg ggg acc ctg gcc acc agc tgc
ctc ctt ctc ttg gcc ctc ttg 101 Leu Met Gly Thr Leu Ala Thr Ser Cys
Leu Leu Leu Leu Ala Leu Leu 15 20 25 gta cag gga gga gca gct gcg
ccc atc agc tcc cac tgc agg ctt gac 149 Val Gln Gly Gly Ala Ala Ala
Pro Ile Ser Ser His Cys Arg Leu Asp 30 35 40 aag tcc aac ttc cag
cag ccc tat atc acc aac cgc acc ttc atg ctg 197 Lys Ser Asn Phe Gln
Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu 45 50 55 gct aag gag
gct agc ttg gct gat aac aac aca gac gtt cgt ctc att 245 Ala Lys Glu
Ala Ser Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile 60 65 70 75 ggg
gag aaa ctg ttc cac gga gtc agt atg agt gag cgc tgc tat ctg 293 Gly
Glu Lys Leu Phe His Gly Val Ser Met Ser Glu Arg Cys Tyr Leu 80 85
90 atg aag cag gtg ctg aac ttc acc ctt gaa gaa gtg ctg ttc cct caa
341 Met Lys Gln Val Leu Asn Phe Thr Leu Glu Glu Val Leu Phe Pro Gln
95 100 105 tct gat agg ttc cag cct tat atg cag gag gtg gtg ccc ttc
ctg gcc 389 Ser Asp Arg Phe Gln Pro Tyr Met Gln Glu Val Val Pro Phe
Leu Ala 110 115 120 agg ctc agc aac agg cta agc aca tgt cat att gaa
ggt gat gac ctg 437 Arg Leu Ser Asn Arg Leu Ser Thr Cys His Ile Glu
Gly Asp Asp Leu 125 130 135 cat atc cag agg aat gtg caa aag ctg aag
gac aca gtg aaa aag ctt 485 His Ile Gln Arg Asn Val Gln Lys Leu Lys
Asp Thr Val Lys Lys Leu 140 145 150 155 gga gag agt gga gag atc aaa
gca att gga gaa ctg gat ttg ctg ttt 533 Gly Glu Ser Gly Glu Ile Lys
Ala Ile Gly Glu Leu Asp Leu Leu Phe 160 165 170 atg tct ctg aga aat
gcc tgc att tgaccagagc aaagctgaaa aatgaataac 587 Met Ser Leu Arg
Asn Ala Cys Ile 175 taaccccctt tccctgctag aaataacaat tagatgcccc
aaagcgattt tttttaacca 647 aaaggaagat gggaagccaa actccatcat
gatgggtgga ttccaaatga acccctgcgt 707 tagttacaaa ggaaaccaat
gccacttttg tttataagac cagaaggtag actttctaag 767 catagatatt
tattgataac atttcattgt aactggtgtt ctatacacag aaaacaattt 827
attttttaaa taattgtctt tttccataaa aaagattact ttccattcct ttaggggaaa
887 aaacccctaa atagcttcat gtttccataa tcagtacttt atatttataa
atgtatttat 947 tattattata agactgcatt ttatttatat cattttatta
atatggattt atttatagaa 1007 acatcattcg atattgctac ttgagtgtaa
ggctaatatt gatatttatg acaataatta 1067 tagagctata acatgtttat
ttgacctcaa taaacacttg gatatccta 1116 2 179 PRT Homo sapiens 2 Met
Ala Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr Leu 1 5 10
15 Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Gly Gly Ala
20 25 30 Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn
Phe Gln 35 40 45 Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu Ala
Lys Glu Ala Ser 50 55 60 Leu Ala Asp Asn Asn Thr Asp Val Arg Leu
Ile Gly Glu Lys Leu Phe 65 70 75 80 His Gly Val Ser Met Ser Glu Arg
Cys Tyr Leu Met Lys Gln Val Leu 85 90 95 Asn Phe Thr Leu Glu Glu
Val Leu Phe Pro Gln Ser Asp Arg Phe Gln 100 105 110 Pro Tyr Met Gln
Glu Val Val Pro Phe Leu Ala Arg Leu Ser Asn Arg 115 120 125 Leu Ser
Thr Cys His Ile Glu Gly Asp Asp Leu His Ile Gln Arg Asn 130 135 140
Val Gln Lys Leu Lys Asp Thr Val Lys Lys Leu Gly Glu Ser Gly Glu 145
150 155 160 Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu
Arg Asn 165 170 175 Ala Cys Ile 3 167 PRT Homo sapiens 3 Met Gly
Thr Leu Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val 1 5 10 15
