U.S. patent application number 11/539079 was filed with the patent office on 2007-03-01 for antibodies to cytokine receptor zcytor19.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to FRANCIS J. GRANT, JULIA E. NOVAK, SCOTT R. PRESNELL, THEODORE E. WHITMORE, WENFENG XU.
Application Number | 20070048846 11/539079 |
Document ID | / |
Family ID | 26943370 |
Filed Date | 2007-03-01 |
United States Patent
Application |
20070048846 |
Kind Code |
A1 |
PRESNELL; SCOTT R. ; et
al. |
March 1, 2007 |
ANTIBODIES TO CYTOKINE RECEPTOR ZCYTOR19
Abstract
Novel polypeptides, polynucleotides encoding the polypeptides,
and related compositions and methods are disclosed for zcytor19, a
novel class II cytokine receptor. The polypeptides may be used
within methods for detecting ligands that stimulate the
proliferation and/or development of hematopoietic, lymphoid and
myeloid cells in vitro and in vivo. Ligand-binding receptor
polypeptides can also be used to block ligand activity in vitro and
in vivo. The polynucleotides encoding zcytor19, are located on
chromosome 1p36.11, and can be used to identify a region of the
genome associated with human disease states. The present invention
also includes methods for producing the protein, uses therefor and
antibodies thereto.
Inventors: |
PRESNELL; SCOTT R.; (Tacoma,
WA) ; XU; WENFENG; (Mukilteo, WA) ; NOVAK;
JULIA E.; (Bainbridge Island, WA) ; WHITMORE;
THEODORE E.; (Redmond, WA) ; GRANT; FRANCIS J.;
(Seattle, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
26943370 |
Appl. No.: |
11/539079 |
Filed: |
October 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11165141 |
Jun 23, 2005 |
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11539079 |
Oct 5, 2006 |
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09995898 |
Nov 28, 2001 |
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11165141 |
Jun 23, 2005 |
|
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60253561 |
Nov 28, 2000 |
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60267211 |
Feb 7, 2001 |
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Current U.S.
Class: |
435/70.21 ;
530/388.22 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 35/00 20180101; C07K 2319/00 20130101; A61P 19/02 20180101;
C07K 14/47 20130101; A61P 37/02 20180101; A61P 33/00 20180101; A61P
43/00 20180101; A61P 21/04 20180101; A61P 1/00 20180101; A61P 3/10
20180101; A61P 13/12 20180101; A61P 29/00 20180101; A61P 11/06
20180101; Y10T 436/143333 20150115; A61P 25/00 20180101; A61P 31/18
20180101; A61P 31/12 20180101; C07K 14/715 20130101 |
Class at
Publication: |
435/070.21 ;
530/388.22 |
International
Class: |
C12P 21/08 20060101
C12P021/08; C12P 21/04 20060101 C12P021/04; C07K 16/28 20070101
C07K016/28 |
Claims
1. 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 50 to 471 amino
acids, wherein the polypeptide comprises a contiguous sequence of
amino acids in SEQ ID NO:2 from amino acid number 21 (Arg), to
amino acid number 491 (Arg); (b) a polypeptide comprising amino
acid number 21 (Arg) to 119 (Tyr) of SEQ ID NO:2; (c) a polypeptide
comprising amino acid number 125 (Pro) to 223 (Pro) of SEQ ID NO:2;
and wherein the polypeptide elicits an immune response in the
animal to produce the antibody; and isolating the antibody from the
animal.
2. The antibody produced by the method of claim 1, which
specifically binds to a polypeptide of SEQ ID NO:2.
3. The antibody of claim 2, wherein the antibody is a monoclonal
antibody.
4. A method of producing an antibody to a polypeptide comprising:
inoculating an animal with a polypeptide consisting of 50 to 500
amino acids, wherein the polypeptide comprises a contiguous
sequence of amino acids in SEQ ID NO:19 from amino acid number 21
(Arg) to amino acid number 520 (Arg) wherein the polypeptide
elicits an immune response in the animal to produce the antibody;
and isolating the antibody from the animal.
5. The antibody produced by the method of claim 4, which
specifically binds to a polypeptide of SEQ ID NO:19.
6. The antibody of claim 5, wherein the antibody is a monoclonal
antibody.
7. A method of producing an antibody to a polypeptide comprising:
inoculating an animal with a polypeptide consisting of 50 to 500
amino acids, wherein the polypeptide comprises a contiguous
sequence of amino acids in SEQ ID NO:21 acid number 21 (Arg), to
amino acid number 211 (Ser) wherein the polypeptide elicits an
immune response in the animal to produce the antibody; and
isolating the antibody from the animal.
8. The antibody produced by the method of claim 7, which
specifically binds to a polypeptide of SEQ ID NO:21.
9. The antibody of claim 8, wherein the antibody is a monoclonal
antibody.
10. A method of producing an antibody to a polypeptide comprising:
inoculating an animal with a polypeptide consisting of a sequence
of amino acid residues that is selected from the group consisting
of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino
acid number 21 (Arg) to amino acid number 223 (Pro); (b) the amino
acid sequence as shown in SEQ ID NO:2 from amino acid number 21
(Arg) to amino acid number 226 (Asn); (c) the amino acid sequence
as shown in SEQ ID NO:2 from amino acid number 21 (Arg) to amino
acid number 249 (Trp); (d) the amino acid sequence as shown in SEQ
ID NO:2 from amino acid number 250 (Lys) to amino acid number 491
(Arg); (e) the amino acid sequence as shown in SEQ ID NO:19 from
amino acid number 250 (Lys) to 520 (Arg); (f) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) to
amino acid number 491 (Arg); (g) the amino acid sequence as shown
in SEQ ID NO:19 from amino acid number 21 (Arg) to amino acid
number 520 (Arg); (h) the amino acid sequence as shown in SEQ ID
NO:2 from amino acid number 1 (Met) to amino acid number 491 (Arg);
(i) the amino acid sequence as shown in SEQ ID NO:19 from amino
acid number 1 (Met) to amino acid number 520 (Arg); (j) the amino
acid sequence as shown in SEQ ID NO:21 from amino acid number 21
(Arg) to amino acid number 163 (Trp); (k) the amino acid sequence
as shown in SEQ ID NO:21 from amino acid number 21 (Arg) to amino
acid number 211 (Ser); and (l) the amino acid sequence as shown in
SEQ ID NO:21 from amino acid number 1 (Met) to amino acid number
211 (Ser) wherein the polypeptide elicits an immune response in the
animal to produce the antibody; and isolating the antibody from the
animal.
11. The antibody produced by the method of claim 8, which
specifically binds to a polypeptide of SEQ ID NO:2, SEQ ID NO:19,
or SEQ ID NO:21.
12. The antibody of claim 11, wherein the antibody is a monoclonal
antibody.
13. An antibody that specifically binds to a polypeptide comprising
the amino acid sequence of SEQ ID NO:2.
14. An antibody that specifically binds to a polypeptide comprising
the amino acid sequence of SEQ ID NO:19.
15. An antibody that specifically binds to a polypeptide comprising
the amino acid sequence of SEQ ID NO:21.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of co-pending U.S.
application Ser. No. 11/165,141 filed Jun. 23, 2005, which is a
continuation of U.S. application Ser. No. 09/995,898, filed Nov.
28, 2001, now abandoned, both of which are herein incorporated by
reference. U.S. application Ser. No. 09/995,898 claims the benefit
of Provisional Application 60/253,561, filed on Nov. 28, 2000, and
Provisional Application U.S. 60/267,211, filed on Feb. 7, 2001.
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 transcription factors. Of particular
interest are receptors for cytokines, molecules that promote the
proliferation and/or differentiation of cells. Examples of
cytokines include 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.
[0004] The demonstrated in vivo activities of these cytokines
illustrate the enormous clinical potential of, and need for, other
cytokines, cytokine agonists, and cytokine antagonists. The present
invention addresses these needs by providing new a hematopoietic
cytokine receptor, as well as related compositions and methods.
[0005] 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.
DESCRIPTION OF THE INVENTION
[0006] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention.
[0007] Within one aspect, the present invention provides an
isolated polynucleotide that encodes a polypeptide comprising an
amino acid sequence selected from the group consisting of: (a) the
amino acid sequence as shown in SEQ ID NO:2 from amino acid number
21 (Arg) to amino acid number 223 (Pro); (b) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) to
amino acid number 226 (Asn); (c) the amino acid sequence as shown
in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acid number
249 (Trp); (d) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 250 (Lys) to amino acid number 491 (Arg); (e) the
amino acid sequence as shown in SEQ ID NO:19 from amino acid number
250 (Lys) to 520 (Arg); (f) the amino acid sequence as shown in SEQ
ID NO:2 from amino acid number 21 (Arg) to amino acid number 491
(Arg); (g) the amino acid sequence as shown in SEQ ID NO:19 from
amino acid number 21 (Arg) to amino acid number 520 (Arg); (h) the
amino acid sequence as shown in SEQ ID NO:2 from amino acid number
1 (Met) to amino acid number 491 (Arg); (i) the amino acid sequence
as shown in SEQ ID NO:19 from amino acid number 1 (Met) to amino
acid number 520 (Arg); (j) the amino acid sequence as shown in SEQ
ID NO:21 from amino acid number 21 (Arg) to amino acid number 163
(Trp);(k) the amino acid sequence as shown in SEQ ID NO:21 from
amino acid number 21 (Arg) to amino acid number 211 (Ser); and (l)
the amino acid sequence as shown in SEQ ID NO:21 from amino acid
number 1 (Met) to amino acid number 211 (Ser). In one embodiment,
the isolated polynucleotide described above comprises a
polynucleotide sequence selected from the group consisting of: (a)
a polynucleotide comprising a nucleotide sequence as shown in SEQ
ID NO:1 from nucleotide 61 to nucleotide 669; (b) a polynucleotide
comprising a nucleotide sequence as shown in SEQ ID NO:1 from
nucleotide 61 to 678; (c) a polynucleotide comprising a nucleotide
sequence as shown in SEQ ID NO:1 from nucleotide 61 to nucleotide
747; (d) a polynucleotide comprising a nucleotide sequence as shown
in SEQ ID NO:1 from nucleotide 748 to nucleotide 1473; (e) a
polynucleotide comprising a nucleotide sequence as shown in SEQ ID
NO:18 from nucleotide 748 to nucleotide 1560; (f) a polynucleotide
comprising a nucleotide sequence as shown in SEQ ID NO:1 from
nucleotide 61 to nucleotide 1473; (g) a polynucleotide comprising a
nucleotide sequence as shown in SEQ ID NO:18 from nucleotide 61 to
nucleotide 1560; (h) a polynucleotide comprising a nucleotide
sequence as shown in SEQ ID NO:1 from nucleotide 1 to nucleotide
1473; (i) a polynucleotide comprising a nucleotide sequence as
shown in SEQ ID NO:18 from nucleotide 1 to nucleotide 1560; (j) a
polynucleotide comprising a nucleotide sequence as shown in SEQ ID
NO:20 from nucleotide 61 to nucleotide 489; (k) a polynucleotide
comprising a nucleotide sequence as shown in SEQ ID NO:20 from
nucleotide 61 to nucleotide 633; and (l) a polynucleotide
comprising a nucleotide sequence as shown in SEQ ID NO:20 from
nucleotide 1 to nucleotide 633. In another embodiment, the isolated
polynucleotide is as described above, wherein the polynucleotide
encodes a polypeptide that further comprises a transmembrane domain
consisting of residues 227 (Trp) to 249 (Trp) of SEQ ID NO:2. In
another embodiment, the isolated polynucleotide is as described
above, wherein the polynucleotide encodes a polypeptide that
further comprises an intracellular domain consisting of residues
250 (Lys) to 491 (Arg) of SEQ ID NO:2, or 250 (Lys) to 520 (Arg) of
SEQ ID NO:19. In another embodiment, the isolated polynucleotide is
as described above, wherein the polypeptide encoded by the
polynucleotide has activity as measured by cell proliferation,
activation of transcription of a reporter gene, or wherein the
polypeptide encoded by the polynucleotide further binds to an
antibody, wherein the antibody is raised to a polypeptide
comprising a sequence of amino acids from the group consisting of:
(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid
number 21 (Arg) to amino acid number 223 (Pro); (b) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) to
amino acid number 226 (Asn); (c) the amino acid sequence as shown
in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acid number
249 (Trp); (d) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 250 (Lys) to amino acid number 491 (Arg); (e) the
amino acid sequence as shown in SEQ ID NO:19 from amino acid number
250 (Lys) to 520 (Arg); (f) the amino acid sequence as shown in SEQ
ID NO:2 from amino acid number 21 (Arg) to amino acid number 491
(Arg); (g) the amino acid sequence as shown in SEQ ID NO:19 from
amino acid number 21 (Arg) to amino acid number 520 (Arg); (h) the
amino acid sequence as shown in SEQ ID NO:2 from amino acid number
1 (Met) to amino acid number 491 (Arg); (i) the amino acid sequence
as shown in SEQ ID NO:19 from amino acid number 1 (Met) to amino
acid number 520 (Arg); () the amino acid sequence as shown in SEQ
ID NO:21 from amino acid number 21 (Arg) to amino acid number 163
(Trp); (k) the amino acid sequence as shown in SEQ ID NO:21 from
amino acid number 21 (Arg) to amino acid number 211 (Ser); and (l)
the amino acid sequence as shown in SEQ ID NO:21 from amino acid
number 1 (Met) to amino acid number 211 (Ser); and wherein the
binding of the antibody to the isolated polypeptide is measured by
a biological or biochemical assay including radioimmunoassay,
radioimmuno-precipitation, Western blot, or enzyme-linked
immunosorbent assay.
[0008] 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
polypeptide from the group consisting of: (a) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) to
amino acid number 223 (Pro); (b) the amino acid sequence as shown
in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acid number
226 (Asn); (c) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 21 (Arg) to amino acid number 249 (Trp); (d) the
amino acid sequence as shown in SEQ ID NO:2 from amino acid number
250 (Lys) to amino acid number 491 (Arg); (e) the amino acid
sequence as shown in SEQ ID NO:19 from amino acid number 250 (Lys)
to 520 (Arg); (f) the amino acid sequence as shown in SEQ ID NO:2
from amino acid number 21 (Arg) to amino acid number 491 (Arg); (g)
the amino acid sequence as shown in SEQ ID NO:19 from amino acid
number 21 (Arg) to amino acid number 520 (Arg); (h) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to
amino acid number 491 (Arg); (i) the amino acid sequence as shown
in SEQ ID NO:19 from amino acid number 1 (Met) to amino acid number
520 (Arg); (j) the amino acid sequence as shown in SEQ ID NO:21
from amino acid number 21 (Arg) to amino acid number 163 (Trp); (k)
the amino acid sequence as shown in SEQ ID NO:21 from amino acid
number 21 (Arg) to amino acid number 211 (Ser); and (l) the amino
acid sequence as shown in SEQ ID NO:21 from amino acid number 1
(Met) to amino acid number 211 (Ser); 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
described above further comprises a secretory signal sequence
operably linked to the DNA segment.
[0009] Within a third aspect, the present invention provides a
cultured cell comprising an expression vector according to claim 7,
wherein the cell expresses a polypeptide encoded by the DNA
segment. In another embodiment, the expression vector is as
described above, wherein the polypeptide further comprises a
transmembrane domain consisting of residues 227 (Trp) to 249 (Trp)
of SEQ ID NO:2. In another embodiment, the expression vector is as
described above, wherein the polypeptide further comprises an
intracellular domain consisting of residues 250 (Lys) to 491 (Arg)
of SEQ ID NO:2 or 250 (Lys) to 520 (Arg) of SEQ ID NO:19. In
another embodiment, the expression vector is as described above,
comprising the following operably linked elements: a transcription
promoter; a DNA segment encoding a polypeptide from the group
consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2
from amino acid number 21 (Arg) to amino acid number 223 (Pro); (b)
the amino acid sequence as shown in SEQ ID NO:2 from amino acid
number 21 (Arg) to amino acid number 226 (Asn); (c) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) to
amino acid number 249 (Trp);(d) the amino acid sequence as shown in
SEQ ID NO:21 from amino acid number 21 (Arg) to amino acid number
211 (Ser); 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.
[0010] Within another aspect, the present invention provides a
cultured cell comprising an expression vector according to claim 7,
wherein the cell expresses a polypeptide encoded by the DNA segment
cultured cell into which has been introduced an expression vector
according to claim 11, wherein the cell expresses a soluble
receptor polypeptide encoded by the DNA segment.
[0011] Within another 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 of SEQ ID NO:2 from amino acid number 1 (Met),
to amino acid number 20 (Gly); (b) the amino acid sequence as shown
in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acid number
223 (Pro); (c) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 21 (Arg) to amino acid number 226 (Asn); (d) the
amino acid sequence as shown in SEQ ID NO:2 from amino acid number
21 (Arg) to amino acid number 249 (Trp); (e) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 250 (Lys)
to amino acid number 491 (Arg); (f) the amino acid sequence as
shown in SEQ ID NO:19 from amino acid number 250 (Lys) to amino
acid number 520 (Arg); (g) the amino acid sequence as shown in SEQ
ID NO:2 from amino acid number 227 (Trp) to amino acid number 249
(Trp); (h) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 227 (Trp) to amino acid number 491 (Arg); (i) the
amino acid sequence as shown in SEQ ID NO:19 from amino acid number
227 (Trp) to amino acid number 520 (Arg); (j) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) to
amino acid number 491 (Arg); (k) the amino acid sequence as shown
in SEQ ID NO:19 from amino acid number 21 (Arg) to amino acid
number 520 (Arg); (1) the amino acid sequence as shown in SEQ ID
NO:2 from amino acid number 1 (Met) to amino acid number 491 (Arg);
and (m) the amino acid sequence as shown in SEQ ID NO:19 from amino
acid number 1 (Met) to amino acid number 520 (Arg); (n) the amino
acid sequence as shown in SEQ ID NO:21 from amino acid number 21
(Arg) to amino acid number 163 (Trp); (o) the amino acid sequence
as shown in SEQ ID NO:21 from amino acid number 21 (Arg) to amino
acid number 211 (Ser); and (p) the amino acid sequence as shown in
SEQ ID NO:21 from amino acid number 1 (Met) to amino acid number
211 (Ser); 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.
[0012] 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 according to claim 13; 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.
[0013] Within another aspect, the present invention provides a
cultured cell comprising an expression vector according to claim
14, wherein the cell expresses a polypeptide encoded by the DNA
construct.
[0014] Within another aspect, the present invention provides a
method of producing a fusion protein comprising: culturing a cell
according to claim 15; and isolating the polypeptide produced by
the cell.
[0015] Within another aspect, the present invention provides an
isolated 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:2 from amino acid number 21 (Arg) to amino
acid number 223 (Pro); (b) the amino acid sequence as shown in SEQ
ID NO:2 from amino acid number 21 (Arg) to amino acid number 226
(Asn); (c) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 21 (Arg) to amino acid number 249 (Trp); (d) the
amino acid sequence as shown in SEQ ID NO:2 from amino acid number
250 (Lys) to amino acid number 491 (Arg); (e) the amino acid
sequence as shown in SEQ ID NO:19 from amino acid number 250 (Lys)
to 520 (Arg); (f) the amino acid sequence as shown in SEQ ID NO:2
from amino acid number 21 (Arg) to amino acid number 491 (Arg); (g)
the amino acid sequence as shown in SEQ ID NO:19 from amino acid
number 21 (Arg) to amino acid number 520 (Arg); (h) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to
amino acid number 491 (Arg); (i) the amino acid sequence as shown
in SEQ ID NO:19 from amino acid number 1 (Met) to amino acid number
520 (Arg); (j) the amino acid sequence as shown in SEQ ID NO:21
from amino acid number 21 (Arg) to amino acid number 163 (Trp); (k)
the amino acid sequence as shown in SEQ ID NO:21 from amino acid
number 21 (Arg) to amino acid number 211 (Ser); and (l) the amino
acid sequence as shown in SEQ ID NO:21 from amino acid number 1
(Met) to amino acid number 211 (Ser). In one embodiment, the
isolated polypeptide is as described above, wherein the polypeptide
consists of a sequence of amino acid residues that is selected from
the group consisting of: (a) the amino acid sequence as shown in
SEQ ID NO:2 from amino acid number 21 (Arg) to amino acid number
223 (Pro); (b) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 21 (Arg) to amino acid number 226 (Asn); (c) the
amino acid sequence as shown in SEQ ID NO:2 from amino acid number
21 (Arg) to amino acid number 249 (Trp); (d) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 250 (Lys)
to amino acid number 491 (Arg); (e) the amino acid sequence as
shown in SEQ ID NO:19 from amino acid number 250 (Lys) to 520
(Arg); (f) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 21 (Arg) to amino acid number 491 (Arg); (g) the
amino acid sequence as shown in SEQ ID NO:19 from amino acid number
21 (Arg) to amino acid number 520 (Arg); (h) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to
amino acid number 491 (Arg); (i) the amino acid sequence as shown
in SEQ ID NO:19 from amino acid number 1 (Met) to amino acid number
520 (Arg); (j) the amino acid sequence as shown in SEQ ID NO:21
from amino acid number 21 (Arg) to amino acid number 163 (Trp); (k)
the amino acid sequence as shown in SEQ ID NO:21 from amino acid
number 21 (Arg) to amino acid number 211 (Ser); and (l) the amino
acid sequence as shown in SEQ ID NO:21 from amino acid number 1
(Met) to amino acid number 211 (Ser). In another embodiment, the
isolated polypeptide is as described above, wherein the polypeptide
further comprises a transmembrane domain consisting of residues 227
(Trp) to 249 (Trp) of SEQ ID NO:2. In another embodiment, the
isolated polypeptide is as described above, wherein the polypeptide
further comprises an intracellular domain consisting of residues
250 (Lys) to amino acid number 491 (Arg) of SEQ ID NO:2, or 250
(Lys) to amino acid number 520 (Arg) of SEQ ID NO:19. In another
embodiment, the isolated polypeptide is as described above, wherein
the polypeptide has activity as measured by cell proliferation,
activation of transcription of a reporter gene, or wherein the
polypeptide encoded by the polynucleotide further binds to an
antibody, wherein the antibody is raised to a polypeptide
comprising a sequence of amino acids from the group consisting of:
(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid
number 21 (Arg) to amino acid number 223 (Pro); (b) the amino acid
sequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) to
amino acid number 226 (Asn); (c) the amino acid sequence as shown
in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acid number
249 (Trp); (d) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 250 (Lys) to amino acid number 491 (Arg); (e) the
amino acid sequence as shown in SEQ ID NO:19 from amino acid number
250 (Lys) to 520 (Arg); (f) the amino acid sequence as shown in SEQ
ID NO:2 from amino acid number 21 (Arg) to amino acid number 491
(Arg); (g) the amino acid sequence as shown in SEQ ID NO:19 from
amino acid number 21 (Arg) to amino acid number 520 (Arg); (h) the
amino acid sequence as shown in SEQ ID NO:2 from amino acid number
1 (Met) to amino acid number 491 (Arg); (i) the amino acid sequence
as shown in SEQ ID NO:19 from amino acid number 1 (Met) to amino
acid number 520 (Arg); (j) the amino acid sequence as shown in SEQ
ID NO:21 from amino acid number 21 (Arg) to amino acid number 163
(Trp); (k) the amino acid sequence as shown in SEQ ID NO:21 from
amino acid number 21 (Arg) to amino acid number 211 (Ser); and (l)
the amino acid sequence as shown in SEQ ID NO:21 from amino acid
number 1 (Met) to amino acid number 211 (Ser); and wherein the
binding of the antibody to the isolated polypeptide is measured by
a biological or biochemical assay including radioimmunoassay,
radioimmuno-precipitation, Western blot, or enzyme-linked
immunosorbent assay.