Gln Gly Gly Ala Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys 20
25 30 Ser Asn Phe Gln Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu
Ala 35 40 45 Lys Glu Ala Ser Leu Ala Asp Asn Asn Thr Asp Val Arg
Leu Ile Gly 50 55 60 Glu Lys Leu Phe His Gly Val Ser Met Ser Glu
Arg Cys Tyr Leu Met 65 70 75 80 Lys Gln Val Leu Asn Phe Thr Leu Glu
Glu Val Leu Phe Pro Gln Ser 85 90 95 Asp Arg Phe Gln Pro Tyr Met
Gln Glu Val Val Pro Phe Leu Ala Arg 100 105 110 Leu Ser Asn Arg Leu
Ser Thr Cys His Ile Glu Gly Asp Asp Leu His 115 120 125 Ile Gln Arg
Asn Val Gln Lys Leu Lys Asp Thr Val Lys Lys Leu Gly 130 135 140 Glu
Ser Gly Glu Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met 145 150
155 160 Ser Leu Arg Asn Ala Cys Ile 165 4 501 DNA Artificial
Sequence Degenerate polynucleotide of ZCYTO18 4 atgggnacny
tngcnacnws ntgyytnytn ytnytngcny tnytngtnca rggnggngcn 60
gcngcnccna thwsnwsnca ytgymgnytn gayaarwsna ayttycarca rccntayath
120 acnaaymgna cnttyatgyt ngcnaargar gcnwsnytng cngayaayaa
yacngaygtn 180 mgnytnathg gngaraaryt nttycayggn gtnwsnatgw
sngarmgntg ytayytnatg 240 aarcargtny tnaayttyac nytngargar
gtnytnttyc cncarwsnga ymgnttycar 300 ccntayatgc argargtngt
nccnttyytn gcnmgnytnw snaaymgnyt nwsnacntgy 360 cayathgarg
gngaygayyt ncayathcar mgnaaygtnc araarytnaa rgayacngtn 420
aaraarytng gngarwsngg ngarathaar gcnathggng arytngayyt nytnttyatg
480 wsnytnmgna aygcntgyat h 501 5 24 DNA Artificial Sequence
Oligonucleotide primer ZC25840 5 ctggatatgc aggtcatcac cttc 24 6 24
DNA Artificial Sequence Oligonucleotide primer ZC25841 6 tcgagttaga
attgtctgca atgg 24 7 23 DNA Artificial Sequence Oligonucleotide
primer ZC25838 7 aggttctcct tccccagtca cca 23 8 23 DNA Artificial
Sequence Oligonucleotide primer ZC25839 8 tagcctcctt agccagcatg aag
23 9 40 DNA Artificial Sequence Oligonucleotide primer ZC13946 9
ccctgcagtg atcaacatgg ccaagttgac cagtgccgtt 40 10 45 DNA Artificial
Sequence Oligonucleotide primer ZC13945 10 gcccatggac tagtttcgaa
aggtcgagtg tcagtcctgc tcctc 45 11 34 DNA Artificial Sequence
Oligonucleotide primer ZC18698 11 tttttttctc gagacttttt tttttttttt
tttt 34 12 18 DNA Oligonucleotide primer Artificial Sequence
Oligonucleotide primer ZC26414 12 agctgcctcc ttctcttg 18 13 18 DNA
Artificial Sequence Oligonucleotide primer ZC26415 13 tagggctgct
ggaagttg 18 14 6 PRT Artificial Sequence Glu-Glu (CEE) peptide Tag
amino acid sequence 14 Glu Tyr Met Pro Met Glu 1 5 15 8 PRT
Artificial Sequence C-terminal FLAG peptide tag 15 Asp Tyr Lys Asp
Asp Asp Asp Lys 1 5 16 36 DNA Artificial Sequence Oligonucleotide
primer ZC28590 16 ttgggtacct ctgcaatggc cgccctgcag aaatct 36 17 33
DNA Artificial Sequence Oligonucleotide primer ZC28580 17
ttgggatcca atgcaggcat ttctcagaga cat 33 18 2831 DNA Homo sapiens
CDS (34)...(1755) 18 tagaggccaa gggagggctc tgtgccagcc ccg atg agg
acg ctg ctg acc atc 54 Met Arg Thr Leu Leu Thr Ile 1 5 ttg act gtg
gga tcc ctg gct gct cac gcc cct gag gac ccc tcg gat 102 Leu Thr Val
Gly Ser Leu Ala Ala His Ala Pro Glu Asp Pro Ser Asp 10 15 20 ctg
ctc cag cac gtg aaa ttc cag tcc agc aac ttt gaa aac atc ctg 150 Leu
Leu Gln His Val Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu 25 30
35 acg tgg gac agc ggg cca gag ggc acc cca gac acg gtc tac agc atc
198 Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile
40 45 50 55 gag tat aag acg tac gga gag agg gac tgg gtg gca aag aag
ggc tgt 246 Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp Val Ala Lys Lys
Gly Cys 60 65 70 cag cgg atc acc cgg aag tcc tgc aac ctg acg gtg
gag acg ggc aac 294 Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu Thr Val
Glu Thr Gly Asn 75 80 85 ctc acg gag ctc tac tat gcc agg gtc acc
gct gtc agt gcg gga ggc 342 Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr
Ala Val Ser Ala Gly Gly 90 95 100 cgg tca gcc acc aag atg act gac
agg ttc agc tct ctg cag cac act 390 Arg Ser Ala Thr Lys Met Thr Asp
Arg Phe Ser Ser Leu Gln His Thr 105 110 115 acc ctc aag cca cct gat
gtg acc tgt atc tcc aaa gtg aga tcg att 438 Thr Leu Lys Pro Pro Asp
Val Thr Cys Ile Ser Lys Val Arg Ser Ile 120 125 130 135 cag atg att
gtt cat cct acc ccc acg cca atc cgt gca ggc gat ggc 486 Gln Met Ile
Val His Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly 140 145 150 cac
cgg cta acc ctg gaa gac atc ttc cat gac ctg ttc tac cac tta 534 His
Arg Leu Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu 155 160
165 gag ctc cag gtc aac cgc acc tac caa atg cac ctt gga ggg aag cag
582 Glu Leu Gln Val Asn Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln
170 175 180 aga gaa tat gag ttc ttc ggc ctg acc cct gac aca gag ttc
ctt ggc 630 Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro Asp Thr Glu Phe
Leu Gly 185 190 195 acc atc atg att tgc gtt ccc acc tgg gcc aag gag
agt gcc ccc tac 678 Thr Ile Met Ile Cys Val Pro Thr Trp Ala Lys Glu
Ser Ala Pro Tyr 200 205 210 215 atg tgc cga gtg aag aca ctg cca gac
cgg aca tgg acc tac tcc ttc 726 Met Cys Arg Val Lys Thr Leu Pro Asp
Arg Thr Trp Thr Tyr Ser Phe 220 225 230 tcc gga gcc ttc ctg ttc tcc
atg ggc ttc ctc gtc gca gta ctc tgc 774 Ser Gly Ala Phe Leu Phe Ser
Met Gly Phe Leu Val Ala Val Leu Cys 235 240 245 tac ctg agc tac aga
tat gtc acc aag ccg cct gca cct ccc aac tcc 822 Tyr Leu Ser Tyr Arg
Tyr Val Thr Lys Pro Pro Ala Pro Pro Asn Ser 250 255 260 ctg aac gtc
cag cga gtc ctg act ttc cag ccg ctg cgc ttc atc cag 870 Leu Asn Val
Gln Arg Val Leu Thr Phe Gln Pro Leu Arg Phe Ile Gln 265 270 275 gag
cac gtc ctg atc cct gtc ttt gac ctc agc ggc ccc agc agt ctg 918 Glu
His Val Leu Ile Pro Val Phe Asp Leu Ser Gly Pro Ser Ser Leu 280 285
290 295 gcc cag cct gtc cag tac tcc cag atc agg gtg tct gga ccc agg
gag 966 Ala Gln Pro Val Gln Tyr Ser Gln Ile Arg Val Ser Gly Pro Arg
Glu 300 305 310 ccc gca gga gct cca cag cgg cat agc ctg tcc gag atc
acc tac tta 1014 Pro Ala Gly Ala Pro Gln Arg His Ser Leu Ser Glu
Ile Thr Tyr Leu 315 320 325 ggg cag cca gac atc tcc atc ctc cag ccc
tcc aac gtg cca cct ccc 1062 Gly Gln Pro Asp Ile Ser Ile Leu Gln
Pro Ser Asn Val Pro Pro Pro 330 335 340 cag atc ctc tcc cca ctg tcc
tat gcc cca aac gct gcc cct gag gtc 1110 Gln Ile Leu Ser Pro Leu
Ser Tyr Ala Pro Asn Ala Ala Pro Glu Val 345 350 355 ggg ccc cca tcc
tat gca cct cag gtg acc ccc gaa gct caa ttc cca 1158 Gly Pro Pro
Ser Tyr Ala Pro Gln Val Thr Pro Glu Ala Gln Phe Pro 360 365 370 375
ttc tac gcc cca cag gcc atc tct aag gtc cag cct tcc tcc tat gcc
1206 Phe Tyr Ala Pro Gln Ala Ile Ser Lys Val Gln Pro Ser Ser Tyr