[0016] Within another aspect, the present invention provides a
method of producing a polypeptide comprising: culturing a cell
according to claim 8; and isolating the polypeptide produced by the
cell.
[0017] Within another aspect, the present invention provides an
isolated polypeptide comprising an amino acid segment selected from
the group consisting of: (a) the amino acid sequence as shown in
SEQ ID NO:2 from amino acid number 21 (Arg) to amino acid number
226 (Asn)); (b) the amino acid sequence as shown in SEQ ID NO:4;
(c) the amino acid sequence as shown in SEQ ID NO:21 from amino
acid number 21 (Arg) to amino acid number 211 (Ser); and (c)
sequences that are at least 90% identical to (a), (b) or (c),
wherein the polypeptide is substantially free of transmembrane and
intracellular domains ordinarily associated with hematopoietic
receptors.
[0018] Within another aspect, the present invention provides a
method of producing a polypeptide comprising: culturing a cell
according to claim 12; and isolating the polypeptide produced by
the cell.
[0019] 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 50 to 471 amino
acids, wherein the polypeptide comprises a contiguous sequence of
amino acids in SEQ ID NO:2 from amino acid number 21 (Arg), to
amino acid number 491 (Arg); (b) a polypeptide consisting of 50 to
500 amino acids, wherein the polypeptide comprises a contiguous
sequence of amino acids in SEQ ID NO:19 from amino acid number 21
(Arg), to amino acid number 520 (Arg); (c) a polypeptide consisting
of 50 to 191 amino acids, wherein the polypeptide comprises a
contiguous sequence of amino acids in SEQ ID NO:21 from amino acid
number 21 (Arg), to amino acid number 211 (Ser); (d) a polypeptide
according to claim 18; (e) a polypeptide comprising amino acid
number 21 (Arg) to 119 (Tyr) of SEQ ID NO:2; (f) a polypeptide
comprising amino acid number 125 (Pro) to 223 (Pro) of SEQ ID NO:2;
(g) a polypeptide comprising a hydrophilic peptide of SEQ ID NO:2
as predicted from a hydrophobicity plot using a Hopp/Woods
hydrophilicity profile based on a sliding six-residue window, with
buried G, S, and T residues and exposed H, Y, and W residues
ignored; and wherein the polypeptide elicits an immune response in
the animal to produce the antibody; and isolating the antibody from
the animal.
[0020] Within another aspect, the present invention provides an
antibody produced by the method of claim 25, which specifically
binds to a polypeptide of SEQ ID NO:2, SEQ ID NO:19 or SEQ ID
NO:21. In one embodiment, the antibody described above is a
monoclonal antibody.
[0021] Within another aspect, the present invention provides an
antibody that specifically binds to a polypeptide as disclosed
above.
[0022] Within another aspect, the present invention provides a
method of detecting, in a test sample, the presence of a modulator
of the activity of a cytokine receptor protein comprising:
culturing a cell into which has been introduced an expression
vector according to claim 6, wherein the cell expresses the protein
encoded by the DNA segment in the presence and absence of a test
sample; and comparing levels of activity of the protein in the
presence and absence of a test sample, by a biological or
biochemical assay; and determining from the comparison, the
presence of modulator the cytokine receptor protein activity in the
test sample.
[0023] Within another aspect, the present invention provides a
method for detecting a cytokine receptor ligand within a test
sample, comprising: contacting a test sample with a cytokine
receptor polypeptide comprising an amino acid sequence from the
group consisting of: (a) the amino acid sequence as shown in SEQ ID
NO:4; (b) the amino acid sequence as shown in SEQ ID NO:2 from
amino acid number 21 (Arg) to amino acid number 226 (Asn); and (c)
the amino acid sequence as shown in SEQ ID NO:21 from amino acid
number 21 (Arg) to amino acid number 211 (Ser); and detecting the
binding of the cytokine receptor polypeptide to a ligand in the
sample. In one embodiment, the method for detecting a cytokine
receptor ligand is as disclosed above, wherein the cytokine
receptor polypeptide is membrane bound within a cultured cell, and
the detecting step comprises measuring a biological response in the
cultured cell. In another embodiment, the method for detecting a
cytokine receptor ligand is as disclosed above, wherein the
biological response is cell proliferation or activation of
transcription of a reporter gene.
[0024] 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, SEQ ID NO:18 or SEQ ID NO:20 or the complement of SEQ ID
NO:1, SEQ ID NO:18 or SEQ ID NO:20, 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.
[0025] 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 of claim 29 under conditions
wherein the antibody binds to its complementary polypeptide in the
tissue or biological sample; visualizing the antibody bound in the
tissue or biological sample; and comparing levels of antibody bound
in the tissue or biological sample from the patient to a normal
control tissue or biological sample, wherein an increase in the
level of antibody bound to the patient tissue or biological sample
relative to the normal control tissue or biological sample is
indicative of a cancer in the patient.
[0026] Within another aspect, the present invention provides a
method for detecting a cancer in a patient, comprising:
[0027] obtaining a tissue or biological sample from a patient;
[0028] labeling a polynucleotide comprising at least 14 contiguous
nucleotides of SEQ ID NO:1, SEQ ID NO:18 or SEQ ID NO:20 or the
complement of SEQ ID NO:1, SEQ ID NO:18 or SEQ ID NO:20;
[0029] incubating the tissue or biological sample with under
conditions wherein the polynucleotide will hybridize to
complementary polynucleotide sequence;
[0030] visualizing the labeled polynucleotide in the tissue or
biological sample; and
[0031] comparing the level of labeled polynucleotide hybridization
in the tissue or biological sample from the patient to a normal
control tissue or biological sample,
[0032] wherein an increase in the labeled polynucleotide
hybridization to the patient tissue or biological sample relative
to the normal control tissue or biological sample is indicative of
a cancer in the patient.
[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-complement pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0038] The term "complements of a polynucleotide molecule" is 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 "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).
[0040] 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.
[0041] 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).
[0042] 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, multimers, or alternatively
glycosylated or derivatized forms.
[0043] 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.
[0044] 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.
[0045] "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.
[0046] 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.
[0047] 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".
[0048] "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.
[0049] 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.
[0050] 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.
[0051] The term "receptor" is used herein to denote a
cell-associated protein, or a polypeptide subunit of such a
protein, that binds to a bioactive molecule (the "ligand") and
mediates the effect of the ligand on the cell. Binding of ligand to
receptor results in a conformational change in the receptor (and,
in some cases, receptor multimerization, i.e., association of
identical or different receptor subunits) that causes interactions
between the effector domain(s) and other molecule(s) in the cell.
These interactions in turn lead to alterations in the metabolism of
the cell. Metabolic events that are linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, cell proliferation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. Cell-surface cytokine receptors are
characterized by a multi-domain structure as discussed in more
detail below. These receptors are anchored in the cell membrane by
a transmembrane domain characterized by a sequence of hydrophobic
amino acid residues (typically about 21-25 residues), which is
commonly flanked by positively charged residues (Lys or Arg). In
general, receptors can be membrane bound, cytosolic or nuclear;
monomeric (e.g., thyroid stimulating hormone receptor,
beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,
growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF
receptor, erythropoietin receptor and IL-6 receptor). The term
"receptor polypeptide" is used to denote complete receptor
polypeptide chains and portions thereof, including isolated
functional domains (e.g., ligand-binding domains). The terms
"ligand-binding domain(s)" and "cytokine-binding domain(s)" can be
used interchangeably.
[0052] A "secretory signal sequence" is 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
peptide is commonly cleaved to remove the secretory peptide during
transit through the secretory pathway.
[0053] A "soluble receptor" is a receptor polypeptide that is not
bound to a cell membrane. Soluble receptors are most commonly
ligand-binding receptor polypeptides that lack transmembrane and
cytoplasmic domains. Soluble receptors can comprise additional
amino acid residues, such as affinity tags that provide for
purification of the polypeptide or provide sites for attachment of
the polypeptide to a substrate, or immunoglobulin constant region
sequences. Many cell-surface receptors have naturally occurring,
soluble counterparts that are produced by proteolysis. Soluble
receptor polypeptides are said to be substantially free of
transmembrane and intracellular polypeptide segments when they lack
sufficient portions of these segments to provide membrane anchoring
or signal transduction, respectively.
[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] Cytokine receptor subunits are characterized by a
multi-domain structure comprising a ligand-binding domain and an
effector domain that is typically involved in signal transduction.
Multimeric cytokine receptors include homodimers (e.g., PDGF
receptor .alpha..alpha. and .beta..beta. isoforms, erythropoietin
receptor, MPL (thrombopoietin receptor), and G-CSF receptor);
heterodimers whose subunits each have ligand-binding and effector
domains (e.g., PDGF receptor .alpha..beta. isoform); and multimers
having component subunits with disparate functions (e.g., IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor
subunits are common to a plurality of receptors. For example, the
AIC2B subunit, which cannot bind ligand on its own but includes an
intracellular signal transduction domain, is a component of IL-3
and GM-CSF receptors. Many cytokine receptors can be placed into
one of four related families on the basis of their structures and
functions. Class I hematopoietic receptors, for example, are
characterized by the presence of a domain containing conserved
cysteine residues and the WSXWS motif (SEQ ID NO:5). Additional
domains, including protein kinase domains; fibronectin type III
domains; and immunoglobulin domains, which are characterized by
disulfide-bonded loops, are present in certain hematopoietic
receptors. Cytokine receptor structure has been reviewed by Urdal,
Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine
5:95-106, 1993. It is generally believed that under selective
pressure for organisms to acquire new biological functions, new
receptor family members arose from duplication of existing receptor
genes leading to the existence of multi-gene families. Family
members thus contain vestiges of the ancestral gene, and these
characteristic features can be exploited in the isolation and
identification of additional family members.
[0058] Cell-surface cytokine receptors are further characterized by
the presence of additional domains. These receptors are anchored in
the cell membrane by a transmembrane domain characterized by a
sequence of hydrophobic amino acid residues (typically about 21-25
residues), which is commonly flanked by positively charged residues
(Lys or Arg). On the opposite end of the protein from the
extracellular domain and separated from it by the transmembrane
domain is an intracellular domain.
[0059] The Zcytor19 receptor of the present invention is a class II
cytokine receptor. These receptors usually bind to
four-helix-bundle cytokines. Interleukin-10 and the interferons
have receptors in this class (e.g., interferon-gamma, alpha and
beta chains and the interferon-alpha/beta receptor alpha and beta
chains). Class II cytokine receptors are characterized by the
presence of one or more cytokine receptor modules (CRM) in their
extracellular domains. Other class II cytokine receptors include
zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4 (Genbank
Accession No. Z17227), IL-10R (Genbank Accession No.s U00672 and
NM.sub.--001558), DIRS1, zcytor7 (commonly owned U.S. Pat. No.
5,945,511), zcytor16, tissue factor, and the like. The CRMs of
class II cytokine receptors are somewhat different than the
better-known CRMs of class I cytokine receptors. While the class II
CRMs contain two type-III fibronectin-like domains, they differ in
organization.
[0060] Zcytor19, like all known class II receptors except
interferon-alpha/beta receptor alpha chain, has only a single class
II CRM in its extracellular domain. Zcytor19 is a receptor for a
helical cytokine of the interferon/IL-10 class. As was stated
above, Zcytor19 is similar to other Class II cytokine receptors
such as zcytor11 and zcytor16. Analysis of a human cDNA clone
encoding Zcytor19 (SEQ ID NO:1) revealed an open reading frame
encoding 491 amino acids (SEQ ID NO:2) comprising a secretory
signal sequence (residues 1 (Met) to 20 (Gly) of SEQ ID NO:2) and a
mature zcytor19 cytokine receptor polyptide (residues 21 (Arg) to
491 (Arg) of SEQ ID NO:2) an extracellular ligand-binding domain of
approximately 206 amino acid residues (residues 21 (Arg) to 226
(Asn) of SEQ ID NO:2), a transmembrane domain of approximately 23
amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID
NO:2), and an intracellular domain of approximately 242 amino acid
residues (residues 250 (Lys) to 491 (Arg) of SEQ ID NO:2). Within
the extracellular ligand-binding domain, there are two fibronectin
type III domains and a linker region. The first fibronectin type
III domain comprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:2,
the linker comprises residues 120 (Leu) to 124 (Glu) of SEQ ID
NO:2, and the second fibronectin type III domain is short, and
comprises residues 125 (Pro) to 223 (Pro) of SEQ ID NO:2. Thus, a
polypeptide comprising amino acids 21 (Arg) to 223 (Pro) of SEQ ID
NO:2 (SEQ ID NO:4) is considered a ligand binding fragment. In
addition as typically conserved in class II receptors, there are
conserved Tryptophan residues comprising residues 43 (Trp) and 68
(Trp) as shown in SEQ ID NO:2, and conserved Cysteine residues at
positions 74, 82, 195, 217 of SEQ ID NO:2.
[0061] In addition, the present invention includes a variant of
zcytor19 receptor that includes an approximately 30 amino acid
insertion in the intracellular domain of the polyeptide (in
reference ot SEQ ID NO:2). Analysis of a human cDNA clone encoding
Zcytor19 (SEQ ID NO:18) revealed an open reading frame encoding 520
amino acids (SEQ ID NO:19) comprising a secretory signal sequence
(residues 1 (Met) to 20 (Gly) of SEQ ID NO:19) and a mature
zcytor19 cytokine receptor polypeptide (residues 21 (Arg) to 520
(Arg) of SEQ ID NO:19) an extracellular ligand-binding domain of
approximately 206 amino acid residues (residues 21 (Arg) to 226
(Asn) of SEQ ID NO:19), a transmembrane domain of approximately 23
amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID
NO:19), and an intracellular domain of approximately 271 amino acid
residues (residues 250 (Lys) to 520 (Arg) of SEQ ID NO: 19). Within
the extracellular ligand-binding domain, there are two fibronectin
type III domains and a linker region. The first fibronectin type
III domain comprises residues 21 (Arg) to 119 (Tyr) of SEQ ID
NO:19, the linker comprises residues 120 (Leu) to 124 (Glu) of SEQ
ID NO:19, and the second fibronectin type III domain comprises
residues 125 (Pro) to 223 (Pro) of SEQ ID NO:19. Thus, a
polypeptide comprising amino acids 21 (Arg) to 223 (Pro) of SEQ ID
NO:19 (SEQ ID NO:4) is considered a ligand binding fragment. In
addition as typically conserved in class II receptors, there are
conserved Tryptophan residues comprising residues 43 (Trp) and 68
(Trp) as shown in SEQ ID NO:19, and conserved Cysteine residues at
positions 74, 82, 195, 217 of SEQ ID NO:19.
[0062] Moreover, a truncated soluble form of the zcytor19 receptor
polypeptide appears to be naturally expressed. Analysis of a human
cDNA clone encoding the truncated soluble Zcytor19 (SEQ ID NO:20)
revealed an open reading frame encoding 211 amino acids (SEQ ID
NO:21) comprising a secretory signal sequence (residues 1 (Met) to
20 (Gly) of SEQ ID NO:21) and a mature truncated soluble zcytor19
receptor polyptide (residues 21 (Arg) to 211 (Ser) of SEQ ID NO:21)
a truncated extracellular ligand-binding domain of approximately
143 amino acid residues (residues 21 (Arg) to 163 (Trp) of SEQ ID
NO:21), no transmembrane domain, but an additional domain of
approximately 48 amino acid residues (residues 164 (Lys) to 211
(Ser) of SEQ ID NO:21). Within the truncated extracellular
ligand-binding domain, there are two fibronectin type III domains
and a linker region. The first fibronectin type III domain
comprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:21, the
linker comprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:21,
and the second fibronectin type III domain comprises residues 125
(Pro) to 163 (Trp) of SEQ ID NO:21. Thus, a polypeptide comprising
amino acids 21 (Arg) to 163 (Trp) of SEQ ID NO:21 is considered a
ligand binding fragment. In addition as typically conserved in
class II receptors, there are conserved Tryptophan residues
comprising residues 43 (Trp) and 68 (Trp) as shown in SEQ ID NO:21,
and conserved Cysteine residues in this truncated soluble form of
the zcytor19 receptor are at positions 74, and 82 of SEQ ID
NO:21.
[0063] Moreover, the zcytor19 polypeptide of the present invention
can be naturally expressed wherein the extracellular ligand binding
domain comprises an additional 5-15 amino acid residues at the
N-terminus of the mature polypeptide, or extracellular cytokine
binding domain or cytokine binding fragment, as described
above.
[0064] Those skilled in the art will recognize that these domain
boundaries are approximate and are based on alignments with known
proteins and predictions of protein folding. Deletion of residues
from the ends of the domains is possible. Moreover the regions,
domains and motifs described above in reference to SEQ ID NO:2 are
also as shown in SEQ ID NO:1; domains and motifs described above in
reference to SEQ ID NO:19 are also as shown in SEQ ID NO:18; and
domains and motifs described above in reference to SEQ ID NO:21 are
also as shown in SEQ ID NO:20.
[0065] The presence of transmembrane regions, and conserved and low
variance motifs generally correlates with or defines important
structural regions in proteins. Regions of low variance (e.g.,
hydrophobic clusters) are generally present in regions of
structural importance (Sheppard, P. et al., Gene 150:163-167,
1994). Such regions of low variance often contain rare or
infrequent amino acids, such as Tryptophan. The regions flanking
and between such conserved and low variance motifs may be more
variable, but are often functionally significant because they may
relate to or define important structures and activities such as
binding domains, biological and enzymatic activity, signal
transduction, cell-cell interaction, tissue localization domains
and the like.
[0066] The regions of conserved amino acid residues in zcytor19,
described above, can be used as tools to identify new family
members. For instance, reverse transcription-polymerase chain
reaction (RT-PCR) can be used to amplify sequences encoding the
conserved regions from RNA obtained from a variety of tissue
sources or cell lines. In particular, highly degenerate primers
designed from the zcytor19 sequences are useful for this purpose.
Designing and using such degenerate primers may be readily
performed by one of skill in the art.
[0067] The present invention provides polynucleotide molecules,
including DNA and RNA molecules that encode the zcytor19
polypeptides disclosed herein. Those skilled in the art will
recognize that, in view of the degeneracy of the genetic code,
considerable sequence variation is possible among these
polynucleotide molecules. SEQ ID NO:3 is a degenerate DNA sequence
that encompass all DNAs that encode the zcytor19 polypeptide of SEQ
ID NO:2; SEQ ID NO:28 is a degenerate DNA sequence that encompass
all DNAs that encode the zcytor19 polypeptide of SEQ ID NO:19; and
SEQ ID NO:29 is a degenerate DNA sequence that encompass all DNAs
that encode the zcytor19 polypeptide of SEQ ID NO:21. Those skilled
in the art will recognize that the degenerate sequences of SEQ ID
NO:3, SEQ ID NO:28, and SEQ ID NO:29 also provide all RNA sequences
encoding SEQ ID NO:2, SEQ ID NO:19, and SEQ ID NO:21 by
substituting U for T. Thus, zcytor19 polypeptide-encoding
polynucleotides comprising nucleotide 1 to nucleotide 1473 of SEQ
ID NO:3, 1 to nucleotide 1560 of SEQ ID NO:28, 1 to nucleotide 633
of SEQ ID NO:29, and their RNA equivalents are contemplated by the
present invention. Moreover, subfragments of these degenerate
sequences such as the mature forms of the polypeptides,
extracellular, cytokine binding domains, intracellular domains, and
the like, as described herein are included in the present
invention. One of skill in the art upon reference to SEQ ID NO:2,
SEQ ID NO:19 and SEQ ID NO:21 and the subfragments thereof
described herein could readily determine the respective nucleotides
in SEQ ID NO:3, SEQ ID NO:28 or SEQ ID NO:29, that encode those
subfragments. Table 1 sets forth the one-letter codes used within
SEQ ID NO:3, to denote degenerate nucleotide positions.
"Resolutions" are the nucleotides denoted by a code letter.
"Complement" indicates the code for the complementary
nucleotide(s). For example, the code Y denotes either C or T, and
its complement R denotes A or G, A being complementary to T, and G
being complementary to C. TABLE-US-00001 TABLE 1 Nucleotide
Resolution Complement Resolution A A T T C C G G G G C C T T A A R
A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W
A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T
N A|C|G|T N A|C|G|T
[0068] The degenerate codons used in SEQ ID NO:3, encompassing all
possible codons for a given amino acid, are set forth in Table 2.
TABLE-US-00002 TABLE 2 One Amino Letter Degenerate Acid Code Codons
Codon Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA
ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN
Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E
GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA
CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT
ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe
F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR
Asn|Asp B RAY Glu|Gln Z SAR Any X NNN
[0069] 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:2,
SEQ ID NO:19, and/or SEQ ID NO:21. Variant sequences can be readily
tested for functionality as described herein.
[0070] 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:3 serves as templates for optimizing
expression of zcytor19 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.
[0071] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NO:1, SEQ ID NO:18, or SEQ ID NO:20, or a sequence complementary
thereto, under stringent conditions. In general, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. 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 (e.g., >50 base pairs) is
performed at temperatures of about 20-25.degree. C. below the
calculated T.sub.m. For smaller probes (e.g., <50 base pairs)
hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below. This allows for the maximum rate of
hybridization for DNA-DNA and DNA-RNA hybrids. 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. Suitable
stringent hybridization conditions are equivalent to about a 5 h to
overnight incubation at about 42.degree. C. in a solution
comprising: about 40-50% formamide, up to about 6.times.SSC, about
5.times. Denhardt's solution, zero up to about 10% dextran sulfate,
and about 10-20 .mu.g/ml denatured commercially-available carrier
DNA. 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; hybridization is then followed by
washing filters in up to about 2.times.SSC. For example, a suitable
wash stringency is equivalent to 0.1.times.SSC to 2.times.SSC, 0.1%
SDS, at 55.degree. C. to 65.degree. C. 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. Stringent hybridization and wash conditions
depend on the length of the probe, reflected in the Tm,
hybridization and wash solutions used, and are routinely determined
empirically by one of skill in the art.
[0072] 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 zcytor19 RNA. Such
tissues and cells are identified by Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include PBLs,
spleen, thymus, bone marrow, and lymph tissues, human
erythroleukemia cell lines, acute monocytic leukemia cell lines,
B-cell and T-cell leukemia tissue or cell lines, other lymphoid and
hematopoietic cell lines, and the like. Total RNA can be prepared
using guanidinium isothiocyanate extraction followed by isolation
by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry
18:52-94, 1979). Poly (A).sup.+ RNA is prepared from total RNA
using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA
69:1408-12, 1972). Complementary DNA (cDNA) is prepared from
poly(A).sup.+ RNA using known methods. In the alternative, genomic
DNA can be isolated. Polynucleotides encoding zcytor19 polypeptides
are then identified and isolated by, for example, hybridization or
polymerase chain reaction (PCR) (Mullis, U.S. Pat. No.
4,683,202).
[0073] A full-length clone encoding zcytor19 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 zcytor19, receptor fragments, or other specific
binding partners.
[0074] The polynucleotides of the present invention can also be
synthesized using DNA synthesis machines. Currently the method of
choice is the phosphoramidite method. If chemically synthesized
double stranded DNA is required for an application such as the
synthesis of a gene or a gene fragment, then each complementary
strand is made separately. The production of short polynucleotides
(60 to 80 bp) is technically straightforward and can be
accomplished by synthesizing the complementary strands and then
annealing them. However, for producing longer polynucleotides
(>300 bp), special strategies are usually employed, because the
coupling efficiency of each cycle during chemical DNA synthesis is
seldom 100%. To overcome this problem, synthetic genes
(double-stranded) are assembled in modular form from
single-stranded fragments that are from 20 to 100 nucleotides in
length.
[0075] An alternative way to prepare a full-length gene is to
synthesize a specified set of overlapping oligonucleotides (40 to
100 nucleotides). After the 3' and 5' short overlapping
complementary regions (6 to 10 nucleotides) are annealed, large
gaps still remain, but the short base-paired regions are both long
enough and stable enough to hold the structure together. The gaps
are filled and the DNA duplex is completed via enzymatic DNA
synthesis by E. coli DNA polymerase I. After the enzymatic
synthesis is completed, the nicks are sealed with T4 DNA ligase.
Double-stranded constructs are sequentially linked to one another
to form the entire gene sequence which is verified by DNA sequence
analysis. See Glick and Pasternak, Molecular Biotechnology
Principles & Applications of Recombinant DNA, (ASM Press,
Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53:
323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA
87:633-7, 1990. Moreover, other sequences are generally added that
contain signals for proper initiation and termination of
transcription and translation.
[0076] 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 zcytor19
polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human zcytor19 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 zcytor19 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
zcytor19-encoding cDNA can then be isolated by a variety of
methods, such as by probing with a complete or partial human cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using PCR (Mullis,
supra.), using primers designed from the representative human
zcytor19 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 zcytor19 polypeptide. Similar techniques can also be
applied to the isolation of genomic clones.
[0077] Cytokine receptor subunits are characterized by a
multi-domain structure comprising an extracellular domain, a
transmembrane domain that anchors the polypeptide in the cell
membrane, and an intracellular domain. The extracellular domain is
typically a ligand-binding domain, and the intracellular domain is
typically an effector domain involved in signal transduction,
although ligand-binding and effector functions may reside on
separate subunits of a multimeric receptor. The ligand-binding
domain may itself be a multi-domain structure. Multimeric receptors
include homodimers (e.g., PDGF receptor .alpha..alpha. and
.beta..beta. isoforms, erythropoietin receptor, MPL, and G-CSF
receptor), heterodimers whose subunits each have ligand-binding and
effector domains (e.g., PDGF receptor up isoform), and multimers
having component subunits with disparate functions (e.g., IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor
subunits are common to a plurality of receptors. For example, the
AIC2B subunit, which cannot bind ligand on its own but includes an
intracellular signal transduction domain, is a component of IL-3
and GM-CSF receptors. Many cytokine receptors can be placed into
one of four related families on the basis of the structure and
function. Hematopoietic receptors, for example, are characterized
by the presence of a domain containing conserved cysteine residues
and the WSXWS motif (SEQ ID NO:5). Cytokine receptor structure has
been reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228, 1991
and Cosman, Cytokine 5:95-106, 1993. Under selective pressure for
organisms to acquire new biological functions, new receptor family
members likely arise from duplication of existing receptor genes
leading to the existence of multi-gene families. Family members
thus contain vestiges of the ancestral gene, and these
characteristic features can be exploited in the isolation and
identification of additional family members. Thus, the cytokine
receptor superfamily is subdivided into several families, for
example, the immunoglobulin family (including CSF-1, MGF, IL-1, and
PDGF receptors); the hematopoietin family (including IL-2 receptor
.beta.-subunit, GM-CSF receptor .alpha.-subunit, GM-CSF receptor
.beta.-subunit; and G-CSF, EPO, IL-3, IL-4, IL-5, IL-6, IL-7, and
IL-9 receptors); TNF receptor family (including TNF (p80) TNF (p60)
receptors, CD27, CD30, CD40, Fas, and NGF receptor).
[0078] Analysis of the zcytor19 sequence suggests that it is a
member of the same receptor subfamily as the class II cytokine
receptors, for example, interferon-gamma, alpha and beta chains and
the interferon-alpha/beta receptor alpha and beta chains, zcytor11
(commonly owned U.S. Pat. No. 5,965,704), CRF2-4 (Genbank Accession
No. Z17227), DIRS1, zcytor7 (commonly owned U.S. Pat. No.
5,945,511) receptors. Receptors in this subfamily may associate to
form homodimers that transduce a signal. Several members of the
subfamily (e.g., receptors that bind interferon, IL-10, IL-19, and
IL-TIF) combine with a second subunit (termed a .beta.-subunit) to
bind ligand and transduce a signal. Specific .beta.-subunits
associate with a plurality of specific cytokine receptor subunits.
For example, with class II cytokine receptors commonly owned
zcytor11 (U.S. Pat. No. 5,965,704) and CRF2-4 receptor
heterodimerize to bind the cytokine IL-TIF (See, WIPO publication
WO 00/24758; Dumontier et al., J. Immunol. 164:1814-1819, 2000;
Spencer, S D et al., J. Exp. Med. 187:571-578, 1998; Gibbs, V C and
Pennica Gene 186:97-101, 1997 (CRF2-4 cDNA); Xie, M H et al., J.
Biol. Chem. 275: 31335-31339, 2000; and Kotenko, S V et al., J.
Biol. Chem. manuscript in press M007837200). Moreover, IL-10.beta.
receptor may be involved as a receptor for IL-TIF, and it is
believed to be synonymous with CRF2-4 (Dumoutier, L. et al., Proc.
Nat'l. Acad. Sci. 97:10144-10149, 2000; Liu Y et al, J. Immunol.
152; 1821-1829, 1994 (IL-10R cDNA). As such, class II receptor
complexes can be heterodimeric, or multimeric. Thus, monomeric,
homodimeric, heterodimeric and multimeric receptors comprising a
zcytor19 subunit are encompassed by the present invention.
[0079] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 represents
one allele of human zcytor19 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, SEQ ID
NO:18 or SEQ ID NO:20 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:2, SEQ ID NO:19 or SEQ ID
NO:21. cDNAs generated from alternatively spliced mRNAs, which
retain the properties of the zcytor19 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.
[0080] The present invention also provides isolated zcytor19
polypeptides that are substantially similar to the polypeptides of
SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 and their orthologs. The
term "substantially similar" is used herein to denote polypeptides
having at least 70%, more preferably at least 80%, sequence
identity to the sequences shown in SEQ ID NO:2, SEQ ID NO:19 or SEQ
ID NO:21 or their orthologs. Such polypeptides will more preferably
be at least 90% identical, and most preferably 95% or more
identical to SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 or its
orthologs.) Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:
603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915-10919, 1992. Briefly, two amino acid sequences are aligned
to optimize the alignment scores using a gap opening penalty of 10,
a gap extension penalty of 1, and the "blosum 62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: Total number of identical matches/[length of
the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences].times.100
TABLE-US-00003 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4
R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0
2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3
-3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3
1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3
-3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1
-2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1
-1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2
-3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2
-2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0081] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0082] 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 zcytor19. 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).
[0083] Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:2, SEQ ID NO:19 or SEQ ID NO:21) 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, with
other parameters set as default. These parameters can be introduced
into a FASTA program by modifying the scoring matrix file
("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol.
183:63 (1990).
[0084] 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.
[0085] The BLOSUM62 table (Table 3) is an amino acid substitution
matrix derived from about 2,000 local multiple alignments of
protein sequence segments, representing highly conserved regions of
more than 500 groups of related proteins (Henikoff and Henikoff,
Proc. Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, the
BLOSUM62 substitution frequencies can be used to define
conservative amino acid substitutions that may be introduced into
the amino acid sequences of the present invention. Although it is
possible to design amino acid substitutions based solely upon
chemical properties (as discussed below), the language
"conservative amino acid substitution" preferably refers to a
substitution represented by a BLOSUM62 value of greater than -1.
For example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
According to this system, preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 1
(e.g., 1, 2 or 3), while more preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 2
(e.g., 2 or 3).
[0086] Variant zcytor19 polypeptides or substantially homologous
zcytor19 polypeptides are characterized as having one or more amino
acid substitutions, deletions or additions. These changes are
preferably of a minor nature, that is conservative amino acid
substitutions (see Table 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
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide of up to
about 20-25 residues, or an affinity tag. The present invention
thus includes polypeptides that comprise a sequence that is at
least 80%, preferably at least 90%, and more preferably 95% or more
identical to the corresponding region of SEQ ID NO:2, SEQ ID NO:19
or SEQ ID NO:21, excluding the tags, extension, linker sequences
and the like. Polypeptides comprising affinity tags can further
comprise a proteolytic cleavage site between the zcytor19
polypeptide and the affinity tag. Suitable sites include thrombin
cleavage sites and factor Xa cleavage sites. TABLE-US-00004 TABLE 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
[0087] The present invention further provides a variety of other
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a zcytor19 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-zcytor19 polypeptide fusions can be
expressed in genetically engineered cells to produce a variety of
multimeric zcytor19 analogs. Auxiliary domains can be fused to
zcytor19 polypeptides to target them to specific cells, tissues, or
macromolecules (e.g., collagen). A zcytor19 polypeptide can be
fused to two or more moieties, such as an affinity tag for
purification and a targeting domain. Polypeptide fusions can also
comprise one or more cleavage sites, particularly between domains.
See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
[0088] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is carried out in a cell-free system
comprising an E. coli S30 extract and commercially available
enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991;
Chung et al., Science 259:806-9, 1993; and Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,
translation is carried out in Xenopus oocytes by microinjection of
mutated mRNA and chemically aminoacylated suppressor tRNAs
(Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third
method, E. coli cells are cultured in the absence of a natural
amino acid that is to be replaced (e.g., phenylalanine) and in the
presence of the desired non-naturally occurring amino acid(s)
(e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine,
or 4-fluorophenylalanine). The non-naturally occurring amino acid
is incorporated into the protein in place of its natural
counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.
Naturally occurring amino acid residues can be converted to
non-naturally occurring species by in vitro chemical modification.
Chemical modification can be combined with site-directed
mutagenesis to further expand the range of substitutions (Wynn and
Richards, Protein Sci. 2:395-403, 1993).
[0089] 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 zcytor19 amino acid residues.
[0090] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass
et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991). In the
latter technique, single alanine mutations are introduced at every
residue in the molecule, and the resultant mutant molecules are
tested for biological activity (e.g. ligand binding and signal
transduction) as disclosed below to identify amino acid residues
that are critical to the activity of the molecule. See also, Hilton
et al., J. Biol. Chem. 271:4699-4708, 1996. Sites of
ligand-receptor, protein-protein or other biological interaction
can also be determined by physical analysis of structure, as
determined by such techniques as nuclear magnetic resonance,
crystallography, electron diffraction or photoaffinity labeling, in
conjunction with mutation of putative contact site amino acids.
See, for example, de Vos et al., Science 255:306-312, 1992; Smith
et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS
Lett. 309:59-64, 1992. The identities of essential amino acids can
also be inferred from analysis of homologies with related
receptors.
[0091] Determination of amino acid residues that are within regions
or domains that are critical to maintaining structural integrity
can be determined. Within these regions one can determine specific
residues that will be more or less tolerant of change and maintain
the overall tertiary structure of the molecule. Methods for
analyzing sequence structure include, but are not limited to,
alignment of multiple sequences with high amino acid or nucleotide
identity and computer analysis using available software (e.g., the
Insight 11C viewer and homology modeling tools; MSI, San Diego,
Calif.), secondary structure propensities, binary patterns,
complementary packing and buried polar interactions (Barton,
Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al.,
Current Opin. Struct. Biol. 6:3-10, 1996). In general, when
designing modifications to molecules or identifying specific
fragments determination of structure will be accompanied by
evaluating activity of modified molecules.
[0092] Amino acid sequence changes are made in zcytor19
polypeptides so as to minimize disruption of higher order structure
essential to biological activity. For example, when the zcytor19
polypeptide comprises one or more structural domains, such as
Fibronectin Type III domains, changes in amino acid residues will
be made so as not to disrupt the domain structure and geometry and
other components of the molecule where changes in conformation
ablate some critical function, for example, binding of the molecule
to its binding partners. The effects of amino acid sequence changes
can be predicted by, for example, computer modeling as disclosed
above or determined by analysis of crystal structure (see, e.g.,
Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Other
techniques that are well known in the art compare folding of a
variant protein to a standard molecule (e.g., the native protein).
For example, comparison of the cysteine pattern in a variant and
standard molecules can be made. Mass spectrometry and chemical
modification using reduction and alkylation provide methods for
determining cysteine residues which are associated with disulfide
bonds or are free of such associations (Bean et al., Anal. Biochem.
201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and
Patterson et al., Anal. Chem. 66:3727-3732, 1994). It is generally
believed that if a modified molecule does not have the same
disulfide bonding pattern as the standard molecule folding would be
affected. Another well known and accepted method for measuring
folding is circular dichroism (CD). Measuring and comparing the CD
spectra generated by a modified molecule and standard molecule is
routine (Johnson, Proteins 7:205-214, 1990). Crystallography is
another well known method for analyzing folding and structure.
Nuclear magnetic resonance (NMR), digestive peptide mapping and
epitope mapping are also known methods for analyzing folding and
structural similarities between proteins and polypeptides (Schaanan
et al., Science 257:961-964, 1992).
[0093] A Hopp/Woods hydrophilicity profile of the zcytor19 protein
sequence as shown in SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 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 zcytor19,
hydrophilic regions include amino acid residues 295 through 300 of
SEQ ID NO:2; 451 through 456 of SEQ ID NO:2; 301 through 306 of SEQ
ID NO:2; 244 through 299 of SEQ ID NO:2; and 65 through 70 of SEQ
ID NO:2. Moreover, one of skill in the art would recognize that
zcytor19 hydrophilic regions including antigenic epitope-bearing
polypeptides can be predicted by a Jameson-Wolf plot, e.g., using
DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.).
[0094] 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 zcytor19 polypeptide,
so as not to disrupt the overall structural and biological profile.
Of particular interest for replacement are hydrophobic residues
selected from the group consisting of Val, Leu and Ile or the group
consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example,
residues tolerant of substitution could include such residues as
shown in SEQ ID NO:2. However, Cysteine residues at positions 74,
82, 195, and 217 of SEQ ID NO:2 or SEQ ID NO:19, and corresponding
Cys residues in SEQ ID NO:4 are relatively intolerant of
substitution. Moreover, Cysteine residues at positions 74, 82, of
SEQ ID NO:21 are relatively intolerant of substitution.
[0095] The identities of essential amino acids can also be inferred
from analysis of sequence similarity between class II cytokine
receptor family members with zcytor19. 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 zcytor19 polynucleotide on the basis of
structure is to determine whether a nucleic acid molecule encoding
a potential variant zcytor19 polynucleotide can hybridize to a
nucleic acid molecule having the nucleotide sequence of SEQ ID
NO:1, SEQ ID NO:18, or SEQ ID NO:20 as discussed above.
[0096] 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. Such mutagenesis and screening
methods are routine in the art. See also, Hilton et al., J Biol.
Chem. 271:4699 (1996).
[0097] The present invention also includes functional fragments of
zcytor19 polypeptides and nucleic acid molecules encoding such
functional fragments. A "functional" zcytor19 or fragment thereof
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-zcytor19 antibody or zcytor19 ligand (either soluble or
immobilized). Moreover, functional fragments also include the
signal peptide, intracellular signaling domain, and the like. As
previously described herein, zcytor19 is characterized by a class
II cytokine receptor structure. Thus, the present invention further
provides fusion proteins encompassing: (a) polypeptide molecules
comprising an extracellular domain, cytokine-binding domain, or
intracellular domain described herein; and (b) functional fragments
comprising one or more of these domains. The other polypeptide
portion of the fusion protein may be contributed by another class
II cytokine receptor, for example, interferon-gamma, alpha and beta
chains and the interferon-alpha/beta receptor alpha and beta
chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4,
DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511), and the
like, or by a non-native and/or an unrelated secretory signal
peptide that facilitates secretion of the fusion protein.
[0098] Routine deletion analyses of nucleic acid molecules can be
performed to obtain functional fragments of a nucleic acid molecule
that encodes a zcytor19 polypeptide. As an illustration, DNA
molecules having the nucleotide sequence of SEQ ID NO:1, SEQ ID
NO:18, or SEQ ID NO:20 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 zcytor19 activity, or for the ability to bind
anti-zcytor19 antibodies or zcytor19 ligand. One alternative to
exonuclease digestion is to use oligonucleotide-directed
mutagenesis to introduce deletions or stop codons to specify
production of a desired zcytor19 fragment. Alternatively,
particular fragments of a zcytor19 polynucleotide can be
synthesized using the polymerase chain reaction.
[0099] 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 l, Boynton et al., (eds.) pages 169-199
(Academic Press 1985); Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995); and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0100] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner
et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO
92/062045) and region-directed mutagenesis (Derbyshire et al., Gene
46:145, 1986; Ner et al., DNA 7:127, 1988).
[0101] Variants of the disclosed zcytor19 DNA and polypeptide
sequences can be generated through DNA shuffling as disclosed by
Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci.
USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly,
variant DNAs are generated by in vitro homologous recombination by
random fragmentation of a parent DNA followed by reassembly using
PCR, resulting in randomly introduced point mutations. This
technique can be modified by using a family of parent DNAs, such as
allelic variants or DNAs from different species, to introduce
additional variability into the process. Selection or screening for
the desired activity, followed by additional iterations of
mutagenesis and assay provides for rapid "evolution" of sequences
by selecting for desirable mutations while simultaneously selecting
against detrimental changes.
[0102] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized zcytor19 receptor polypeptides in host cells.
Preferred assays in this regard include cell proliferation assays
and biosensor-based ligand-binding assays, which are described
below. Mutagenized DNA molecules that encode active receptors or
portions thereof (e.g., ligand-binding fragments, signaling
domains, and the like) can be recovered from the host cells and
rapidly sequenced using modern equipment. These methods allow the
routine and rapid determination of the importance of individual
amino acid residues in a polypeptide of interest.
[0103] In addition, the proteins of the present invention (or
polypeptide fragments thereof) can be joined to other bioactive
molecules, particularly cytokine receptors, to provide
multi-functional molecules. For example, one or more domains from
zcytor19 soluble receptor can be joined to other cytokine soluble
receptors to enhance their biological properties or efficiency of
production.
[0104] The present invention thus provides a series of novel,
hybrid molecules in which a segment comprising one or more of the
domains of zcytor19 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.