Ala 380 385 390 cct caa gcc act ccg gac agc tgg cct ccc tcc tat ggg
gta tgc atg 1254 Pro Gln Ala Thr Pro Asp Ser Trp Pro Pro Ser Tyr
Gly Val Cys Met 395 400 405 gaa ggt tct ggc aaa gac tcc ccc act ggg
aca ctt tct agt cct aaa 1302 Glu Gly Ser Gly Lys Asp Ser Pro Thr
Gly Thr Leu Ser Ser Pro Lys 410 415 420 cac ctt agg cct aaa ggt cag
ctt cag aaa gag cca cca gct gga agc 1350 His Leu Arg Pro Lys Gly
Gln Leu Gln Lys Glu Pro Pro Ala Gly Ser 425 430 435 tgc atg tta ggt
ggc ctt tct ctg cag gag gtg acc tcc ttg gct atg 1398 Cys Met Leu
Gly Gly Leu Ser Leu Gln Glu Val Thr Ser Leu Ala Met 440 445 450 455
gag gaa tcc caa gaa gca aaa tca ttg cac cag ccc ctg ggg att tgc
1446 Glu Glu Ser Gln Glu Ala Lys Ser Leu His Gln Pro Leu Gly Ile
Cys 460 465 470 aca gac aga aca tct gac cca aat gtg cta cac agt ggg
gag gaa ggg 1494 Thr Asp Arg Thr Ser Asp Pro Asn Val Leu His Ser
Gly Glu Glu Gly 475 480 485 aca cca cag tac cta aag ggc cag ctc ccc
ctc ctc tcc tca gtc cag 1542 Thr Pro Gln Tyr Leu Lys Gly Gln Leu
Pro Leu Leu Ser Ser Val Gln 490 495 500 atc gag ggc cac ccc atg tcc
ctc cct ttg caa cct cct tcc ggt cca 1590 Ile Glu Gly His Pro Met
Ser Leu Pro Leu Gln Pro Pro Ser Gly Pro 505 510 515 tgt tcc ccc tcg
gac caa ggt cca agt ccc tgg ggc ctg ctg gag tcc 1638 Cys Ser Pro
Ser Asp Gln Gly Pro Ser Pro Trp Gly Leu Leu Glu Ser 520 525 530 535
ctt gtg tgt ccc aag gat gaa gcc aag agc cca gcc cct gag acc tca
1686 Leu Val Cys Pro Lys Asp Glu Ala Lys Ser Pro Ala Pro Glu Thr
Ser 540 545 550 gac ctg gag cag ccc aca gaa ctg gat tct ctt ttc aga
ggc ctg gcc 1734 Asp Leu Glu Gln Pro Thr Glu Leu Asp Ser Leu Phe
Arg Gly Leu Ala 555 560 565 ctg act gtg cag tgg gag tcc tgaggggaat
gggaaaggct tggtgcttcc 1785 Leu Thr Val Gln Trp Glu Ser 570
tccctgtccc tacccagtgt cacatccttg gctgtcaatc ccatgcctgc ccatgccaca
1845 cactctgcga tctggcctca gacgggtgcc cttgagagaa gcagagggag
tggcatgcag 1905 ggcccctgcc atgggtgcgc tcctcaccgg aacaaagcag
catgataagg actgcagcgg 1965 gggagctctg gggagcagct tgtgtagaca
agcgcgtgct cgctgagccc tgcaaggcag 2025 aaatgacagt gcaaggagga
aatgcaggga aactcccgag gtccagagcc ccacctccta 2085 acaccatgga
ttcaaagtgc tcagggaatt tgcctctcct tgccccattc ctggccagtt 2145
tcacaatcta gctcgacaga gcatgaggcc cctgcctctt ctgtcattgt tcaaaggtgg
2205 gaagagagcc tggaaaagaa ccaggcctgg aaaagaacca gaaggaggct
gggcagaacc 2265 agaacaacct gcacttctgc caaggccagg gccagcagga
cggcaggact ctagggaggg 2325 gtgtggcctg cagctcattc ccagccaggg
caactgcctg acgttgcacg atttcagctt 2385 cattcctctg atagaacaaa
gcgaaatgca ggtccaccag ggagggagac acacaagcct 2445 tttctgcagg
caggagtttc agaccctatc ctgagaatgg ggtttgaaag gaaggtgagg 2505
gctgtggccc ctggacgggt acaataacac actgtactga tgtcacaact ttgcaagctc
2565 tgccttgggt tcagcccatc tgggctcaaa ttccagcctc accactcaca
agctgtgtga 2625 cttcaaacaa atgaaatcag tgcccagaac ctcggtttcc
tcatctgtaa tgtggggatc 2685 ataacaccta cctcatggag ttgtggtgaa
gatgaaatga agtcatgtct ttaaagtgct 2745 taatagtgcc tggtacatgg
gcagtgccca ataaacggta gctatttaaa aaaaaaaaaa 2805 aaaaaaaaaa
atagcggccg cctcga 2831 19 574 PRT Homo sapiens 19 Met Arg Thr Leu
Leu Thr Ile Leu Thr Val Gly Ser Leu Ala Ala His 1 5 10 15 Ala Pro
Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser 20 25 30
Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr 35