[0105] Using the methods discussed herein, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptide
fragments or variants of SEQ ID NO:2 or SEQ ID NO:19 that retain
the signal transduction or ligand binding activity. For example,
one can make a zcytor19 "soluble receptor" by preparing a variety
of polypeptides that are substantially homologous to the
extracellular cytokine-binding domain (residues 21 (Arg) to 226
(Asn) of SEQ ID NO:2 or SEQ ID NO:19), a cytokine-binding fragment
(e.g., residues 21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ ID
NO:19; SEQ ID NO:4) or allelic variants or species orthologs
thereof) and retain ligand-binding activity of the wild-type
zcytor19 protein. Moreover, variant zcytor19 soluble receptors can
be isolated. Such polypeptides may include additional amino acids
from, for example, part or all of the transmembrane and
intracellular domains. Such polypeptides may also include
additional polypeptide segments as generally disclosed herein such
as labels, affinity tags, and the like.
[0106] For any zcytor19 polypeptide, including variants, soluble
receptors, and fusion polypeptides or proteins, one of ordinary
skill in the art can readily generate a fully degenerate
polynucleotide sequence encoding that variant using the information
set forth in Tables 1 and 2 above.
[0107] The zcytor19 polypeptides of the present invention,
including full-length polypeptides, biologically active fragments,
and fusion polypeptides, can be produced in genetically engineered
host cells according to conventional techniques. Suitable host
cells are those cell types that can be transformed or transfected
with exogenous DNA and grown in culture, and include bacteria,
fungal cells, and cultured higher eukaryotic cells. Eukaryotic
cells, particularly cultured cells of multicellular organisms, are
preferred. Techniques for manipulating cloned DNA molecules and
introducing exogenous DNA into a variety of host cells are
disclosed by Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocols in
Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
[0108] In general, a DNA sequence encoding a zcytor19 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.
[0109] To direct a zcytor19 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
zcytor19, 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 zcytor19 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).
[0110] Alternatively, the secretory signal sequence contained in
the polypeptides of the present invention is used to direct other
polypeptides into the secretory pathway. The present invention
provides for such fusion polypeptides. A signal fusion polypeptide
can be made wherein a secretory signal sequence derived from amino
acid 1 (Met) to amino acid 20 (Gly) of SEQ ID NO:2 or SEQ ID NO:19
is operably linked to another polypeptide using methods known in
the art and disclosed herein. The secretory signal sequence
contained in the fusion polypeptides of the present invention is
preferably fused amino-terminally to an additional peptide to
direct the additional peptide into the secretory pathway. Such
constructs have numerous applications known in the art. For
example, these novel secretory signal sequence fusion constructs
can direct the secretion of a polypeptide fragment or 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. Moreover, such fusion constructs allow for the expression,
secretion, and purification of zcytor19 polypepitde fragments that
can be used to inoculate an animal and generate antibodies, as
described herein.
[0111] Cultured mammalian cells are suitable hosts within the
present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et
al., ibid.), and liposome-mediated transfection (Hawley-Nelson et
al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and
viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989;
Wang and Finer, Nature Med. 2:714-716, 1996). The production of
recombinant polypeptides in cultured mammalian cells is disclosed,
for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et
al., U.S. Pat. No. 4,784,950; Palmiter et al, U.S. Pat. No.
4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured
mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC
No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL
10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. 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 (ATCC), Rockville, Md. In general, strong
transcription promoters are preferred, such as promoters from SV-40
or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other
suitable promoters include those from metallothionein genes (U.S.
Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late
promoter.
[0112] 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.
[0113] Other higher eukaryotic cells can also be used as hosts,
including plant cells, insect cells and avian cells. The use of
Agrobacterium rhizogenes as a vector for expressing genes in plant
cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore)
11:47-58, 1987. Transformation of insect cells and production of
foreign polypeptides therein is disclosed by Guarino et al., U.S.
Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells
can be infected with recombinant baculovirus, commonly derived from
Autographa californica nuclear polyhedrosis virus (AcNPV). See,
King, L. A. and Possee, R. D., The Baculovirus Expression System: A
Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. et
al., Baculovirus Expression Vectors: A Laboratory Manual, New York,
Oxford University Press., 1994; and, Richardson, C. D., Ed.,
Baculovirus Expression Protocols, Methods in Molecular Biology,
Totowa, N.J., Humana Press, 1995. A second method of making
recombinant zcytor19 baculovirus utilizes a transposon-based system
described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79,
1993). This system, which utilizes transfer vectors, is sold in the
Bac-to-Bac.TM. kit (Life Technologies, Rockville, Md.). This system
utilizes a transfer vector, pFastBacI.TM. (Life Technologies)
containing a Tn7 transposon to move the DNA encoding the zcytor19
polypeptide into a baculovirus genome maintained in E. coli as a
large plasmid called a "bacmid." See, Hill-Perkins, M. S. and
Possee, R. D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J
Gen Virol 75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport,
B., J Biol Chem 270:1543-9, 1995. In addition, transfer vectors can
include an in-frame fusion with DNA encoding an epitope tag at the
C- or N-terminus of the expressed zcytor19 polypeptide, for
example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc.
Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known in the
art, a transfer vector containing zcytor19 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 zcytor19 is
subsequently produced. Recombinant viral stocks are made by methods
commonly used in the art.
[0114] 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 Sf90
II.TM. (Life Technologies) or ESF 921.TM. (Expression Systems) for
the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences, Lenexa, KS) or
Express FiveO.TM. (Life Technologies) for the T. ni cells.
Procedures used are generally described in available laboratory
manuals (King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et
al., ibid.; Richardson, C. D., ibid.). Subsequent purification of
the zcytor19 polypeptide from the supernatant can be achieved using
methods described herein.
[0115] 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.
132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0116] The use of Pichia methanolica as host for the production of
recombinant proteins is disclosed in WIPO Publications WO 97/17450,
WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in
transforming P. methanolica will commonly be prepared as
double-stranded, circular plasmids, which are preferably linearized
prior to transformation. For polypeptide production in P.
methanolica, it is preferred that the promoter and terminator in
the plasmid be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A preferred selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), which allows ade2 host cells to grow in the absence of
adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, it is preferred to use
host cells in which both methanol utilization genes (AUG1 and AUG2)
are deleted. For production of secreted proteins, host cells
deficient in vacuolar protease genes (PEP4 and PRB1) are preferred.
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. It is preferred to transform P. methanolica
cells by electroporation using an exponentially decaying, pulsed
electric field having a field strength of from 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (t) of from 1 to
40 milliseconds, most preferably about 20 milliseconds.
[0117] 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 zcytor19 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.
[0118] 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% Bacto.TM. yeast extract (Difco
Laboratories), 0.004% adenine and 0.006% L-leucine).
[0119] Within one aspect of the present invention, a zcytor19
cytokine receptor (including transmembrane and intracellular
domains) is produced by a cultured cell, and the cell is used to
screen for ligands for the receptor, including the natural ligand,
as well as agonists and antagonists of the natural ligand. To
summarize this approach, a cDNA or gene encoding the receptor is
combined with other genetic elements required for its expression
(e.g., a transcription promoter), and the resulting expression
vector is inserted into a host cell. Cells that express the DNA and
produce functional receptor are selected and used within a variety
of screening systems.
[0120] Mammalian cells suitable for use in expressing the novel
receptors of the present invention and transducing a
receptor-mediated signal include cells that express a
.beta.-subunit, such as a class II cytokine receptor subunit, for
example, interferon-gamma, alpha and beta chains and the
interferon-alpha/beta receptor alpha and beta chains, zcytor11
(commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7
(commonly owned U.S. Pat. No. 5,945,511) receptors. Such subunits
can either naturally be expressed in the cells, or be
co-transfected with zcytor19 receptor. An exemplary cell system for
class I cytokine receptors is to use cells that express gp130, and
cells that co-express gp130 and LIF receptor (Gearing et al., EMBO
J. 10:2839-2848, 1991; Gearing et al., U.S. Pat. No. 5,284,755). In
this regard it is generally preferred to employ a cell that is
responsive to other cytokines that bind to receptors in the same
subfamily, such as IL-6 or LIF, because such cells will contain the
requisite signal transduction pathway(s). Preferred cells of this
type include BaF3 cells (Palacios and Steinmetz, Cell 41: 727-734,
1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986),
the human TF-1 cell line (ATCC number CRL-2003) and the DA-1 cell
line (Branch et al., Blood 69:1782, 1987; Broudy et al., Blood
75:1622-1626, 1990). In the alternative, suitable host cells can be
engineered to produce a .beta.-subunit or other cellular component
needed for the desired cellular response. For example, the murine
cell line BaF3 (Palacios and Steinmetz, Cell 41:727-734, 1985;
Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), a baby
hamster kidney (BHK) cell line, or the CTLL-2 cell line (ATCC
TIB-214) can be transfected to express individual class II subunits
such as, interferon-gamma, alpha and beta chains and the
interferon-alpha/beta receptor alpha and beta chains, zcytor11
(commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7
(commonly owned U.S. Pat. No. 5,945,511) receptors in addition to
zcytor19. It is generally preferred to use a host cell and
receptor(s) from the same species, however this approach allows
cell lines to be engineered to express multiple receptor subunits
from any species, thereby overcoming potential limitations arising
from species specificity. In the alternative, species homologs of
the human receptor cDNA can be cloned and used within cell lines
from the same species, such as a mouse cDNA, in the BaF3 cell line.
Cell lines that are dependent upon one hematopoietic growth factor,
such as IL-3, can thus be engineered to become dependent upon a
zcytor19 ligand or anti-zcytor19 antibody.
[0121] Cells expressing functional zcytor19 are used within
screening assays. A variety of suitable assays are known in the
art. These assays are based on the detection of a biological
response in the target cell. One such assay is a cell proliferation
assay. Cells are cultured in the presence or absence of a test
compound, and cell proliferation is detected by, for example,
measuring incorporation of tritiated thymidine or by colorimetric
assay based on the reduction or metabolic breakdown of Alymar
Blue.TM. (AccuMed, Chicago, Ill.) or
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
(Mosman, J. Immunol. Meth. 65: 55-63, 1983). An alternative assay
format uses cells that are further engineered to express a reporter
gene. The reporter gene is linked to a promoter element that is
responsive to the receptor-linked pathway, e.g, JAK/STAT pathway,
and the assay detects activation of transcription of the reporter
gene. A preferred promoter element in this regard is a serum
response element, SRE (see, for example, Shaw et al., Cell
56:563-572, 1989). A preferred such reporter gene is a luciferase
gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of
the luciferase gene is detected by luminescence using methods known
in the art (e.g., Baumgartner et al., J. Biol. Chem.
269:19094-29101, 1994; Schenborn and Goiffin, Promega Notes 41:11,
1993). Luciferase assay kits are commercially available from, for
example, Promega Corp., Madison, Wis. Target cell lines of this
type can be used to screen libraries of chemicals, cell-conditioned
culture media, fungal broths, soil samples, water samples, and the
like. For example, a bank of cell- or tissue-conditioned media
samples can be assayed on a target cell to identify cells that
produce ligand. Positive cells are then used to produce a cDNA
library in a mammalian cell expression vector, which is divided
into pools, transfected into host cells, and expressed. Media
samples from the transfected cells are then assayed, with
subsequent division of pools, retransfection, subculturing, and
re-assay of positive cells to isolate a clonal cell line expressing
the ligand. Alternatively, media samples from the transfected cells
can be assayed, with subsequent division of pools, retransfection,
and re-assay to isolate a bacterial clone expressing the ligand
cDNA. Media samples conditioned by kidney, liver, spleen, thymus,
other lymphoid tissues, B-cells, T-cells, or leukemia cell lines
are preferred sources of ligand for use in screening
procedures.
[0122] A natural ligand for zcytor19 can also be identified by
mutagenizing a cytokine-dependent cell line expressing zcytor19 and
culturing it under conditions that select for autocrine growth. See
WIPO publication WO 95/21930. Within a typical procedure, cells
expressing zcytor19 are mutagenized, such as with EMS. The cells
are then allowed to recover in the presence of the required
cytokine, then transferred to a culture medium lacking the
cytokine. Surviving cells are screened for the production of a
ligand for zcytor19, such as by adding soluble receptor polypeptide
comprising the zcytor19 extracellular cytokine-binding domain, or
cytokine-binding fragment described herein to the culture medium to
compete against the ligand or by assaying conditioned media on
wild-type cells compared to transfected cells expressing the
zcytor19 receptor. Preferred cell lines for use within this method
include cells that are transfected to express gp130 or gp130 in
combination with LIF receptor. Preferred such host cell lines
include transfected CTLL-2 cells (Gillis and Smith, Nature
268:154-156, 1977) and transfected BaF3 cells.
[0123] Moreover, a secretion trap method employing zcytor19 soluble
receptor polypeptide can be used to isolate a zcytor19 ligand
(Aldrich, et al, Cell 87: 1161-1169, 1996). A cDNA expression
library prepared from a known or suspected ligand source is
transfected into COS-7 cells. The cDNA library vector generally has
an SV40 origin for amplification in COS-7 cells, and a CMV promoter
for high expression. The transfected COS-7 cells are grown in a
monolayer and then fixed and permeabilized. Tagged or
biotin-labeled zcytor19 soluble receptor, described herein, is then
placed in contact with the cell layer and allowed to bind cells in
the monolayer that express an anti-complementary molecule, i.e., a
zcytor19 ligand. A cell expressing a ligand will thus be bound with
receptor molecules. An anti-tag antibody (anti-Ig for Ig fusions,
M2 or anti-FLAG for FLAG-tagged fusions, streptavidin, anti-Glu-Glu
tag, and the like) which is conjugated with horseradish peroxidase
(HRP) is used to visualize these cells to which the tagged or
biotin-labeled zcytor19 soluble receptor has bound. The HRP
catalyzes deposition of a tyramide reagent, for example,
tyramide-FITC. A commercially-available kit can be used for this
detection (for example, Renaissance TSA-Direct.TM. Kit; NEN Life
Science Products, Boston, Mass.). Cells which express zcytor19
receptor ligand will be identified under fluorescence microscopy as
green cells and picked for subsequent cloning of the ligand using
procedures for plasmid rescue as outlined in Aldrich, et al,
supra., followed by subsequent rounds of secretion trap assay, or
conventional screening of cDNA library pools, until single clones
are identified.
[0124] As a receptor, the activity of zcytor19 polypeptide can be
measured by a silicon-based biosensor microphysiometer which
measures the extracellular acidification rate or proton excretion
associated with receptor binding and subsequent physiologic
cellular responses. An exemplary device is the Cytosensor.TM.
Microphysiometer manufactured by Molecular Devices, Sunnyvale,
Calif. A variety of cellular responses, such as cell proliferation,
ion transport, energy production, inflammatory response, regulatory
and receptor activation, and the like, can be measured by this
method. See, for example, McConnell, H. M. et al., Science
257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.
228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,
1998; Van Liefde, I. Et al., Eur. J. Pharmacol. 346:87-95, 1998.
The microphysiometer can be used for assaying eukaryotic,
prokaryotic, adherent or non-adherent cells. By measuring
extracellular acidification changes in cell media over time, the
microphysiometer directly measures cellular responses to various
stimuli, including agonists, ligands, or antagonists of the
zcytor19 polypeptide. Preferably, the microphysiometer is used to
measure responses of a zcytor19-expressing eukaryotic cell,
compared to a control eukaryotic cell that does not express
zcytor19 polypeptide. Zcytor19-expressing eukaryotic cells comprise
cells into which zcytor19 has been transfected or infected via
adenovirus vector, and the like, as described herein, creating a
cell that is responsive to zcytor19-modulating stimuli, or are
cells naturally expressing zcytor19, such as zcytor19-expressing
cells derived from lymphoid, spleen, thymus tissue or PBLs.
Differences, measured by an increase or decrease in extracellular
acidification, in the response of cells expressing zcytor19,
relative to a control, are a direct measurement of
zcytor19-modulated cellular responses. Moreover, such
zcytor19-modulated responses can be assayed under a variety of
stimuli. Also, using the microphysiometer, there is provided a
method of identifying agonists and antagonists of zcytor19
polypeptide, comprising providing cells expressing a zcytor19
polypeptide, culturing a first portion of the cells in the absence
of a test compound, culturing a second portion of the cells in the
presence of a test compound, and detecting an increase or a
decrease in a cellular response of the second portion of the cells
as compared to the first portion of the cells. Antagonists and
agonists, including the natural ligand for zcytor19 polypeptide,
can be rapidly identified using this method.
[0125] Additional assays provided by the present invention include
the use of hybrid receptor polypeptides. These hybrid polypeptides
fall into two general classes. Within the first class, the
intracellular domain of zcytor19, comprising approximately residues
250 (Lys) to 491 (Arg) of SEQ ID NO:2 or residues 250 (Lys) to 520
(Arg) of SEQ ID NO:19), is joined to the ligand-binding domain of a
second receptor. It is preferred that the second receptor be a
hematopoietic cytokine receptor, such as mpl receptor (Souyri et
al., Cell 63:1137-1147, 1990). The hybrid receptor will further
comprise a transmembrane domain, which may be derived from either
receptor. A DNA construct encoding the hybrid receptor is then
inserted into a host cell. Cells expressing the hybrid receptor are
cultured in the presence of a ligand for the binding domain and
assayed for a response. This system provides a means for analyzing
signal transduction mediated by zcytor19 while using readily
available ligands. This system can also be used to determine if
particular cell lines are capable of responding to signals
transduced by zcytor19. A second class of hybrid receptor
polypeptides comprise the extracellular (ligand-binding)
cytokine-binding domain (residues 21 (Arg) to 226 (Asn) of SEQ ID
NO:2 or SEQ ID NO:19), or cytokine-binding fragment (e.g., residues
21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ ID NO:19; SEQ ID NO:4)
with a cytoplasmic domain of a second receptor, preferably a
cytokine receptor, and a transmembrane domain. The transmembrane
domain may be derived from either receptor. Hybrid receptors of
this second class are expressed in cells known to be capable of
responding to signals transduced by the second receptor. Together,
these two classes of hybrid receptors enable the use of a broad
spectrum of cell types within receptor-based assay systems.
[0126] Cells found to express a ligand for zcytor19 are then used
to prepare a cDNA library from which the ligand-encoding cDNA may
be isolated as disclosed above. The present invention thus
provides, in addition to novel receptor polypeptides, methods for
cloning polypeptide ligands for the receptors.
[0127] The zcytor19 structure and tissue expression suggests a role
in early hematopoietic or thymocyte development and immune response
regulation. These processes involve stimulation of cell
proliferation and differentiation in response to the binding of one
or more cytokines to their cognate receptors. In view of the tissue
distribution observed for this receptor, agonists (including the
natural ligand) and antagonists have enormous potential in both in
vitro and in vivo applications. Compounds identified as receptor
agonists are useful for stimulating proliferation and development
of target cells in vitro and in vivo. For example, agonist
compounds or antizcytor19 antibodies, are useful as components of
defined cell culture media, and may be used alone or in combination
with other cytokines and hormones to replace serum that is commonly
used in cell culture. Agonists are thus useful in specifically
promoting the growth and/or development of T-cells, B-cells, and
other cells of the lymphoid and myeloid lineages, and hematopoietic
cells in culture.
[0128] Agonist ligands for zcytor19, or anti-zcytor19 antibodies,
may be useful in stimulating cell-mediated immunity and for
stimulating lymphocyte proliferation, such as in the treatment of
infections involving immunosuppression, including certain viral
infections. Additional uses include tumor suppression, where
malignant transformation results in tumor cells that are antigenic.
Agonist ligands or anti-zcytor19 antibodies could be used to induce
cytotoxicity, which may be mediated through activation of effector
cells such as T-cells, NK (natural killer) cells, or LAK (lymphoid
activated killer) cells, or induced directly through apoptotic
pathways. For example, zcytor19 antibodies could be used for
stimulating cytotoxicity or ADCC on zcytor19-bearing cancer cells.
Agonist ligands may also be useful in treating leukopenias by
increasing the levels of the affected cell type, and for enhancing
the regeneration of the T-cell repertoire after bone marrow
transplantation.
[0129] Antagonist ligands, compounds, soluble zcytor19 receptors,
or anti-zcytor19 antibodies may find utility in the suppression of
the immune system, such as in the treatment of autoimmune diseases,
including rheumatoid arthritis, multiple sclerosis, diabetes
mellitis, inflammatory bowel disease, Crohn's disease, etc. Immune
suppression can also be used to reduce rejection of tissue or organ
transplants and grafts and to treat T-cell specific leukemias or
lymphomas by inhibiting proliferation of the affected cell
type.
[0130] The present invention contemplates the use of naked
anti-zcytor19 antibodies (or naked antibody fragments thereof), as
well as the use of immunoconjugates to effect treatment of various
disorders, including B-cell malignancies and other cancers
described herein wherein zcytor19 is expressed. Such
immunoconjugates as well as anti-zcytor19 antibodies can be used
for stimulating cytotoxicity or ADCC on zcytor19-bearing cancer
cells. Immunoconjugates can be prepared using standard techniques.
For example, immunoconjugates can be produced by indirectly
conjugating a therapeutic agent to an antibody component (see, for
example, Shih et al., Int. J Cancer 41:832-839 (1988); Shih et al.,
Int. J. Cancer 46:1101-1106 (1990); and Shih et al., U.S. Pat. No.
5,057,313). Briefly, one standard approach involves reacting an
antibody component having an oxidized carbohydrate portion with a
carrier polymer that has at least one free amine function and that
is loaded with a plurality of drug, toxin, chelator, boron addends,
or other therapeutic agent. This reaction results in an initial
Schiff base (imine) linkage, which can be stabilized by reduction
to a secondary amine to form the final conjugate.
[0131] The carrier polymer can be an aminodextran or polypeptide of
at least 50 amino acid residues, although other substantially
equivalent polymer carriers can also be used. Preferably, the final
immunoconjugate is soluble in an aqueous solution, such as
mammalian serum, for ease of administration and effective targeting
for use in therapy. Thus, solubilizing functions on the carrier
polymer will enhance the serum solubility of the final
immunoconjugate.
[0132] In an alternative approach for producing immunoconjugates
comprising a polypeptide therapeutic agent, the therapeutic agent
is coupled to aminodextran by glutaraldehyde condensation or by
reaction of activated carboxyl groups on the polypeptide with
amines on the aminodextran. Chelators can be attached to an
antibody component to prepare immunoconjugates comprising
radiometals or magnetic resonance enhancers. Illustrative chelators
include derivatives of ethylenediaminetetraacetic acid and
diethylenetriaminepentaacetic acid. Boron addends, such as
carboranes, can be attached to antibody components by conventional
methods.
[0133] Immunoconjugates can also be prepared by directly
conjugating an antibody component with a therapeutic agent. The
general procedure is analogous to the indirect method of
conjugation except that a therapeutic agent is directly attached to
an oxidized antibody component.
[0134] As a further illustration, a therapeutic agent can be
attached at the hinge region of a reduced antibody component via
disulfide bond formation. For example, the tetanus toxoid peptides
can be constructed with a single cysteine residue that is used to
attach the peptide to an antibody component. As an alternative,
such peptides can be attached to the antibody component using a
heterobifunctional cross-linker, such as N-succinyl
3-(2-pyridyldithio)proprionate. Yu et al., Int. J. Cancer 56:244
(1994). General techniques for such conjugation are well-known in
the art. See, for example, Wong, Chemistry Of Protein Conjugation
And Cross-Linking (CRC Press 1991); Upeslacis et al., "Modification
of Antibodies by Chemical Methods," in Monoclonal Antibodies:
Principles And Applications, Birch et al. (eds.), pages 187-230
(Wiley-Liss, Inc. 1995); Price, "Production and Characterization of
Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering And Clinical Application, Ritter et al.
(eds.), pages 60-84 (Cambridge University Press 1995).
[0135] As described above, carbohydrate moieties in the Fc region
of an antibody can be used to conjugate a therapeutic agent.
However, the Fc region is absent if an antibody fragment is used as
the antibody component of the immunoconjugate. Nevertheless, it is
possible to introduce a carbohydrate moiety into the light chain
variable region of an antibody or antibody fragment. See, for
example, Leung et al., J. Immunol. 154:5919 (1995); Hansen et al.,
U.S. Pat. No. 5,443,953 (1995). The engineered carbohydrate moiety
is then used to attach a therapeutic agent.
[0136] In addition, those of skill in the art will recognize
numerous possible variations of the conjugation methods. For
example, the carbohydrate moiety can be used to attach
polyethyleneglycol in order to extend the half-life of an intact
antibody, or antigen-binding fragment thereof, in blood, lymph, or
other extracellular fluids. Moreover, it is possible to construct a
divalent immunoconjugate by attaching therapeutic agents to a
carbohydrate moiety and to a free sulfhydryl group. Such a free
sulfhydryl group may be located in the hinge region of the antibody
component.
[0137] One type of immunoconjugate comprises an antibody component
and a polypeptide cytotoxin. An example of a suitable polypeptide
cytotoxin is a ribosome-inactivating protein. Type I
ribosome-inactivating proteins are single-chain proteins, while
type II ribosome-inactivating proteins consist of two nonidentical
subunits (A and B chains) joined by a disulfide bond (for a review,
see Soria et al., Targeted Diagn. Ther. 7:193 (1992)). Useful type
I ribosome-inactivating proteins include polypeptides from
Saponaria officinalis (e.g., saporin-1, saporin-2, saporin-3,
saporin-6), Momordica charantia (e.g, momordin), Byronia dioica
(e.g., bryodin, bryodin-2), Trichosanthes kirilowii (e.g.,
trichosanthin, trichokirin), Gelonium multiflorum (e.g., gelonin),
Phytolacca americana (e.g., pokeweed antiviral protein, pokeweed
antiviral protein-II, pokeweed antiviral protein-S), Phytolacca
dodecandra (e.g., dodecandrin, Mirabilis antiviral protein), and
the like. Ribosome-inactivating proteins are described, for
example, by Walsh et al., U.S. Pat. No. 5,635,384.
[0138] Suitable type II ribosome-inactivating proteins include
polypeptides from Ricinus communis (e.g., ricin), Abrus precatorius
(e.g., abrin), Adenia digitata (e.g., modeccin), and the like.
Since type II ribosome-inactiving proteins include a B chain that
binds galactosides and a toxic A chain that depurinates adensoine,
type II ribosome-inactivating protein conjugates should include the
A chain. Additional useful ribosome-inactivating proteins include
bouganin, clavin, maize ribosome-inactivating proteins, Vaccaria
pyramidata ribosome-inactivating proteins, nigrine b, basic nigrine
1, ebuline, racemosine b, luffin-a, luffin-b, luffin-S, and other
ribosome-inactivating proteins known to those of skill in the art.
See, for example, Bolognesi and Stirpe, international publication
No. WO98/55623, Colnaghi et al., international publication No.
WO97/49726, Hey et al., U.S. Pat. No. 5,635,384, Bolognesi and
Stirpe, international publication No. WO95/07297, Arias et al.,
international publication No. WO94/20540, Watanabe et al., J.
Biochem. 106:6 977 (1989); Islam et al., Agric. Biol. Chem. 55:229
(1991), and Gao et al., FEBS Lett. 347:257 (1994).
[0139] Analogs and variants of naturally-occurring
ribosome-inactivating proteins are also suitable for the targeting
compositions described herein, and such proteins are known to those
of skill in the art. Ribosome-inactivating proteins can be produced
using publicly available amino acid and nucleotide sequences. As an
illustration, a nucleotide sequence encoding saporin-6 is disclosed
by Lorenzetti et al., U.S. Pat. No. 5,529,932, while Walsh et al.,
U.S. Pat. No. 5,635,384, describe maize and barley
ribosome-inactivating protein nucleotide and amino acid sequences.
Moreover, ribosome-inactivating proteins are also commercially
available.
[0140] Additional polypeptide cytotoxins include ribonuclease,
DNase I, Staphylococcal enterotoxin-A, diphtheria toxin,
Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,
Pastan et al., Cell 47:641 (1986), and Goldenberg, C A--A Cancer
Journal for Clinicians 44:43 (1994).
[0141] Another general type of useful cytotoxin is a tyrosine
kinase inhibitor. Since the activation of proliferation by tyrosine
kinases has been suggested to play a role in the development and
progression of tumors, this activation can be inhibited by
anti-zcytor19 antibody components that deliver tyrosine kinase
inhibitors. Suitable tyrosine kinase inhibitors include
isoflavones, such as genistein (5,7,4'-trihydroxyisoflavone),
daidzein (7,4'-dihydroxyisoflavone), and biochanin A
(4-methoxygenistein), and the like. Methods of conjugating tyrosine
inhibitors to a growth factor are described, for example, by Uckun,
U.S. Pat. No. 5,911,995.
[0142] Another group of useful polypeptide cytotoxins includes
immunomodulators. As used herein, the term "immunomodulator"
includes cytokines, stem cell growth factors, lymphotoxins,
co-stimulatory molecules, hematopoietic factors, and the like, as
well as synthetic analogs of these molecules. Examples of
immunomodulators include tumor necrosis factor, interleukins (e.g.,
interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, and IL-20), colony stimulating factors (e.g.,
granulocyte-colony stimulating factor and granulocyte
macrophage-colony stimulating factor), interferons (e.g.,
interferons-.alpha., -.beta., -.gamma., -.omega., -.epsilon., and
-.tau.), the stem cell growth factor designated "S1 factor,"
erythropoietin, and thrombopoietin. Illustrative immunomodulator
moieties include IL-2, IL-6, IL-10, interferon-.quadrature.,
TNF-.quadrature., and the like.
[0143] Immunoconjugates that include an immunomodulator provide a
means to deliver an immunomodulator to a target cell, and are
particularly useful against tumor cells. The cytotoxic effects of
immunomodulators are well known to those of skill in the art. See,
for example, Klegerman et al., "Lymphokines and Monokines," in
Biotechnology And Pharmacy, Pessuto et al. (eds.), pages 53-70
(Chapman & Hall 1993). As an illustration, interferons can
inhibit cell proliferation by inducing increased expression of
class I histocompatibility antigens on the surface of various cells
and thus, enhance the rate of destruction of cells by cytotoxic T
lymphocytes. Furthermore, tumor necrosis factors, such as tumor
necrosis factor-a, are believed to produce cytotoxic effects by
inducing DNA fragmentation.
[0144] The present invention also includes immunocongugates that
comprise a nucleic acid molecule encoding a cytotoxin. As an
example of this approach, Hoganson et al., Human Gene Ther. 9:2565
(1998), describe FGF-2 mediated delivery of a saporin gene by
producing an FGF-2-polylysine conjugate which was condensed with an
expression vector comprising a saporin gene.
[0145] Other suitable toxins are known to those of skill in the
art.
[0146] Conjugates of cytotoxic polypeptides and antibody components
can be prepared using standard techniques for conjugating
polypeptides. For example, Lam and Kelleher, U.S. Pat. No.
5,055,291, describe the production of antibodies conjugated with
either diphtheria toxin fragment A or ricin toxin. The general
approach is also illustrated by methods of conjugating fibroblast
growth factor with saporin, as described by Lappi et al., Biochem.
Biophys. Res. Commun. 160:917 (1989), Soria et al., Targeted Diagn.
Ther. 7:193 (1992), Buechler et al., Eur. J. Biochem. 234:706
(1995), Behar-Cohen et al., Invest. Ophthalmol. Vis. Sci. 36:2434
(1995), Lappi and Baird, U.S. Pat. No. 5,191,067, Calabresi et al.,
U.S. Pat. No. 5,478,804, and Lappi and Baird, U.S. Pat. No.
5,576,288. Also see, Ghetie and Vitteta, "Chemical Construction of
Immunotoxins," in Drug Targeting: Strategies, Principles, and
Applications, Francis and Delgado (Eds.), pages 1-26 (Humana Press,
Inc. 2000), Hall (Ed.), Immunotoxin Methods and Protocols (Humana
Press, Inc. 2000), and Newton and Rybak, "Construction of
Ribonuclease-Antibody Conjugates for Selective Cytotoxicity," in
Drug Targeting: Strategies, Principles, and Applications, Francis
and Delgado (Eds.), pages 27-35 (Humana Press, Inc. 2000).
[0147] Alternatively, fusion proteins comprising an antibody
component and a cytotoxic polypeptide can be produced using
standard methods. Methods of preparing fusion proteins comprising a
cytotoxic polypeptide moiety are well-known in the art of
antibody-toxin fusion protein production. For example, antibody
fusion proteins comprising an interleukin-2 moiety are described by
Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al., Cancer
Gene Ther. 2:161 (1995), Becker et al., Proc. Natl Acad. Sci. USA
93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and
Hu et al., Cancer Res. 56:4998 (1996). In addition, Yang et al.,
Hum. Antibodies Hybridomas 6:129 (1995), describe a fusion protein
that includes an F(ab').sub.2 fragment and a tumor necrosis factor
alpha moiety. Antibody-Pseudomonas exotoxin A fusion proteins have
been described by Chaudhary et al., Nature 339:394 (1989),
Brinkmann et al., Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra
et al., Proc. Nat'l Acad. Sci. USA 89:5867 (1992), Friedman et al.,
J. Immunol. 150:3054 (1993), Wels et al., Int. J. Can. 60:137
(1995), Fominaya et al., J. Biol. Chem. 271:10560 (1996), Kuan et
al., Biochemistry 35:2872 (1996), and Schmidt et al., Int. J. Can.
65:538 (1996). Antibody-toxin fusion proteins containing a
diphtheria toxin moiety have been described by Kreitman et al.,
Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem. 268:5302
(1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), and
Vallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor
Targeting 1:177 (1995), have described an antibody-toxin fusion
protein having an RNase moiety, while Linardou et al., Cell
Biophys. 24-25:243 (1994), produced an antibody-toxin fusion
protein comprising a DNase I component. Gelonin was used as the
toxin moiety in the antibody-toxin fusion protein of Better et al.,
J. Biol. Chem. 270:14951 (1995). As a further example, Dohlsten et
al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994), reported an
antibody-toxin fusion protein comprising Staphylococcal
enterotoxin-A. Also see, Newton and Rybak, "Preparation of
Recombinant RNase Single-Chain Antibody Fusion Proteins," in Drug
Targeting: Strategies, Principles, and Applications, Francis and
Delgado (Eds.), pages 77-95 (Humana Press, Inc. 2000).
[0148] As an alternative to a polypeptide cytotoxin,
immunoconjugates can comprise a radioisotope as the cytotoxic
moiety. For example, an immunoconjugate can comprise an
anti-zcytor19 antibody component and an .alpha.-emitting
radioisotope, a .beta.-emitting radioisotope, a .gamma.-emitting
radioisotope, an Auger electron emitter, a neutron capturing agent
that emits .alpha.-particles or a radioisotope that decays by
electron capture. Suitable radioisotopes include .sup.198Au,
.sup.199Au, .sup.32P, .sup.33P, .sup.125I, .sup.131I, .sup.123I,
.sup.90Y, .sup.186Re, .sup.188Re, .sup.67Cu, .sup.211At, .sup.47Sc,
.sup.103Pb, .sup.109Pd, .sup.212Pb, .sup.71Ge, .sup.77As,
.sup.105Rh, .sup.113Ag, .sup.119Sb, .sup.121Sn, .sup.131Cs,
.sup.143Pr, .sup.161Tb, .sup.177Lu, .sup.191Os, .sup.193Pt,
.sup.197Hg, and the like.
[0149] A radioisotope can be attached to an antibody component
directly or indirectly, via a chelating agent. For example,
.sup.67Cu, which provides .beta.-particles and .gamma.-rays, can be
conjugated to an antibody component using the chelating agent,
p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid. Chase and
Shapiro, "Medical Applications of Radioisotopes," in Gennaro (Ed.),
Remington: The Science and Practice of Pharmacy, 19th Edition,
pages 843-865 (Mack Publishing Company 1995). As an alternative,
.sup.90Y, which emits an energetic .beta.-particle, can be coupled
to an antibody component using diethylenetriaminepentaacetic acid.
Moreover, an exemplary suitable method for the direct radiolabeling
of an antibody component with .sup.131I is described by Stein et
al., Antibody Immunoconj. Radiopharm. 4:703 (1991). Alternatively,
boron addends such as carboranes can be attached to antibody
components, using standard techniques.
[0150] Another type of suitable cytotoxin for the preparation of
immunoconjugates is a chemotherapeutic drug. Illustrative
chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates,
nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs,
purine analogs, antibiotics, epipodophyllotoxins, platinum
coordination complexes, and the like. Specific examples of
chemotherapeutic drugs include methotrexate, doxorubicin,
daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin,
bleomycin, melphalan, chlorambucil, maytansinoids, calicheamicin,
taxol, and the like. Suitable chemotherapeutic agents are described
in Remington: The Science and Practice of Pharmacy, 19th Edition
(Mack Publishing Co. 1995), and in Goodman And Gilman's The
Pharmacological Basis Of Therapeutics, 9th Ed. (MacMillan
Publishing Co. 1995). Other suitable chemotherapeutic agents are
known to those of skill in the art.
[0151] In another approach, immunoconjugates are prepared by
conjugating photoactive agents or dyes to an antibody component.
Fluorescent and other chromogens, or dyes, such as porphyrins
sensitive to visible light, have been used to detect and to treat
lesions by directing the suitable light to the lesion. This type of
"photoradiation," "phototherapy," or "photodynamic" therapy is
described, for example, by Mew et al., J. Immunol. 130:1473 (1983),
Jori et al. (eds.), Photodynamic Therapy Of Tumors And Other
Diseases (Libreria Progetto 1985), Oseroff et al., Proc. Natl.
Acad. Sci. USA 83:8744 (1986), van den Bergh, Chem. Britain 22:430
(1986), Hasan et al., Prog. Clin. Biol. Res. 288:471 (1989),
Tatsuta et al., Lasers Surg. Med. 9:422 (1989), and Pelegrin et
al., Cancer 67:2529 (1991).
[0152] The approaches described above can also be used to prepare
multispecific antibody compositions that comprise an
immunoconjugate.
[0153] Anti-zcytor19 antibodies and multispecific antibody
compositions can be used to modulate the immune system by
preventing the binding of zcytor19 ligands with endogenous zcytor19
receptors. Such antibodies can be administered to any subject in
need of treatment, and the present invention contemplates both
veterinary and human therapeutic uses. Illustrative subjects
include mammalian subjects, such as farm animals, domestic animals,
and human patients.
[0154] Multispecific antibody compositions and dual reactive
antibodies that bind zcytor19 can be used for the treatment of
autoimmune diseases, B cell cancers, immunomodulation, and other
pathologies (e.g., ITCP, T cell-mediated diseases, cattleman's
disease, autoimmune disease, myelodysplastic syndrome, and the
like), renal diseases, graft rejection, and graft versus host
disease. The antibodies of the present invention can be targeted to
specifically regulate B cell responses during the immune response.
Additionally, the antibodies of the present invention can be used
to modulate B cell development, antigen presentation by B cells,
antibody production, and cytokine production.
[0155] Antagonistic anti-zcytor19 antibodies can be useful to
neutralize the effects of zcytor19 ligands for treating B cell
lymphomas and leukemias, chronic or acute lymphocytic leukemia,
myelomas such as multiple myeloma, plasma cytomas, and lymphomas
such as non-Hodgkins lymphoma, for which an increase in zcytor19
ligand polypeptides is associated, or where zcytor19 ligand is a
survival factor or growth factor. Anti-zcytor19 antibodies can also
be used to treat Epstein Barr virus-associated lymphomas arising in
immunocompromised patients (e.g., AIDS or organ transplant).
[0156] Anti-zcytor19 antibodies that induce a signal by binding
with zcytor19 may inhibit the growth of lymphoma and leukemia cells
directly via induction of signals that lead to growth inhibition,
cell cycle arrest, apoptosis, or tumor cell death. Zcytor19
antibodies that initiate a signal are preferred antibodies to
directly inhibit or kill cancer cells. In addition, agonistic
anti-zcytor19 monoclonal antibodies may activate normal B cells and
promote an anticancer immune response. Anti-zcytor19 antibodies may
directly inhibit the growth of leukemias, lymphomas, and multiple
myelomas, and the antibodies may engage immune effector functions.
Anti-zcytor19 monoclonal antibodies may enable antibody-dependent
cellular cytotoxicity, complement dependent cytotoxicity, and
phagocytosis.
[0157] zcytor19 ligand may be expressed in neutrophils, monocytes,
dendritic cells, and activated monocytes. In certain autoimmune
disorders (e.g., myasthenia gravis, and rheumatoid arthritis), B
cells might exacerbate autoimmunity after activation by zcytor19
ligand. Immunosuppressant proteins that selectively block the
action of B-lymphocytes would be of use in treating disease.
Autoantibody production is common to several autoimmune diseases
and contributes to tissue destruction and exacerbation of disease.
Autoantibodies can also lead to the occurrence of immune complex
deposition complications and lead to many symptoms of systemic
lupus erythematosus, including kidney failure, neuralgic symptoms
and death. Modulating antibody production independent of cellular
response would also be beneficial in many disease states. B cells
have also been shown to play a role in the secretion of
arthritogenic immunoglobulins in rheumatoid arthritis. As such,
inhibition of zcytor19 ligand antibody production would be
beneficial in treatment of autoimmune diseases such as myasthenia
gravis and rheumatoid arthritis. Immunosuppressant therapeutics
such as anti-zcytor19 antibodies that selectively block or
neutralize the action of B-lymphocytes would be useful for such
purposes.
[0158] The invention provides methods employing anti-zcytor19
antibodies, or multispecific antibody compositions, for selectively
blocking or neutralizing the actions of B-cells in association with
end stage renal diseases, which may or may not be associated with
autoimmune diseases. Such methods would also be useful for treating
immunologic renal diseases. Such methods would be would be useful
for treating glomerulonephritis associated with diseases such as
membranous nephropathy, IgA nephropathy or Berger's Disease, IgM
nephropathy, Goodpasture's Disease, post-infectious
glomerulonephritis, mesangioproliferative disease, chronic
lymphocytic leukemia, minimal-change nephrotic syndrome. Such
methods would also serve as therapeutic applications for treating
secondary glomerulonephritis or vasculitis associated with such
diseases as lupus, polyarteritis, Henoch-Schonlein, Scleroderma,
HIV-related diseases, amyloidosis or hemolytic uremic syndrome. The
methods of the present invention would also be useful as part of a
therapeutic application for treating interstitial nephritis or
pyelonephritis associated with chronic pyelonephritis, analgesic
abuse, nephrocalcinosis, nephropathy caused by other agents,
nephrolithiasis, or chronic or acute interstitial nephritis.
[0159] The present invention also provides methods for treatment of
renal or urological neoplasms, multiple myelomas, lymphomas,
leukemias, light chain neuropathy, or amyloidosis.
[0160] The invention also provides methods for blocking or
inhibiting activated B cells using anti-zcytor19 antibodies, or
multispecific antibody compositions, for the treatment of asthma
and other chronic airway diseases such as bronchitis and
emphysema.
[0161] Also provided are methods for inhibiting or neutralizing a T
cell response using anti-zcytor19 antibodies, or multispecific
antibody compositions, for immunosuppression, in particular for
such therapeutic use as for graft-versus-host disease and graft
rejection. Moreover, anti-zcytor19 antibodies, or multispecific
antibody compositions, would be useful in therapeutic protocols for
treatment of such autoimmune diseases as insulin dependent diabetes
mellitus (IDDM), multiple sclerosis, rheumatoid arthritis, systemic
lupus erythematosus, inflammatory bowel disease (IBD), and Crohn's
Disease. Methods of the present invention would have additional
therapeutic value for treating chronic inflammatory diseases, in
particular to lessen joint pain, swelling, anemia and other
associated symptoms as well as treating septic shock.
[0162] B cell responses are important in fighting infectious
diseases including bacterial, viral, protozoan and parasitic
infections. Antibodies against infectious microorganisms can
immobilize the pathogen by binding to antigen followed by
complement mediated lysis or cell mediated attack. Agonistic, or
signaling, anti-zcytor19 antibodies may serve to boost the humoral
response and would be a useful therapeutic for individuals at risk
for an infectious disease or as a supplement to vaccination.