40 45 Pro Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg
Asp 50 55 60 Trp Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys
Ser Cys Asn 65 70 75 80 Leu Thr Val Glu Thr Gly Asn Leu Thr Glu Leu
Tyr Tyr Ala Arg Val 85 90 95 Thr Ala Val Ser Ala Gly Gly Arg Ser
Ala Thr Lys Met Thr Asp Arg 100 105 110 Phe Ser Ser Leu Gln His Thr
Thr Leu Lys Pro Pro Asp Val Thr Cys 115 120 125 Ile Ser Lys Val Arg
Ser Ile Gln Met Ile Val His Pro Thr Pro Thr 130 135 140 Pro Ile Arg
Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile Phe 145 150 155 160
His Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln 165
170 175 Met His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu
Thr 180 185 190 Pro Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val
Pro Thr Trp 195 200 205 Ala Lys Glu Ser Ala Pro Tyr Met Cys Arg Val
Lys Thr Leu Pro Asp 210 215 220 Arg Thr Trp Thr Tyr Ser Phe Ser Gly
Ala Phe Leu Phe Ser Met Gly
225 230 235 240 Phe Leu Val Ala Val Leu Cys Tyr Leu Ser Tyr Arg Tyr
Val Thr Lys 245 250 255 Pro Pro Ala Pro Pro Asn Ser Leu Asn Val Gln
Arg Val Leu Thr Phe 260 265 270 Gln Pro Leu Arg Phe Ile Gln Glu His
Val Leu Ile Pro Val Phe Asp 275 280 285 Leu Ser Gly Pro Ser Ser Leu
Ala Gln Pro Val Gln Tyr Ser Gln Ile 290 295 300 Arg Val Ser Gly Pro
Arg Glu Pro Ala Gly Ala Pro Gln Arg His Ser 305 310 315 320 Leu Ser
Glu Ile Thr Tyr Leu Gly Gln Pro Asp Ile Ser Ile Leu Gln 325 330 335
Pro Ser Asn Val Pro Pro Pro Gln Ile Leu Ser Pro Leu Ser Tyr Ala 340
345 350 Pro Asn Ala Ala Pro Glu Val Gly Pro Pro Ser Tyr Ala Pro Gln
Val 355 360 365 Thr Pro Glu Ala Gln Phe Pro Phe Tyr Ala Pro Gln Ala
Ile Ser Lys 370 375 380 Val Gln Pro Ser Ser Tyr Ala Pro Gln Ala Thr
Pro Asp Ser Trp Pro 385 390 395 400 Pro Ser Tyr Gly Val Cys Met Glu
Gly Ser Gly Lys Asp Ser Pro Thr 405 410 415 Gly Thr Leu Ser Ser Pro
Lys His Leu Arg Pro Lys Gly Gln Leu Gln 420 425 430 Lys Glu Pro Pro
Ala Gly Ser Cys Met Leu Gly Gly Leu Ser Leu Gln 435 440 445 Glu Val
Thr Ser Leu Ala Met Glu Glu Ser Gln Glu Ala Lys Ser Leu 450 455 460
His Gln Pro Leu Gly Ile Cys Thr Asp Arg Thr Ser Asp Pro Asn Val 465
470 475 480 Leu His Ser Gly Glu Glu Gly Thr Pro Gln Tyr Leu Lys Gly
Gln Leu 485 490 495 Pro Leu Leu Ser Ser Val Gln Ile Glu Gly His Pro
Met Ser Leu Pro 500 505 510 Leu Gln Pro Pro Ser Gly Pro Cys Ser Pro
Ser Asp Gln Gly Pro Ser 515 520 525 Pro Trp Gly Leu Leu Glu Ser Leu
Val Cys Pro Lys Asp Glu Ala Lys 530 535 540 Ser Pro Ala Pro Glu Thr
Ser Asp Leu Glu Gln Pro Thr Glu Leu Asp 545 550 555 560 Ser Leu Phe
Arg Gly Leu Ala Leu Thr Val Gln Trp Glu Ser 565 570 20 39 DNA
Artificial Sequence Oligonucleotide primer ZC26665 20 cacacaggcc
ggccaccatg gccgccctgc agaaatctg 39 21 37 DNA Artificial Sequence
Oligonucleotide primer ZC26666 21 cacacaggcg cgcctcaaat gcaggcattt
ctcagag 37 22 18 DNA Artificial Sequence Oligonucleotide primer
ZC14666 22 agccaccaag atgactga 18 23 22 DNA Artificial Sequence
Oligonucleotide primer ZC14742 23 tgcatttggt aggtgcggtt ga 22 24 23
DNA Artificial Sequence Oligonucleotide primer ZC25963 24
agtcaacgca tgagtctctg aag 23 25 23 DNA Artificial Sequence
Oligonucleotide primer ZC28354 25 accaacaaag agccattgac ttg 23 26
23 DNA Artificial Sequence Oligonucleotide primer ZC21195 26