[0163] Well established animal models are available to test in vivo
efficacy of anti-zcytor19 antibodies, or multispecific antibody
compositions, of the present invention in certain disease states.
As an illustration, anti-zcytor19 antibodies can be tested in vivo
in a number of animal models of autoimmune disease, such as
MRL-lpr/lpr or NZB.times.NZW F1 congenic mouse strains which serve
as a model of systemic lupus erythematosus. Such animal models are
known in the art.
[0164] Offspring of a cross between New Zealand Black (NZB) and New
Zealand White (NZW) mice develop a spontaneous form of systemic
lupus erythematosus that closely resembles systemic lupus
erythematosus in humans. The offspring mice, known as NZBW begin to
develop IgM autoantibodies against T-cells at one month of age, and
by 5-7 months of age, Ig anti-DNA autoantibodies are the dominant
immunoglobulin. Polyclonal B-cell hyperactivity leads to
overproduction of autoantibodies. The deposition of these
autoantibodies, particularly ones directed against single stranded
DNA is associated with the development of glomerulonephritis, which
manifests clinically as proteinuria, azotemia, and death from renal
failure. Kidney failure is the leading cause of death in mice
affected with spontaneous systemic lupus erythematosus, and in the
NZBW strain, this process is chronic and obliterative. The disease
is more rapid and severe in females than males, with mean survival
of only 245 days as compared to 406 days for the males. While many
of the female mice will be symptomatic (proteinuria) by 7-9 months
of age, some can be much younger or older when they develop
symptoms. The fatal immune nephritis seen in the NZBW mice is very
similar to the glomerulonephritis seen in human systemic lupus
erythematosus, making this spontaneous murine model useful for
testing of potential systemic lupus erythematosus therapeutics.
[0165] Murine models of experimental allergic encephalomyelitis
have been used as tools to investigate both the mechanisms of
immune-mediated disease, and methods of potential therapeutic
intervention. The model resembles human multiple sclerosis, and
produces demyelination as a result of T-cell activation to neural
proteins such as myelin basic protein, or proteolipid protein.
Inoculation with antigen leads to induction of CD4+, class II
MHC-restricted T-cells. Changes in the protocol for experimental
allergic encephalomyelitis can produce acute, chronic-relapsing, or
passive-transfer variants of the model.
[0166] In the collagen-induced arthritis model, mice develop
chronic inflammatory arthritis, which closely resembles human
rheumatoid arthritis. Since collagen-induced arthritis shares
similar immunological and pathological features with rheumatoid
arthritis, this makes it an ideal model for screening potential
human anti-inflammatory compounds. Another advantage in using the
collagen-induced arthritis model is that the mechanisms of
pathogenesis are known. The T and B cell epitopes on type II
collagen have been identified, and various immunological
(delayed-type hypersensitivity and anti-collagen antibody) and
inflammatory (cytokines, chemokines, and matrix-degrading enzymes)
parameters relating to immune-mediating arthritis have been
determined, and can be used to assess test compound efficacy in the
models.
[0167] Myasthenia gravis is another autoimmune disease for which
murine models are available. Myasthenia gravis is a disorder of
neuromuscular transmission involving the production of
autoantibodies directed against the nicotinic acetylcholine
receptor. Myasthenia gravis is acquired or inherited with clinical
features including abnormal weakness and fatigue on exertion. A
mouse model of myasthenia gravis have been established.
Experimental autoimmune myasthenia gravis is an antibody mediated
disease characterized by the presence of antibodies to
acetylcholine receptor. These antibodies destroy the receptor
leading to defective neuromuscular electrical impulses, resulting
in muscle weakness. In the experimental autoimmune myasthenia
gravis model, mice are immunized with the nicotinic acetylcholine
receptor. Clinical signs of myasthenia gravis become evident weeks
after the second immunization. Experimental autoimmune myasthenia
gravis is evaluated by several methods including measuring serum
levels of acetylcholine receptor antibodies by radioimmunoassay,
measuring muscle acetylcholine receptor, or electromyography.
[0168] Generally, the dosage of administered anti-zcytor19
antibodies, or multispecific antibody compositions, will vary
depending upon such factors as the subject's age, weight, height,
sex, general medical condition and previous medical history. As an
illustration, anti-zcytor19 antibodies, or multispecific antibody
compositions, can be administered at low protein doses, such as 20
to 100 milligrams protein per dose, given once, or repeatedly.
Alternatively, anti-zcytor19 antibodies, or multispecific antibody
compositions, can be administered in doses of 30 to 90 milligrams
protein per dose, or 40 to 80 milligrams protein per dose, or 50 to
70 milligrams protein per dose, although a lower or higher dosage
also may be administered as circumstances dictate.
[0169] Administration of antibody components to a subject can be
intravenous, intraarterial, intraperitoneal, intramuscular,
subcutaneous, intrapleural, intrathecal, by perfusion through a
regional catheter, or by direct intralesional injection. When
administering therapeutic proteins by injection, the administration
may be by continuous infusion or by single or multiple boluses.
Additional routes of administration include oral, mucosal-membrane,
pulmonary, and transcutaneous.
[0170] A pharmaceutical composition comprising an anti-zcytor19
antibody, or bispecific antibody components, can be formulated
according to known methods to prepare pharmaceutically useful
compositions, whereby the therapeutic proteins are combined in a
mixture with a pharmaceutically acceptable carrier. A composition
is said to be a "pharmaceutically acceptable carrier" if its
administration can be tolerated by a recipient patient. Sterile
phosphate-buffered saline is one example of a pharmaceutically
acceptable carrier. Other suitable carriers are well-known to those
in the art. See, for example, Gennaro (ed.), Remington's
Pharmaceutical Sciences, 19th Edition (Mack Publishing Company
1995).
[0171] For purposes of therapy, anti-zcytor19 antibodies, or
bispecific antibody components, and a pharmaceutically acceptable
carrier are administered to a patient in a therapeutically
effective amount. A combination of anti-zcytor19 antibodies, or
bispecific antibody components, and a pharmaceutically acceptable
carrier is said to be administered in a "therapeutically effective
amount" if the amount administered is physiologically significant.
An agent is physiologically significant if its presence results in
a detectable change in the physiology of a recipient patient. For
example, an agent used to treat inflammation is physiologically
significant if its presence alleviates the inflammatory response.
As another example, an agent used to inhibit the growth of tumor
cells is physiologically significant if the administration of the
agent results in a decrease in the number of tumor cells, decreased
metastasis, a decrease in the size of a solid tumor, or increased
necrosis of a tumor.
[0172] A pharmaceutical composition comprising anti-zcytor19
antibodies, or bispecific antibody components, can be furnished in
liquid form, in an aerosol, or in solid form. Liquid forms, are
illustrated by injectable solutions and oral suspensions. Exemplary
solid forms include capsules, tablets, and controlled-release
forms. The latter form is illustrated by miniosmotic pumps and
implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade,
"Implants in Drug Delivery," in Drug Delivery Systems, Ranade and
Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,
"Protein Delivery with Infusion Pumps," in Protein Delivery:
Physical Systems, Sanders and Hendren (eds.), pages 239-254 (Plenum
Press 1997); Yewey et al., "Delivery of Proteins from a Controlled
Release Injectable Implant," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).
[0173] Those of skill in the art can devise various pharmaceutical
compositions using standard techniques. See, for example, Lieberman
et al., (Eds.), Pharmaceutical Dosage Forms: Tablets, Vol. 1, 2nd
Edition (Marcel Dekker, Inc. 1989), Lieberman et al., (Eds.),
Pharmaceutical Dosage Forms: Tablets, Vol. 2, 2nd Edition (Marcel
Dekker, Inc. 1990), Lieberman et al., (Eds.), Pharmaceutical Dosage
Forms: Tablets, Vol. 3, 2nd Edition (Marcel Dekker, Inc. 1990),
Lieberman et al., (Eds.), Pharmaceutical Dosage Forms: Disperse
Systems, Vol. 1, 2nd Edition (Marcel Dekker, Inc. 1996), Lieberman
et al., (Eds.), Pharmaceutical Dosage Forms: Disperse Systems, Vol.
2, 2nd Edition (Marcel Dekker, Inc. 1996), Lieberman et al.,
(Eds.), Pharmaceutical Dosage Forms: Disperse Systems, Vol. 3, 2nd
Edition (Marcel Dekker, Inc. 1998), Avis et al., (Eds.),
Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1, 2nd
Edition (Marcel Dekker, Inc. 1991), Lieberman et al., (Eds.),
Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 2, 2nd
Edition (Marcel Dekker, Inc. 1992), and Avis et al., (Eds.),
Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 3, 2nd
Edition (Marcel Dekker, Inc. 1993).
[0174] As another example, liposomes provide a means to deliver
anti-zcytor19 antibodies, or bispecific antibody components, to a
subject intravenously, intraperitoneally, intrathecally,
intramuscularly, subcutaneously, or via oral administration,
inhalation, or intranasal administration. Liposomes are microscopic
vesicles that consist of one or more lipid bilayers surrounding
aqueous compartments (see, generally, Bakker-Woudenberg et al.,
Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61 (1993),
Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug Delivery
Using Liposomes as Carriers," in Drug Delivery Systems, Ranade and
Hollinger (Eds.), pages 3-24 (CRC Press 1995)). Liposomes are
similar in composition to cellular membranes and as a result,
liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0175] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents (Scherphof et al.,
Ann. N.Y Acad. Sci. 446:368 (1985)). After intravenous
administration, small liposomes (0.1 to 1.0 .mu.m) are typically
taken up by cells of the reticuloendothelial system, located
principally in the liver and spleen, whereas liposomes larger than
3.0 .mu.m are deposited in the lung. This preferential uptake of
smaller liposomes by the cells of the reticuloendothelial system
has been used to deliver chemotherapeutic agents to macrophages and
to tumors of the liver.
[0176] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means (Claassen et al., Biochim. Biophys. Acta 802:428 (1984)). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system (Allen et al., Biochim. Biophys. Acta
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993)).
[0177] As an alternative to administering liposomes that comprise
an anti-zcytor19 antibody component, target cells can be prelabeled
with biotinylated anti-zcytor19 antibodies. After plasma
elimination of free antibody, streptavidin-conjugated liposomes are
administered. This general approach is described, for example, by
Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998). Such an
approach can also be used to prepare multispecific antibody
compositions.
[0178] Polypeptides comprising an anti-zcytor19 antibody component,
or bispecific antibody components, can be encapsulated within
liposomes, or attached to the exterior of liposomes, using standard
techniques (see, for example, Anderson et al., Infect. Immun.
31:1099 (1981), Wassef et al., Meth. Enzymol. 149:124 (1987),
Anderson et al., Cancer Res. 50:1853 (1990), Cohen et al., Biochim.
Biophys. Acta 1063:95 (1991), Alving et al. "Preparation and Use of
Liposomes in Immunological Studies," in Liposome Technology, 2nd
Edition, Vol. III, Gregoriadis (Ed.), page 317 (CRC Press 1993),
and Ansell et al., "Antibody Conjugation Methods for Active
Targeting of Liposomes," in Drug Targeting: Strategies, Principles,
and Applications, Francis and Delgado (Eds.), pages 51-68 (Humana
Press, Inc. 2000)). As noted above, therapeutically useful
liposomes may contain a variety of components. For example,
liposomes may comprise lipid derivatives of poly(ethylene glycol)
(Allen et al., Biochim. Biophys. Acta 1150:9 (1993)).
[0179] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (Eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney
and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin.
Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
nanospheres can also provide carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0180] The present invention also contemplates chemically modified
antibody components, in which an antibody component is linked with
a polymer. Typically, the polymer is water soluble so that an
antibody component does not precipitate in an aqueous environment,
such as a physiological environment. An example of a suitable
polymer is one that has been modified to have a single reactive
group, such as an active ester for acylation, or an aldehyde for
alkylation. In this way, the degree of polymerization can be
controlled. An example of a reactive aldehyde is polyethylene
glycol propionaldehyde, or mono-(C.sub.1-C.sub.10) alkoxy, or
aryloxy derivatives thereof (see, for example, Harris, et al., U.S.
Pat. No. 5,252,714). The polymer may be branched or unbranched.
Moreover, a mixture of polymers can be used to produce conjugates
with antibody components.
[0181] Suitable water-soluble polymers include polyethylene glycol
(PEG), monomethoxy-PEG, mono-(C.sub.1-C.sub.10)alkoxy-PEG,
aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG,
PEG propionaldehyde, bis-succinimidyl carbonate PEG, propylene
glycol homopolymers, a polypropylene oxide/ethylene oxide
co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl
alcohol, dextran, cellulose, or other carbohydrate-based polymers.
Suitable PEG may have a molecular weight from about 600 to about
60,000, including, for example, 5,000, 12,000, 20,000 and 25,000. A
conjugate can also comprise a mixture of such water-soluble
polymers.
[0182] As an illustration, a polyalkyl oxide moiety can be attached
to the N-terminus of antibody component. PEG is one suitable
polyalkyl oxide. As an illustration, an antibody component can be
modified with PEG, a process known as "PEGylation." PEGylation of
an antibody component can be carried out by any of the PEGylation
reactions known in the art (see, for example, EP 0 154 316, Delgado
et al., Critical Reviews in Therapeutic Drug Carrier Systems 9:249
(1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994),
and Francis et al., Int J Hematol 68:1 (1998)). For example,
PEGylation can be performed by an acylation reaction or by an
alkylation reaction with a reactive polyethylene glycol molecule.
In an alternative approach, antibody component conjugates are
formed by condensing activated PEG, in which a terminal hydroxy or
amino group of PEG has been replaced by an activated linker (see,
for example, Karasiewicz et al., U.S. Pat. No. 5,382,657).
[0183] PEGylation by acylation typically requires reacting an
active ester derivative of PEG with an antibody component. An
example of an activated PEG ester is PEG esterified to
N-hydroxysuccinimide. As used herein, the term "acylation" includes
the following types of linkages between an antibody component and a
water soluble polymer: amide, carbamate, urethane, and the like.
Methods for preparing PEGylated anti-zcytor19 antibody components
by acylation will typically comprise the steps of (a) reacting an
antibody component with PEG (such as a reactive ester of an
aldehyde derivative of PEG) under conditions whereby one or more
PEG groups attach to the antibody component, and (b) obtaining the
reaction product(s). Generally, the optimal reaction conditions for
acylation reactions will be determined based upon known parameters
and desired results. For example, the larger the ratio of
PEG:antibody component, the greater the percentage of polyPEGylated
antibody component product.
[0184] The product of PEGylation by acylation is typically a
polyPEGylated antibody component product, wherein the lysine
8-amino groups are PEGylated via an acyl linking group. An example
of a connecting linkage is an amide. Typically, the resulting
antibody component will be at least 95% mono-, di-, or
tri-pegylated, although some species with higher degrees of
PEGylation may be formed depending upon the reaction conditions.
PEGylated species can be separated from unconjugated antibody
component using standard purification methods, such as dialysis,
ultrafiltration, ion exchange chromatography, affinity
chromatography, and the like.
[0185] PEGylation by alkylation generally involves reacting a
terminal aldehyde derivative of PEG with antibody component in the
presence of a reducing agent. PEG groups can be attached to the
polypeptide via a --CH.sub.2--NH group.
[0186] Derivatization via reductive alkylation to produce a
monoPEGylated product takes advantage of the differential
reactivity of different types of primary amino groups available for
derivatization. Typically, the reaction is performed at a pH that
allows one to take advantage of the pKa differences between the
.epsilon.-amino groups of the lysine residues and the .alpha.-amino
group of the N-terminal residue of the protein. By such selective
derivatization, attachment of a water-soluble polymer that contains
a reactive group such as an aldehyde, to a protein is controlled.
The conjugation with the polymer occurs predominantly at the
N-terminus of the protein without significant modification of other
reactive groups such as the lysine side chain amino groups.
[0187] Reductive alkylation to produce a substantially homogenous
population of monopolymer antibody component conjugate molecule can
comprise the steps of: (a) reacting an antibody component with a
reactive PEG under reductive alkylation conditions at a pH suitable
to permit selective modification of the a-amino group at the amino
terminus of the antibody component, and (b) obtaining the reaction
product(s). The reducing agent used for reductive alkylation should
be stable in aqueous solution and preferably be able to reduce only
the Schiff base formed in the initial process of reductive
alkylation. Preferred reducing agents include sodium borohydride,
sodium cyanoborohydride, dimethylamine borane, trimethylamine
borane, and pyridine borane.
[0188] For a substantially homogenous population of monopolymer
antibody component conjugates, the reductive alkylation reaction
conditions are those which permit the selective attachment of the
water soluble polymer moiety to the N-terminus of the antibody
component. Such reaction conditions generally provide for pKa
differences between the lysine amino groups and the .alpha.-amino
group at the N-terminus. The pH also affects the ratio of polymer
to protein to be used. In general, if the pH is lower, a larger
excess of polymer to protein will be desired because the less
reactive the N-terminal .alpha.-group, the more polymer is needed
to achieve optimal conditions. If the pH is higher, the
polymer:antibody component need not be as large because more
reactive groups are available. Typically, the pH will fall within
the range of 3 to 9, or 3 to 6.
[0189] General methods for producing conjugates comprising a
polypeptide and water-soluble polymer moieties are known in the
art. See, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657,
Greenwald et al., U.S. Pat. No. 5,738, 846, Nieforth et al., Clin.
Pharmacol. Ther. 59:636 (1996), Monkarsh et al., Anal. Biochem.
247:434 (1997)).
[0190] Polypeptide cytotoxins can also be conjugated with a soluble
polymer using the above methods either before or after conjugation
to an antibody component. Soluble polymers can also be conjugated
with antibody fusion proteins.
[0191] Naked anti-zcytor19 antibodies, or antibody fragments, can
be supplemented with immunoconjugate or antibody fusion protein
administration. In one variation, naked anti-zcytor19 antibodies
(or naked antibody fragments) are administered with low-dose
radiolabeled anti-zcytor19 antibodies or antibody fragments. As a
second alternative, naked anti-zcytor19 antibodies (or antibody
fragments) are administered with low-dose radiolabeled
anti-zcytor19 antibodies-cytokine immunoconjugates. As a third
alternative, naked anti-zcytor19 antibodies (or antibody fragments)
are administered with anti-zcytor19-cytokine immunoconjugates that
are not radiolabeled. With regard to "low doses" of
".sup.131I-labeled immunoconjugates, a preferable dosage is in the
range of 15 to 40 mCi, while the most preferable range is 20 to 30
mCi. In contrast, a preferred dosage of .sup.90Y-labeled
immunoconjugates is in the range from 10 to 30 mCi, while the most
preferable range is 10 to 20 mCi. Similarly, bispecific antibody
components can be supplemented with immunoconjugate or antibody
fusion protein administration.
[0192] Immunoconjugates having a boron addend-loaded carrier for
thermal neutron activation therapy will normally be effected in
similar ways. However, it will be advantageous to wait until
non-targeted immunoconjugate clears before neutron irradiation is
performed. Clearance can be accelerated using an antibody that
binds to the immunoconjugate. See U.S. Pat. No. 4,624,846 for a
description of this general principle.
[0193] The present invention also contemplates a method of
treatment in which immunomodulators are administered to prevent,
mitigate or reverse radiation-induced or drug-induced toxicity of
normal cells, and especially hematopoietic cells. Adjunct
immunomodulator therapy allows the administration of higher doses
of cytotoxic agents due to increased tolerance of the recipient
mammal. Moreover, adjunct immunomodulator therapy can prevent,
palliate, or reverse dose-limiting marrow toxicity. Examples of
suitable immunomodulators for adjunct therapy include
granulocyte-colony stimulating factor, granulocyte
macrophage-colony stimulating factor, thrombopoietin, IL-1, IL-3,
IL-12, and the like. The method of adjunct immunomodulator therapy
is disclosed by Goldenberg, U.S. Pat. No. 5,120,525.
[0194] The efficacy of anti-zcytor19 antibody therapy can be
enhanced by supplementing naked antibody components with
immunoconjugates and other forms of supplemental therapy described
herein. In such multimodal regimens, the supplemental therapeutic
compositions can be administered before, concurrently or after
administration of naked anti-zcytor19 antibodies. Multimodal
therapies of the present invention further include immunotherapy
with naked anti-zcytor19 antibody components supplemented with
administration of anti-zcytor19 immunoconjugates. In another form
of multimodal therapy, subjects receive naked anti-zcytor19
antibodies and standard cancer chemotherapy.
[0195] The antibodies, immunoconjugates, and antibody fusion
proteins described herein can also be advantageously supplemented
with antibody components (e.g., naked antibodies, naked antibody
fragments, immunoconjugates, antibody fusion proteins, etc.) that
bind the so-called "stalk region" of the TACI receptor, which
resides between the second cysteine-rich region and the
transmembrane domain. Studies indicate that, to an extent, TACI
proteins are cleaved and shed by cells, leaving a small
extracellular peptide, or stalk on the cell surface. A murine
monoclonal antibody was found to be therapeutically useful in a
lymphoma murine model. Epitope mapping indicates that the antibody
binds with a fragment of the TACI extracellular domain, represented
by amino acid residues 110 to 118 of SEQ ID NO:4. Antibodies can be
generated against a polypeptide representing the region between the
second cysteine-rich domain and the transmembrane domain (amino
acid residues 105 to 166 of SEQ ID NO:4), or to a fragment thereof
(e.g., amino acid residues 110 to 118 of SEQ ID NO:4). Such
antibodies are particularly useful for treatment of TACI-bearing
tumor cells, such as B-lymphoma cells, myeloma cells, and the
like.
[0196] The antibodies and antibody fragments of the present
invention can be used as vaccines to treat the various disorders
and diseases described above. As an example, an antibody component
of a dual reactive TACI/BCMA monoclonal antibody can provide a
suitable basis for a vaccine. Cysteine-rich regions of zcytor19
receptors can also provide useful components for a vaccine. For
example, a vaccine can comprise at least one of the following
polypeptides: a polypeptide comprising amino acid residues 8 to 41
of SEQ ID NO:2, a polypeptide comprising amino acid residues 34 to
66 of SEQ ID NO:4, and a polypeptide comprising amino acid residues
71 to 104 of SEQ ID NO:4.
[0197] The efficacy of an antibody component as a vaccine can be
enhanced by conjugating the antibody component to a soluble
immunogenic carrier protein. Suitable carrier proteins include
tetanus toxin/toxoid, NTHi high molecular weight protein,
diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera
toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens
exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core
antigen, rotavirus VP 7 protein, respiratory syncytial virus F and
G protein, and the like. Methods of preparing conjugated vaccines
are known to those of skill in the art. See, for example, Cruse and
Lewis (Eds.), Conjugate Vaccines (S. Karger Publishing 1989), and
O'Hagan (Ed.), Vaccine Adjuvants (Humana Press, Inc. 2000). A
vaccination composition can also include an adjuvant. Examples of
suitable adjuvants include aluminum hydroxide and lipid. Methods of
formulating vaccine compositions are well-known to those of
ordinary skill in the art. See, for example, Rola, "Immunizing
Agents and Diagnostic Skin Antigens," in Remington: The Science and
Practice of Pharmacy, 19th Edition, Gennaro (Ed.), pages 1417-1433
(Mack Publishing Company 1995).
[0198] Pharmaceutical compositions may be supplied as a kit
comprising a container that comprises anti-zcytor19 antibody
components, or bispecific antibody components. Therapeutic
molecules can be provided in the form of an injectable solution for
single or multiple doses, or as a sterile powder that will be
reconstituted before injection. Alternatively, such a kit can
include a dry-powder disperser, liquid aerosol generator, or
nebulizer for administration of an anti-zcytor19 antibody
component. Such a kit may further comprise written information on
indications and usage of the pharmaceutical composition. Moreover,
such information may include a statement that the composition is
contraindicated in patients with known hypersensitivity to
exogenous antibodies.
[0199] Zcytor19 polypeptides, such as soluble zcytor19 receptors,
may also be used within diagnostic systems for the detection of
circulating levels of ligand. Within a related embodiment,
antibodies or other agents that specifically bind to zcytor19
receptor polypeptides can be used to detect circulating receptor
polypeptides. Elevated or depressed levels of ligand or receptor
polypeptides may be indicative of pathological conditions,
including cancer. Soluble receptor polypeptides may contribute to
pathologic processes and can be an indirect marker of an underlying
disease. For example, elevated levels of soluble IL-2 receptor in
human serum have been associated with a wide variety of
inflammatory and neoplastic conditions, such as myocardial
infarction, asthma, myasthenia gravis, rheumatoid arthritis, acute
T-cell leukemia, B-cell lymphomas, chronic lymphocytic leukemia,
colon cancer, breast cancer, and ovarian cancer (Heaney et al.,
Blood 87:847-857, 1996). Similarly, as zcytor19 is expressed in
B-cell leukemia cells, an increase of zcytor19 expression can even
serve as a marker of an underlying disease, such as leukemia.
[0200] A ligand-binding polypeptide of a zcytor19 receptor, or
"soluble receptor," can be prepared by expressing a truncated DNA
encoding the zcytor19 extracellular cytokine-binding domain
(residues 21 (Arg) to 226 (Asn) of SEQ ID NO:2 or SEQ ID NO:19),
cytokine-binding fragment (e.g., residues 21 (Arg) to 223 (Pro) of
SEQ ID NO:2 or SEQ ID NO:19; SEQ ID NO:4), the soluble version of
zcytor19 variant, or the corresponding region of a non-human
receptor. It is preferred that the extracellular domain be prepared
in a form substantially free of transmembrane and intracellular
polypeptide segments. Moreover, ligand-binding polypeptide
fragments within the zcytor19 cytokine-binding domain, described
above, can also serve as zcytor19 soluble receptors for uses
described herein. To direct the export of a receptor polypeptide
from the host cell, the receptor DNA is linked to a second DNA
segment encoding a secretory peptide, such as a t-PA secretory
peptide or a zcytor19 secretory peptide. To facilitate purification
of the secreted receptor polypeptide, a C-terminal extension, such
as a poly-histidine tag, Glu-Glu tag peptide, substance P, Flag.TM.
peptide (Hopp et al., Bio/Technology 6:1204-1210, 1988; available
from Eastman Kodak Co., New Haven, Conn.) or another polypeptide or
protein for which an antibody or other specific binding agent is
available, can be fused to the receptor polypeptide.
[0201] In an alternative approach, a receptor extracellular domain
can be expressed as a fusion with immunoglobulin heavy chain
constant regions, typically an F.sub.C fragment, which contains two
constant region domains and lacks the variable region. Such fusions
are typically secreted as multimeric molecules wherein the Fc
portions are disulfide bonded to each other and two receptor
polypeptides are arrayed in close proximity to each other. Fusions
of this type can be used to affinity purify the cognate ligand from
solution, as an in vitro assay tool, to block signals in vitro by
specifically titrating out ligand, and as antagonists in vivo by
administering them parenterally to bind circulating ligand and
clear it from the circulation. To purify ligand, a zcytor19-Ig
chimera is added to a sample containing the ligand (e.g.,
cell-conditioned culture media or tissue extracts) under conditions
that facilitate receptor-ligand binding (typically
near-physiological temperature, pH, and ionic strength). The
chimera-ligand complex is then separated by the mixture using
protein A, which is immobilized on a solid support (e.g., insoluble
resin beads). The ligand is then eluted using conventional chemical
techniques, such as with a salt or pH gradient. In the alternative,
the chimera itself can be bound to a solid support, with binding
and elution carried out as above. Collected fractions can be
re-fractionated until the desired level of purity is reached.
[0202] Moreover, zcytor19 soluble receptors can be used as a
"ligand sink," i.e., antagonist, to bind ligand in vivo or in vitro
in therapeutic or other applications where the presence of the
ligand is not desired. For example, in cancers that are expressing
large amount of bioactive zcytor19 ligand, zcytor19 soluble
receptors can be used as a direct antagonist of the ligand in vivo,
and may aid in reducing progression and symptoms associated with
the disease. Moreover, zcytor19 soluble receptor can be used to
slow the progression of cancers that over-express zcytor19
receptors, by binding ligand in vivo that would otherwise enhance
proliferation of those cancers. Similar in vitro applications for a
zcytor19 soluble receptor can be used, for instance, as a negative
selection to select cell lines that grow in the absence of zcytor19
ligand.
[0203] Moreover, zcytor19 soluble receptor can be used in vivo or
in diagnostic applications to detect zcytor19 ligand-expressing
cancers in vivo or in tissue samples. For example, the zcytor19
soluble receptor can be conjugated to a radio-label or fluorescent
label as described herein, and used to detect the presence of the
ligand in a tissue sample using an in vitro ligand-receptor type
binding assay, or fluorescent imaging assay. Moreover, a
radiolabeled zcytor19 soluble receptor could be administered in
vivo to detect ligand-expressing solid tumors through a
radio-imaging method known in the art. Similarly, zcytor19
polynucleotides, polypeptides, anti-zcytor19 andibodies, or peptide
binding fragments can be used to detect zcytor19 receptor
expressing cancers. In a preferred embodiment zcytor19
polynucleotides, polypeptides, anti-zcytor19 andibodies, or peptide
binding fragments can be used to detect leukemias, more preferably
B-cell leukemias, and most preferably pre-B-cell acute
lymphoblastic leukemia.
[0204] 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.
[0205] 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 lymphoid
cells, hematopoietic cells and endothelial cells. Thus molecules of
the present invention, such as soluble zcytor19 receptors,
cytokine-binding fragments, anti-zcytor19 antibodies, sense and
antisense polynucleotides may have use in inhibiting tumor cells,
and particularly lymphoid, hematopoietic, prostate, endothelial,
and thyroid tumor cells.
[0206] 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, zcytor19 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 zcytor19 polypeptide, or its loss of expression in a tissue as
it differentiates, can serve as a marker for differentiation of
tissues. Moreover, since zcytor19 is specifically-expressed in
pre-B cell acute lymphoblastic leukemia cells, as well as several
other cancers as described herein. As such, one of skill in the art
would recognize that the polynucleotides, polypeptides and
antibodies of the present invention can be used as a marker for
these cancers.
[0207] Similarly, direct measurement of zcytor19 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
zcytor19 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 zcytor19 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, or as a B-cell tumor-specific marker, zcytor19 gain
or loss of expression may serve as a diagnostic for lymphoid,
B-cell, endothelial, hematopoietic and other cancers. Moreover,
analogous to the prostate specific antigen (PSA), as a
naturally-expressed tissue-specific marker, increased levels of
zcytor19 polypeptides, or anti-zcytor19 antibodies in a patient,
relative to a normal control can be indicative of disease in normal
tissues where zcytor19 is expressed (See, e.g., Mulders, T M T, et
al., Eur. J. Surgical Oncol. 16:37-41, 1990). Moreover, where
zcytor19 expression appears to be restricted to specific normal
human tissues, lack of zcytor19 expression in those tissues or
strong zcytor19 expression in non-specific tissues would serve as a
diagnostic of an abnormality in the cell or tissue type, of
invasion or metastasis of cancerous tissues into non-cancerous
tissue, and could aid a physician in directing further testing or
investigation, or aid in directing therapy. As zcytor19 is
expressed in esophagus, liver, ovary, rectum, stomach, and uterus
tumors, and melanoma, disgnostic probes have paricular use in
diagnosing and identifying tissues from these cancers.
[0208] In addition, as zcytor19 is tissue-specific, polynucleotide
probes, anti-zcytor19 antibodies, and detection the presence of
zcytor19 polypeptides in tissue can be used to assess whether a
specific tissue is present, for example, after surgery involving
the excision of a diseased or cancerous tissues in which zcytor19
is expressed. As such, the polynucleotides, polypeptides, and
antibodies of the present invention can be used as an aid to
determine whether all tissue is excised after surgery, for example,
after surgery for cancer. In such instances, it is especially
important to remove all potentially diseased tissue to maximize
recovery from the cancer, and to minimize recurrence. Preferred
embodiments include fluorescent, radiolabeled, or calorimetrically
labeled anti-zcytor19 antibodies and zcytor19 polypeptide binding
partners, that can be used histologically or in situ. Specific
tissues in which zcytor19 is exporessed are disclosed herein.
[0209] Moreover, the activity and effect of zcytor19 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 zcytor19, 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., zcytor19, 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 zcytor19. Use
of stable zcytor19 transfectants as well as use of induceable
promoters to activate zcytor19 expression in vivo are known in the
art and can be used in this system to assess zcytor19 induction of
metastasis. Moreover, purified zcytor19 or zcytor19 conditioned
media can be directly injected in to this mouse model, and hence be
used in this system. For general reference see, O'Reilly M S, et
al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models of
Liver Metastasis. Invasion Metastasis 14:349-361, 1995.
[0210] The activity of zcytor19 and its derivatives (conjugates) on
growth and dissemination of tumor cells derived from human
hematologic malignancies can also be measured in vivo in a mouse
Xenograft model. Several mouse models have been developed in which
human tumor cells are implanted into immunodeficient mice,
collectively referred to as xenograft models. See Cattan, A R and
Douglas, E Leuk. Res. 18:513-22, 1994; and Flavell, D J,
Hematological Oncology 14:67-82, 1996. The characteristics of the
disease model vary with the type and quantity of cells delivered to
the mouse. Typically, the tumor cells will proliferate rapidly and
can be found circulating in the blood and populating numerous organ
systems. Therapeutic strategies appropriate for testing in such a
model include antibody induced toxicity, ligand-toxin conjugates or
cell-based therapies. The latter method, commonly referred to
adoptive immunotherapy, involves treatment of the animal with
components of the human immune system (i.e. lymphocytes, NK cells)
and may include ex vivo incubation of cells with zcytor19 or other
immunomodulatory agents.
[0211] The mRNA corresponding to this novel DNA shows expression in
lymphoid tissues, including pre-B cell acute lymphoblastic
leukemia, bone marrow, and may be expressed in spleen, lymph nodes,
and peripheral blood leukocytes. These data indicate a role for the
zcytor19 receptor in leukemia, including B-cell leukemia,
proliferation, differentiation, and/or activation of immune cells,
and suggest a role in development and regulation of immune
responses. The data also suggest that the interaction of zcytor19
with its ligand may stimulate proliferation and development of
myeloid cells and may, like cytoikine receptors IL-2, IL-6, LIF,
IL-11 and OSM (Baumann et al., J. Biol. Chem. 268:8414-8417, 1993),
induce acute-phase protein synthesis in hepatocytes.
[0212] 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.
[0213] Expressed recombinant zcytor19 polypeptides (or zcytor19
chimeric or fusion 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, sulfhydryl activation, hydrazide activation,
and carboxyl and amino derivatives for carbodiimide coupling
chemistries. These and other solid media are well known and widely
used in the art, and are available from commercial suppliers.
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.
[0214] The polypeptides of the present invention can be isolated by
exploitation of their biochemical, structural, and biological
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). 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.
[0215] Moreover, using methods described in the art, polypeptide
fusions, or hybrid zcytor19 proteins, are constructed using regions
or domains of the inventive zcytor19 in combination with those of
other human cytokine receptor family proteins, 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, constrict or expand
the substrate specificity, or alter tissue and cellular
localization of a polypeptide, and can be applied to polypeptides
of unknown structure.
[0216] Fusion polypeptides or 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 one or more 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 domain(s) conferring a biological
function may be swapped between zcytor19 of the present invention
with the functionally equivalent domain(s) from another cytokine
family member. Such domains include, but are not limited to, the
secretory signal sequence, extracellular cytokine binding domain,
cytokine binding fragment, fibronectin type III domains,
transmembrane domain, and intracellular signaling domain, as
disclosed herein. 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 family
proteins, depending on the fusion constructed. Moreover, such
fusion proteins may exhibit other properties as disclosed
herein.
[0217] Standard molecular biological and cloning techniques can be
used to swap the equivalent domains between the zcytor19
polypeptide and those polypeptides to which they are fused.
Generally, a DNA segment that encodes a domain of interest, e.g., a
zcytor19 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 receptor, such
as, interferon-gamma, alpha and beta chains and the
interferon-alpha/beta receptor alpha and beta chains, zcytor11
(commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7
(commonly owned U.S. Pat. No. 5,945,511), or other class II
cytokine receptor), 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 cytokine binding domain, followed by a
transmembrane domain, followed by an intracellular signaling
domain. 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
zcytor19 polypeptide, to be used, for example to inoculate an
animal to generate anti-zcytor19 antibodies as described herein.
For example a secretory signal sequence can be operably linked to
extracellular cytokine binding domain, cytokine binding fragment,
individual fibronectin type III domains, transmembrane domain, and
intracellular signaling domain, as disclosed herein, or a
combination thereof (e.g., operably linked polypeptides comprising
a fibronectin III domain attached to a linker, or zcytor19
polypeptide fragments described herein), to secrete a fragment of
zcytor19 polypeptide that can be purified as described herein and
serve as an antigen to be inoculated into an animal to produce
anti-zcytor19 antibodies, as described herein.
[0218] Zcytor19 polypeptides or fragments thereof may also be
prepared through chemical synthesis. Zcytor19 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.
[0219] Polypeptides of the present invention can also be
synthesized by exclusive solid phase synthesis, partial solid phase
methods, fragment condensation or classical solution synthesis.
Methods for synthesizing polypeptides are well known in the art.
See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963;
Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire
synthesis of the desired peptide on a solid support, the
peptide-resin is with a reagent which cleaves the polypeptide from
the resin and removes most of the side-chain protecting groups.
Such methods are well established in the art.
[0220] The activity of molecules of the present invention can be
measured using a variety of assays that measure cell
differentiation and proliferation. Such assays are well known in
the art and described herein.
[0221] Proteins of the present invention are useful for example, in
treating lymphoid, immune, inflammatory, spleenic, blood or bone
disorders, and can be measured in vitro using cultured cells or in
vivo by administering molecules of the present invention to the
appropriate animal model. For instance, host cells expressing a
zcytor19 soluble receptor polypeptide can be embedded in an
alginate environment and injected (implanted) into recipient
animals. Alginate-poly-L-lysine microencapsulation, permselective
membrane encapsulation and diffusion chambers are a means to entrap
transfected mammalian cells or primary mammalian cells. These types
of non-immunogenic "encapsulations" permit the diffusion of
proteins and other macromolecules secreted or released by the
captured cells to the recipient animal. Most importantly, the
capsules mask and shield the foreign, embedded cells from the
recipient animal's immune response. Such encapsulations can extend
the life of the injected cells from a few hours or days (naked
cells) to several weeks (embedded cells). Alginate threads provide
a simple and quick means for generating embedded cells.
[0222] The materials needed to generate the alginate threads are
known in the art. In an exemplary procedure, 3% alginate is
prepared in sterile H.sub.2O, and sterile filtered. Just prior to
preparation of alginate threads, the alginate solution is again
filtered. An approximately 50% cell suspension (containing about
5.times.10.sup.5 to about 5.times.10.sup.7 cells/ml) is mixed with
the 3% alginate solution. One ml of the alginate/cell suspension is
extruded into a 100 mM sterile filtered CaCl.sub.2 solution over a
time period of 15 min, forming a "thread". The extruded thread is
then transferred into a solution of 50 mM CaCl.sub.2, and then into
a solution of 25 mM CaCl.sub.2. The thread is then rinsed with
deionized water before coating the thread by incubating in a 0.01%
solution of poly-L-lysine. Finally, the thread is rinsed with
Lactated Ringer's Solution and drawn from solution into a syringe
barrel (without needle). A large bore needle is then attached to
the syringe, and the thread is intraperitoneally injected into a
recipient in a minimal volume of the Lactated Ringer's
Solution.
[0223] An in vivo approach for assaying proteins of the present
invention involves 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. 43:161-89, 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 a large number of different
promoters including ubiquitous, tissue specific, and regulatable
promoters. Also, because adenoviruses are stable in the
bloodstream, they can be administered by intravenous injection.
[0224] 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 El
gene has been deleted from the viral vector, and the virus will not
replicate unless the El 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.
[0225] 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.
[0226] The adenovirus system can also be used for protein
production in vitro. By culturing adenovirus-infected non-293 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 Gamier et al., Cytotechnol. 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.
[0227] In view of the tissue distribution observed for zcytor19,
agonists (including the natural ligand/substrate/cofactor/etc.) and
antagonists have enormous potential in both in vitro and in vivo
applications. Compounds identified as zcytor19 agonists are useful
for stimulating growth of immune and hematopoietic cells in vitro
and in vivo. For example, zcytor19 soluble receptors, 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. Moreover, zcytor19
soluble receptor, agonist, or antagonist may be used in vitro in an
assay to measure stimulation of colony formation from isolated
primary bone marrow cultures. Such assays are well known in the
art.
[0228] Antagonists are also useful as research reagents for
characterizing sites of ligand-receptor interaction. Inhibitors of
zcytor19 activity (zcytor19 antagonists) include anti-zcytor19
antibodies and soluble zcytor19 receptors, as well as other
peptidic and non-peptidic agents (including ribozymes).
[0229] Zcytor19 can also be used to identify modulators (e.g,
antagonists) of its activity. Test compounds are added to the
assays disclosed herein to identify compounds that inhibit the
activity of zcytor19. In addition to those assays disclosed herein,
samples can be tested for inhibition of zcytor19 activity within a
variety of assays designed to measure zcytor19 binding,
oligomerization, or the stimulation/inhibition of
zcytor19-dependent cellular responses. For example,
zcytor19-expressing cell lines can be transfected with a reporter
gene construct that is responsive to a zcytor19-stimulated cellular
pathway. Reporter gene constructs of this type are known in the
art, and will generally comprise a zcytor19-DNA response element
operably linked to a gene encoding an assay detectable protein,
such as luciferase. DNA response elements can include, but are not
limited to, cyclic AMP response elements (CRE), hormone response
elements (HRE) insulin response element (IRE) (Nasrin et al., Proc.
Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements
(SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP response
elements are reviewed in Roestler et al., J. Biol. Chem. 263
(19):9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8):1087-94;
1990. Hormone response elements are reviewed in Beato, Cell
56:335-44; 1989. Candidate compounds, solutions, mixtures or
extracts or conditioned media from various cell types are tested
for the ability to enhance the activity of zcytor19 receptor as
evidenced by a increase in zcytor19 stimulation of reporter gene
expression. Assays of this type will detect compounds that directly
stimulate zcytor19 signal transduction activity through binding the
receptor or by otherwise stimulating part of the signal cascade. As
such, there is provided a method of identifying agonists of
zcytor19 polypeptide, comprising providing cells responsive to a
zcytor19 polypeptide, culturing a first portion of the cells in the
absence of a test compound, culturing a second portion of the cells
in the presence of a test compound, and detecting a increase in a
cellular response of the second portion of the cells as compared to
the first portion of the cells. Moreover a third cell, containing
the reporter gene construct described above, but not expressing
zcytor19 receptor, can be used as a control cell to assess
non-specific, or non-zcytor19-mediated, stimulation of the
reporter. Agonists, including the natural ligand, are therefore
useful to stimulate or increase zcytor19 polypeptide function.
[0230] A zcytor19 ligand-binding polypeptide, such as the
extracellular domain or cytokine binding domain disclosed herein,
can also 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.
[0231] 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 may be advantageously employed (e.g., BIAcore.TM.,
Pharmacia Biosensor, Piscataway, N.J.; or SELDI.TM. technology,
Ciphergen, Inc., Palo Alto, Calif.). 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
145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol.
234:554-63, 1993. A receptor, antibody, member or fragment is
covalently attached, using amine or sulfhydryl chemistry, to
dextran fibers that are attached to gold film within the flow cell.
A test sample is passed through the cell. If a ligand, epitope, or
opposite member of the complement/anti-complement pair is present
in the sample, it will bind to the immobilized receptor, antibody
or member, respectively, causing a change in the refractive index
of the medium, which is detected as a change in surface plasmon
resonance of the gold film. This system allows the determination of
on- and off-rates, from which binding affinity can be calculated,
and assessment of stoichiometry of binding.