gaggagacca taacccccga cag 23 27 23 DNA Artificial Sequence
Oligonucleotide primer ZC21196 27 catagctccc accacacgat ttt 23 28
25 DNA Artificial Sequence Oligonucleotide primer ZC14063 28
caccagacat aatagctgac agact 25 29 21 DNA Artificial Sequence
Oligonucleotide primer ZC17574 29 ggtrttgctc agcatgcaca c 21 30 24
DNA Artificial Sequence Oligonucleotide primer ZC17600 30
catgtaggcc atgaggtcca ccac 24 31 23 DNA Artificial Sequence
Oligonucleotide primer ZC25964 31 gttcttgagt accccaacag tct 23 32
2149 DNA Homo sapiens CDS (1)...(693) 32 atg atg cct aaa cat tgc
ttt cta ggc ttc ctc atc agt ttc ttc ctt 48 Met Met Pro Lys His Cys
Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu 1 5 10 15 act ggt gta gca
gga act cag tca acg cat gag tct ctg aag cct cag 96 Thr Gly Val Ala
Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln 20 25 30 agg gta
caa ttt cag tcc cga aat ttt cac aac att ttg caa tgg cag 144 Arg Val
Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln 35 40 45
cct ggg agg gca ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192
Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr 50
55 60 aaa ata tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg
ggt 240 Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp
Gly 65 70 75 80 act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca
gac ata cag 288 Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser
Asp Ile Gln 85 90 95 gaa cct tat tac ggg agg gtg agg gcg gcc tcg
gct ggg agc tac tca 336 Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser
Ala Gly Ser Tyr Ser 100 105 110 gaa tgg agc atg acg ccg cgg ttc act
ccc tgg tgg gaa aca aaa ata 384 Glu Trp Ser Met Thr Pro Arg Phe Thr
Pro Trp Trp Glu Thr Lys Ile 115 120 125 gat cct cca gtc atg aat ata
acc caa gtc aat ggc tct ttg ttg gta 432 Asp Pro Pro Val Met Asn Ile
Thr Gln Val Asn Gly Ser Leu Leu Val 130 135 140 att ctc cat gct cca
aat tta cca tat aga tac caa aag gaa aaa aat 480 Ile Leu His Ala Pro
Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn 145 150 155 160 gta tct
ata gaa gat tac tat gaa cta cta tac cga gtt ttt ata att 528 Val Ser
Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile 165 170 175
aac aat tca cta gaa aag gag caa aag gtt tat gaa ggg gct cac aga 576
Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg 180
185 190 gcg gtt gaa att gaa gct cta aca cca cac tcc agc tac tgt gta
gtg 624 Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val
Val 195 200 205 gct gaa ata tat cag ccc atg tta gac aga aga agt cag
aga agt gaa 672 Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln
Arg Ser Glu 210 215 220 gag aga tgt gtg gaa att cca tgacttgtgg
aatttggcat tcagcaatgt 723 Glu Arg Cys Val Glu Ile Pro 225 230
ggaaattcta aagctccctg agaacaggat gactcgtgtt tgaaggatct tatttaaaat
783 tgtttttgta ttttcttaaa gcaatattca ctgttacacc ttggggactt
ctttgtttat 843 ccattctttt atcctttata tttcatttta aactatattt
gaacgacatt ccccccgaaa 903 aattgaaatg taaagatgag gcagagaata
aagtgttcta tgaaattcag aactttattt 963 ctgaatgtaa catccctaat
aacaaccttc attcttctaa tacagcaaaa taaaaattta 1023 acaaccaagg
aatagtattt aagaaaatgt tgaaataatt tttttaaaat agcattacag 1083
actgaggcgg tcctgaagca atggtttttc actctcttat tgagccaatt aaattgacat
1143 tgctttgaca atttaaaact tctataaagg tgaatatttt tcatacattt
ctattttata 1203 tgaatatact ttttatatat ttattattat taaatatttc
tacttaatga atcaaaattt 1263 tgttttaaag tctactttat gtaaataaga
acaggttttg gggaaaaaaa tcttatgatt 1323 tctggattga tatctgaatt
aaaactatca acaacaagga agtctactct gtacaattgt 1383 ccctcattta
aaagatatat taagcttttc ttttctgttt gtttttgttt tgtttagttt 1443
ttaatcctgt cttagaagaa cttatcttta ttctcaaaat taaatgtaat ttttttagtg
1503 acaaagaaga aaggaaacct cattactcaa tccttctggc caagagtgtc
ttgcttgtgg 1563 cgccttcctc atctctatat aggaggatcc catgaatgat
ggtttattgg gaactgctgg 1623 ggtcgacccc atacagagaa ctcagcttga
agctggaagc acacagtggg tagcaggaga 1683 aggaccggtg ttggtaggtg
cctacagaga ctatagagct agacaaagcc ctccaaactg 1743 gcccctcctg
ctcactgcct ctcctgagta gaaatctggt gacctaaggc tcagtgcggt 1803
caacagaaag ctgccttctt cacttgaggc taagtcttca tatatgttta aggttgtctt
1863 tctagtgagg agatacatat cagagaacat ttgtacaatt ccccatgaaa
attgctccaa 1923 agttgataac aatatagtcg gtgcttctag ttatatgcaa
gtactcagtg ataaatggat 1983 taaaaaatat tcagaaatgt attggggggt
ggaggagaat aagaggcaga gcaagagcta 2043 gagaattggt ttccttgctt
ccctgtatgc tcagaaaaca ttgatttgag catagacgca 2103 gagactgaaa
aaaaaaaaat gctcgagcgg ccgccatatc cttggt 2149 33 231 PRT Homo
sapiens 33 Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe
Phe Leu 1 5 10 15 Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser
Leu Lys Pro Gln 20 25 30 Arg Val Gln Phe Gln Ser Arg Asn Phe His
Asn Ile Leu Gln Trp Gln 35 40 45 Pro Gly Arg Ala Leu Thr Gly Asn
Ser Ser Val Tyr Phe Val Gln Tyr 50 55 60 Lys Ile Tyr Gly Gln Arg
Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly 65 70 75 80 Thr Gln Glu Leu
Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln 85 90 95 Glu Pro
Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser 100 105 110
Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile 115
120 125 Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu
Val 130 135 140 Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys
Glu Lys Asn 145 150 155 160 Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu
Tyr Arg Val Phe Ile Ile 165 170 175 Asn Asn Ser Leu Glu Lys Glu Gln
Lys Val Tyr Glu Gly Ala His Arg 180 185 190 Ala Val Glu Ile Glu Ala
Leu Thr Pro His Ser Ser Tyr Cys Val Val 195 200 205 Ala Glu Ile Tyr
Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu 210 215 220 Glu Arg
Cys Val Glu Ile Pro 225 230 34 29 PRT Artificial Sequence Human
ZCYTO18 peptide 1 (huZCYTO18-1) 34 Lys Glu Ala Ser Leu Ala Asp Asn
Asn Thr Asp Val Arg Leu Ile Gly 1 5 10 15 Glu Lys Leu Phe His Gly
Val Ser Met Ser Glu Arg Cys 20 25 35 21 PRT Artificial Sequence
Human ZCYTO18 peptide 2 (huZCYTO18-2) 35 Glu Glu Val Leu Phe Pro
Gln Ser Asp Arg Phe Gln Pro Tyr Met Gln 1 5 10 15 Glu Val Val Pro
Cys 20 36 24 PRT Artificial Sequence Human ZCYTO18 peptide 3
(huZCYTO18-3) 36 Cys Asn Val Gln Lys Leu Lys Asp Thr Val Lys Lys
Leu Gly Glu Ser 1 5 10 15 Gly Glu Ile Lys Ala Ile Gly Glu 20 37 778
DNA mus musculus CDS (47)...(583) 37 aggctctcct ctcacttatc
aactgttgac acttgtgcga tcggtg atg gct gtc 55 Met Ala Val 1 ctg cag
aaa tct atg agt ttt tcc ctt atg ggg act ttg gcc gcc agc 103 Leu Gln
Lys Ser Met Ser Phe Ser Leu Met Gly Thr Leu Ala Ala Ser 5 10 15 tgc
ctg ctt ctc att gcc ctg tgg gcc cag gag gca aat gcg ctg ccc 151 Cys
Leu Leu Leu Ile Ala Leu Trp Ala Gln Glu Ala Asn Ala Leu Pro 20 25
30 35 gtc aac acc cgg tgc aag ctt gag gtg tcc aac ttc cag cag ccg
tac 199 Val Asn Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln Gln Pro
Tyr 40 45 50 atc gtc aac cgc acc ttt atg ctg gcc aag gag gcc agc
ctt gca gat 247 Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser
Leu Ala Asp 55 60 65 aac aac aca gat gtc cgg ctc atc ggg gag aaa
ctg ttc cga gga gtc 295 Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys
Leu Phe Arg Gly Val 70 75 80 aat gct aag gat cag tgc tac ctg atg
aag cag gtg ctc aac ttc acc 343 Asn Ala Lys Asp Gln Cys Tyr Leu Met
Lys Gln Val Leu Asn Phe Thr 85 90 95 ctg gaa gac gtt ctg ctc ccc
cag tca gac agg ttc cag ccc tac atg 391 Leu Glu Asp Val Leu Leu Pro
Gln Ser Asp Arg Phe Gln Pro Tyr Met 100 105 110 115 cag gag gtg gtg
cct ttc ctg acc aaa ctc agc aat cag ctc agc tcc 439 Gln Glu Val Val
Pro Phe Leu Thr Lys Leu Ser Asn Gln Leu Ser Ser 120 125 130 tgt cac
atc agc ggt gac gac cag aac atc cag aag aat gtc aga agg 487 Cys His
Ile Ser Gly Asp Asp Gln Asn Ile Gln Lys Asn Val Arg Arg 135 140 145
ctg aag gag aca gtg aaa aag ctt gga gag agt gga gag atc aag gcg 535
Leu Lys Glu Thr Val Lys Lys Leu Gly Glu Ser Gly Glu Ile Lys Ala 150
155 160 att ggg gaa ctg gac ctg ctg ttt atg tct ctg aga aat gct tgc
gtc 583 Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn Ala Cys
Val 165 170 175 tgagcgagaa gaagctagaa aacgaagaac tgctccttcc
tgccttctaa aaagaacaat 643 aagatccctg aatggacttt tttactaaag
gaaagtgaga agctaacgtc catcatcatt 703 agaagatttc acatgaaacc
tggctcagtt gaaaaagaaa atagtgtcaa gttgtccatg 763 agaccagagg tagac
778 38 179 PRT mus musculus 38 Met Ala Val Leu Gln Lys Ser Met Ser
Phe Ser Leu Met Gly Thr Leu 1 5 10 15 Ala Ala Ser Cys Leu Leu Leu
Ile Ala Leu Trp Ala Gln Glu Ala Asn 20 25 30 Ala Leu Pro Val Asn
Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln 35 40 45 Gln Pro Tyr
Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser 50 55 60 Leu
Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe 65 70
75 80 Arg Gly Val Asn Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val
Leu 85 90 95 Asn Phe Thr Leu Glu Asp Val Leu Leu Pro Gln Ser Asp
Arg Phe Gln 100 105 110 Pro Tyr Met Gln Glu Val Val Pro Phe Leu Thr
Lys Leu Ser Asn Gln 115 120 125 Leu Ser Ser Cys His Ile Ser Gly Asp
Asp Gln Asn Ile Gln Lys Asn 130 135 140 Val Arg Arg Leu Lys Glu Thr
Val Lys Lys Leu Gly Glu Ser Gly Glu 145 150 155 160 Ile Lys Ala Ile
Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn 165 170 175 Ala Cys
Val 39 32 DNA Artificial Sequence Oligonucleotide primer ZC37125 39
ctatttggcc ggccaccatg gctgtcctgc ag 32 40 32 DNA Artificial
Sequence Oligonucleotide primer ZC37126 40 cgtacgggcg cgcctcagac
gcaagcattt ct 32 41 25 DNA Artificial Sequence Oligonucleotide
primer ZC28348 41 cgggatcccg atggccgccc tgcag 25 42 28 DNA
Artificial Sequence Oligonucleotide primer ZC28345 42 gctctagacc
aatgcaggca tttctcag 28 43 17 DNA Artificial Sequence
Oligonucleotide primer ZC447 43 taacaatttc acacagg 17 44 18 DNA
Artificial Sequence Oligonucleotide primer ZC976 44 cgttgtaaaa
cgacggcc 18
* * * * *
References