[0232] 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: 660-672, 1949) and calorimetric
assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et
al., Science 245:821-25, 1991).
[0233] Zcytor19 polypeptides can also be used to prepare antibodies
that bind to zcytor19 epitopes, peptides or polypeptides. The
zcytor19 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 zcytor19 polypeptide (e.g., SEQ
ID NO:2, SEQ ID NO:19 or SEQ ID NO:21). Polypeptides comprising a
larger portion of a zcytor19 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 zcytor19 polypeptide encoded by SEQ
ID NO:2 from amino acid number 21 (Arg) to amino acid number 491
(Arg), or a contiguous 9 to 471 amino acid fragment thereof.
Suitable antigens also include the zcytor19 polypeptide encoded by
SEQ ID NO:19 from amino acid number 21 (Arg) to amino acid number
520 (Arg), or a contiguous 9 to 500 amino acid fragment thereof;
and the truncated soluble zcytor19 polypeptide encoded by SEQ ID
NO:21 from amino acid number 21 (Arg) to amino acid number 211
(Ser), or a contiguous 9 to 191 amino acid fragment thereof.
Preferred peptides to use as antigens are the extracellular
cytokine binding domain, cytokine binding fragment, fibronectin
type III domains, intracellular signaling domain, or other domains
and motifs disclosed herein, or a combination thereof; and zcytor19
hydrophilic peptides such as those predicted by one of skill in the
art from a hydrophobicity plot, determined for example, from a
Hopp/Woods hydrophilicity profile based on a sliding six-residue
window, with buried G, S, and T residues and exposed H, Y, and W
residues ignored. Zcytor19 hydrophilic peptides include peptides
comprising amino acid sequences selected from the group consisting
of: (1) residues 295 through 300 of SEQ ID NO:2; (2) residues 451
through 456 of SEQ ID NO:2; (3) residues 301 through 306 of SEQ ID
NO:2; (4) residues 294 through 299 of SEQ ID NO:2; and (5) residues
65 through 70 of SEQ ID NO:2. In addition, zcytor19 antigenic
epitopes as predicted by a Jameson-Wolf plot, e.g., using DNASTAR
Protean program (DNASTAR, Inc., Madison, Wis.) are suitable
antigens. In addition, conserved motifs, and variable regions
between conserved motifs of zcytor19 are suitable antigens.
Antibodies generated from this immune response 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, NY, 1989; and Hurrell, J. G.
R., Ed., Monoclonal Hybridoma Antibodies: Techniques and
Applications, CRC Press, Inc., Boca Raton, Fla., 1982.
[0234] 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 zcytor19 polypeptide or a
fragment thereof. The immunogenicity of a zcytor19 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 zcytor19 or a portion thereof with
an immunoglobulin polypeptide or with maltose binding protein. The
polypeptide immunogen may be a full-length molecule or a portion
thereof. If the polypeptide portion is "hapten-like", such portion
may be advantageously joined or linked to a macromolecular carrier
(such as keyhole limpet hemocyanin (KLH), bovine serum albumin
(BSA) or tetanus toxoid) for immunization.
[0235] 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.
[0236] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to zcytor19 protein or peptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled zcytor19 protein or peptide). Genes encoding
polypeptides having potential zcytor19 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 zcytor19 sequences disclosed
herein to identify proteins which bind to zcytor19. These "binding
peptides" which interact with zcytor19 polypeptides can be used for
tagging cells, e.g., such as those in which zcytor19 is
specifically expressed; for isolating homolog polypeptides by
affinity purification; they can be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like. These
binding peptides can also be used in analytical methods such as for
screening expression libraries and neutralizing activity. The
binding peptides can also be used for diagnostic assays for
determining circulating levels of zcytor19 polypeptides; for
detecting or quantitating soluble zcytor19 polypeptides as marker
of underlying pathology or disease. These binding peptides can also
act as zcytor19 "antagonists" to block zcytor19 binding and signal
transduction in vitro and in vivo. These anti-zcytor19 binding
peptides would be useful for inhibiting the action of a ligand that
binds with zcytor19.
[0237] 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-zcytor19
antibodies herein bind to a zcytor19 polypeptide, peptide or
epitope with an affinity at least 10-fold greater than the binding
affinity to control (non-zcytor19) polypeptide. It is preferred
that the antibodies exhibit a binding affinity (K.sub.a) of
10.sup.6 M.sup.-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).
[0238] Whether anti-zcytor19 antibodies do not significantly
cross-react with related polypeptide molecules is shown, for
example, by the antibody detecting zcytor19 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 (e.g.,
class II cytokine receptors, for example, interferon-gamma, alpha
and beta chains and the interferon-alpha/beta receptor alpha and
beta chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704),
CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511)
receptors). Screening can also be done using non-human zcytor19,
and zcytor19 mutant polypeptides. Moreover, using routine methods,
antibodies can be "screened against" known related polypeptides, to
isolate a population that specifically binds to the zcytor19
polypeptides. For example, antibodies raised to zcytor19 are
adsorbed to related polypeptides adhered to insoluble matrix;
antibodies specific to zcytor19 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-zcytor19 antibodies can be detected by a
number of methods in the art, and disclosed below.
[0239] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which specifically bind to zcytor19
proteins or peptides. 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
zcytor19 protein or polypeptide.
[0240] Antibodies to zcytor19 may be used for tagging cells that
express zcytor19; for isolating zcytor19 by affinity purification;
for diagnostic assays for determining circulating levels of
zcytor19 polypeptides; for detecting or quantitating soluble
zcytor19 as marker of underlying pathology or disease; for
detecting or quantitating in a histologic, biopsy, or tissue sample
zcytor19 receptor as marker of underlying pathology or disease; for
stimulating cytotoxicity or ADCC on zcytor19-bearing cancer cells;
in analytical methods employing FACS; for screening expression
libraries; for generating anti-idiotypic antibodies; and as
neutralizing antibodies or as antagonists to block zcytor19
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 zcytor19 or fragments thereof may be used
in vitro to detect denatured zcytor19 or fragments thereof in
assays, for example, Western Blots or other assays known in the
art.
[0241] Antibodies to zcytor19 are useful for tagging cells that
express the receptor and assaying Zcytorl 9 expression levels, for
affinity purification, within diagnostic assays for determining
circulating levels of soluble receptor polypeptides, analytical
methods employing fluorescence-activated cell sorting. Divalent
antibodies may be used as agonists to mimic the effect of a
zcytor19 ligand.
[0242] Antibodies herein can also be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like, and these
conjugates used for in vivo diagnostic or therapeutic applications.
For instance, antibodies or binding polypeptides which recognize
zcytor19 of the present invention can be used to identify or treat
tissues or organs that express a corresponding anti-complementary
molecule (i.e., a zcytor19 receptor). More specifically,
anti-zcytor19 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 zcytor19 molecule. A preferred use of such conjugated
antibodies is to target the drug to cancers that express the
zcytor19 receptor. For example, such antibodies can be used to
target lymphoid, B-cell, and pre-B-cell acute lymphoblastic
leukemia cancers, and esophagus, liver, ovary, rectum, stomach, and
uterus tumors, and melanoma,
[0243] Suitable detectable molecules may be directly or indirectly
attached to polypeptides that bind zcytor19 ("binding
polypeptides," including binding peptides disclosed above),
antibodies, or bioactive fragments or portions thereof. Suitable
detectable molecules include radionuclides, enzymes, substrates,
cofactors, inhibitors, fluorescent markers, chemiluminescent
markers, magnetic particles and the like. Suitable cytotoxic
molecules may be directly or indirectly attached to the polypeptide
or antibody, and include bacterial or plant toxins (for instance,
diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like),
as well as therapeutic radionuclides, such as iodine-131,
rhenium-188 or yttrium-90 (either directly attached to the
polypeptide or antibody, or indirectly attached through means of a
chelating moiety, for instance). Binding polypeptides or antibodies
may also be conjugated to cytotoxic drugs, such as adriamycin. For
indirect attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule can be conjugated with a member of
a complementary/anticomplementary pair, where the other member is
bound to the binding polypeptide or antibody portion. For these
purposes, biotin/streptavidin is an exemplary
complementary/anticomplementary pair.
[0244] In another embodiment, binding polypeptide-toxin fusion
proteins or antibody-toxin fusion proteins can be used for targeted
cell or tissue inhibition or ablation (for instance, to treat
cancer cells or tissues, e.g., such as those specific tissues and
tumors wherein zcytor19 is expressed). Alternatively, if the
binding polypeptide has multiple functional domains (i.e., an
activation domain or a ligand binding domain, plus a targeting
domain), a fusion protein including only the targeting domain may
be suitable for directing a detectable molecule, a cytotoxic
molecule or a complementary molecule to a cell or tissue type of
interest. In instances where the fusion protein including only a
single domain includes a complementary molecule, the
anti-complementary molecule can be conjugated to a detectable or
cytotoxic molecule. Such domain-complementary molecule fusion
proteins thus represent a generic targeting vehicle for
cell/tissue-specific delivery of generic
anti-complementary-detectable/cytotoxic molecule conjugates.
[0245] Similarly, in another embodiment, zcytor19 binding
polypeptide-cytokine or antibody-cytokine fusion proteins can be
used for enhancing in vivo killing of target tissues (for example,
blood, lymphoid, colon, and bone marrow cancers, or other cancers
described herein wherin zcytor19 is expressed), if the binding
polypeptide-cytokine or anti-zcytor19 antibody targets the
hyperproliferative cell (See, generally, Homick et al., Blood
89:4437-47, 1997). They described fusion proteins enable targeting
of a cytokine to a desired site of action, thereby providing an
elevated local concentration of cytokine. Suitable anti-zcytor19
antibodies target an undesirable cell or tissue (i.e., a tumor or a
leukemia), and the fused cytokine mediates improved target cell
lysis by effector cells. Suitable cytokines for this purpose
include interleukin 2 and granulocyte-macrophage colony-stimulating
factor (GM-CSF), for instance.
[0246] Alternatively, zcytor19 binding polypeptide or antibody
fusion proteins described herein can be used for enhancing in vivo
killing of target tissues by directly stimulating a
zcytor19-modulated apoptotic pathway, resulting in cell death of
hyperproliferative cells expressing zcytor19.
[0247] The bioactive binding polypeptide or antibody conjugates
described herein can be delivered orally, intravenously,
intraarterially or intraductally, or may be introduced locally at
the intended site of action.
[0248] Moreover, anti-zcytor19 antibodies and binding fragments can
be used for tagging and sorting cells that specifically-express
Zcytor19, such as bone marrow and thyroid cells, and other cells,
described herein. Such methods of cell tagging and sorting are well
known in the art (see, e.g., "Molecular Biology of the Cell",
3.sup.rd Ed., Albert, B. et al. (Garland Publishing, London &
New York, 1994). One of skill in the art would recognize the
importance of separating cell tissue types to study cells, and the
use of antibodies to separate specific cell tissue types.
Basically, antibodies that bind to the surface of a cell type are
coupled to various matrices such as collagen, polysaccharide beads,
or plastic to form an affinity surface to which only cells
recognized by the antibodies will adhere. The bound cells are then
recovered by conventional techniques. Other methods involve
separating cells by flow cytometry, or using a
fluorescence-activated cell sorter (FACS). In this technique one
labels cells with antibodies that are coupled to a fluorescent dye.
The labeled cells are then separated from unlabeled cells in a FACS
machine. In FACS sorting individual cells traveling in single file
pass through a laser beam and the fluorescence of each cell is
measured. Slightly further down-stream, tiny droplets, most
containing either one or no cells, are formed by a vibrating
nozzle. The droplets containing a single cell are automatically
give a positive or negative charge at the moment of formation,
depending on whether the cell they contain is fluorescent, and then
deflected by a strong electric field into an appropriate container.
Such machines can select 1 cell in 1000 and sort about 5000 cells
each second. This produces a uniform population of cells for cell
culture.
[0249] One of skill in the art would recognize that the antibodies
to the Zcytor19 polypeptides of the present invention are useful,
because not all tissue types express the Zcytor19 receptor and
because it is important that biologists be able to separate
specific cell types for further study and/or therapeutic
re-implantation into the body. This is particularly relevant in
cells such as immune cells, wherein zcytor19 is expressed.
[0250] Four-helix bundle cytokines that bind to cytokine receptors
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 and diabetes. Zcytor19 receptor
antagonists or agonists, including zcytor19 soluble receptors,
anti-receptor antibodies, and the natural ligand, 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, and sepsis. There may
be a role of zcytor19 antagonists or agonists, including soluble
receptors, anti-receptor antibodies and the natural ligand, in
mediating tumorgenesis, and therefore would be useful in the
treatment of cancer. Zcytor19 antagonists or agonists, including
soluble receptors anti-receptor antibodies and the natural ligand,
may be a potential therapeutic in suppressing the immune system
which would be important for reducing graft rejection or in
prevention of graft vs. host disease.
[0251] Alternatively, zcytor19 antagonists or agonists, including
soluble receptors, anti-zcytor19 receptor antibodies and the
natural ligand may activate the immune system which would be
important in boosting immunity to infectious diseases, treating
immunocompromised patients, such as HIV+ patient, or in improving
vaccines. In particular, zcytor19 antagonists or agonists,
including soluble receptors, anti-receptor antibodies, and the
natural ligand can modulate, stimulate or expand NK cells, or their
progenitors, and 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).
[0252] Polynucleotides encoding zcytor19 polypeptides are useful
within gene therapy applications where it is desired to increase or
inhibit zcytor19 activity. If a mammal has a mutated or absent
zcytor19 gene, the zcytor19 gene can be introduced into the cells
of the mammal. In one embodiment, a gene encoding a zcytor19
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).
[0253] In another embodiment, a zcytor19 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.
[0254] It is possible to remove the target cells from the body; to
introduce the vector as a naked DNA plasmid; and then to re-implant
the transformed cells into the body. Naked DNA vectors for gene
therapy can be introduced into the desired host cells by methods
known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun or use of a DNA vector
transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992;
Wu et al., J. Biol. Chem. 263:14621-4, 1988.
[0255] Antisense methodology can be used to inhibit zcytor19 gene
transcription, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a
zcytor19-encoding polynucleotide (e.g., a polynucleotide as set
forth in SEQ ID NO:1 SEQ ID NO: 18, or SEQ ID NO:20) are designed
to bind to zcytor19-encoding mRNA and to inhibit translation of
such mRNA. Such antisense polynucleotides are used to inhibit
expression of zcytor19 polypeptide-encoding genes in cell culture
or in a subject.
[0256] In addition, as a cell surface molecule, zcytor19
polypeptides can be used as a target to introduce gene therapy into
a cell. This application would be particularly appropriate for
introducing therapeutic genes into cells in which zcytor19 is
normally expressed, such as lymphoid tissue, bone marrow, prostate,
thyroid, and PBLs, or cancer cells which express zcytor19
polypeptide. For example, viral gene therapy, such as described
above, can be targeted to specific cell types in which express a
cellular receptor, such as zcytor19 polypeptide, rather than the
viral receptor. Antibodies, or other molecules that recognize
zcytor19 molecules on the target cell's surface can be used to
direct the virus to infect and administer gene therapeutic material
to that target cell. See, Woo, S. L. C, Nature Biotech. 14:1538,
1996; Wickham, T. J. et al, Nature Biotech. 14:1570-1573, 1996;
Douglas, J. T et al., Nature Biotech. 14:1574-1578, 1996; Rihova,
B., Crit. Rev. Biotechnol. 17:149-169, 1997; and Vile, R. G. et
al., Mol. Med. Today 4:84-92, 1998. For example, a bispecific
antibody containing a virus-neutralizing Fab fragment coupled to a
zcytor19-specific antibody can be used to direct the virus to cells
expressing the zcytor19 receptor and allow efficient entry of the
virus containing a genetic element into the cells. See, for
example, Wickham, T. J., et al., J. Virol. 71:7663-7669, 1997; and
Wickham, T. J., et al., J. Virol. 70:6831-6838, 1996.
[0257] The present invention also provides reagents which will find
use in diagnostic applications. For example, the zcytor19 gene, a
probe comprising zcytor19 DNA or RNA or a subsequence thereof can
be used to determine if the zcytor19 gene is present on chromosome
1 or if a mutation has occurred. Zcytor19 is located at the 1p36.11
region of chromosome 1. Detectable chromosomal aberrations at the
zcytor19 gene locus include, but are not limited to, aneuploidy,
gene copy number changes, 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, fluorescence in situ hybridization
methods, 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).
[0258] 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.
[0259] The zcytor19 gene is located at the 1p36.11 region of
chromosome 1. One of skill in the art would recognize that
chromosomal aberrations in and around the 1p36 region are involved
in several cancers including neuroblastoma, melanoma, breast,
colon, prostate and other cancers. Such aberrations include gross
chromosomal abnormalities such as translocations, loss of
heterogeneity (LOH) and the like in and around 1p36. Thus, a marker
in the 1p36.11 locus, such as provided by the polynucleotides of
the present invention, would be useful in detecting translocations,
aneuploidy, rearrangements, LOH other chromosomal abnormalities
involving this chromosomal region that are present in cancers. For
example, zcytor19 polynucleotide probes can be used to detect
abnormalities or genotypes associated with neuroblastoma, wherein
LOH between 1p36.1 and 1p36.3 is prevalent, and a breakpoint at
1p36.1 is evident. At least 70% of neuroblastomas have
cytogenetically visible chromosomal aberrations in 1p, including
translocation and deletion, and that the abnormality is most likely
due to complex translocation and deletion mechanisms. See, for
example Ritke, M K et al., Cytogenet. Cell Genet. 50:84-90, 1989;
and Weith, A et al., Genes Chromosomes Cancer 1:159-166, 1989). As
zcytor19 is localized to 1p36.11, and falls directly within the
region wherin aberrations are prevalent in neuroblastoma, one of
skill in the art would apprecitate that the polynucleotides of the
present invention could serve as a diagnostic for neuroblastoma, as
well as aid in the elucidation of translocation and deletion
mechanisms that give rise to neuroblastoma. In addition, LOH at
1p36 is evident in melanoma (Dracopoli, N C et al, Am. J. Hum.
Genet. 45 (suppl.):Al9, 1989; Dracopoli, N C et al, Proc. Nat.
Acad. Sci. 86:4614-4618, 1989; Goldstein, A M et al., Am. J. Hum.
Genet. 52:537-550, 1993); as well as prostate cancer in families
with a history of both prostate and brain cancer (1p36, LOH)
(Gibbs, M et al., Am. J. Hum. Genet. 64:776-787, 1999); and breast
cancer, wherin deletions and duplications of chromosome 1 are the
most common aberrations in breast carcinoma (1p36) (Kovacs, G. Int.
J. Cancer 21:688-694, 1978; Rodgers, C et al., Cancer Genet.
Cytogent. 13:95-119, 1984; and Genuardi, M et al., Am. J. Hum.
Genet. 45:73-82, 1989). Since translocation, LOH and other
aberrations in this region of human chromosome 1 are so prevalent
in human cancers, and the zcytor19 gene is specifically localized
to 1p36.11, the polynucleitides of the present invention have use
in detecting such aberrations that are clearly associated with
human disease, as deacribed herein.
[0260] Moreover, there is further evidence for cancer resulting
from mutations in the 1p36 region wherein zcytor19 is located, and
polynucleotide probes can be used to detect abnormalities or
genotypes associated therewith: P73, a potential tumor suppressor
maps to 1p36 a region frequently deleted in neuroblastoma and other
cancers (Kaghad, M et al., Cell 90:809-819, 1997);
rhabdomyosarcoma, which involves a translocation at the
1p36.2-p36.12 region of chromosome 1 that results in a fusion of
the PAX7 gene from chromosome 1 with FKHR gene on choromosome 13;
Leukemia-associated Protein (LAP) (1p36.1-p35) is increased in the
cells of various types of leukemia; heparin sulfate proteoglycan
(Perlecan) (1p36.1) associated with tumors, and wherein
translocations are seen; and colon cancer (1p36-p35). Further,
zcytor19 polynucleotide probes can be used to detect abnormalities
or genotypes associated with chromosome 1p36.11 deletions and
translocations associated with human diseases, and prefereably
cancers, as described above. Moreover, amongst other genetic loci,
those for C1q complement components (C1QA, B, and G)
(1p36.3-p34.1); dyslexia (1p36-p34); lymphoid activation antigen
CD30 (1p36); sodium channel non-voltage-gated type 1
(1p36.3-p36.2); tumor necrosis factor receptors (TNFRSF1b and
TNFRS12) (1p36.3-p36.2) which like zcytor19 are cytokine receptors;
phospholipase A2 (PLA2) (1p35); rigid spine muscular dystrophy
(1p36-p35) 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.TM., National Center for
Biotechnology Information, National Library of Medicine. Bethesda,
Md.) gene map, and references therein, for this region of human
chromosome 1 on a publicly available world wide web server
(http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?chromosome=1p36).
All of these serve as possible candidate genes for an inheritable
disease which show linkage to the same chromosomal region as the
zcytor19 gene. Thus, zcytor19 polynucleotide probes can be used to
detect abnormalities or genotypes associated with these
defects.
[0261] Similarly, defects in the zcytor19 gene itself may result in
a heritable human disease state. The zcytor19 gene (1p36.11) is
located near another class II receptor, the zcytor11 cytokine
receptor gene (1p35.1) (commonly owned U.S. Pat. No. 5,965,704), as
well as TNF receptors (1p36.3-p36.2), suggesting that this
chromosomal region is commonly regulated, and/or important for
immune function. Moreover, one of skill in the art would appreciate
that defects in cytokine receptors are known to cause disease
states in humans. For example, growth hormone receptor mutation
results in dwarfism (Amselem, S et al., New Eng. J. Med. 321:
989-995, 1989), IL-2 receptor gamma mutation results in severe
combined immunodeficiency (SClD) (oguchi, M et al., Cell 73:
147-157, 1993), c-Mpl mutation results in thrombocytopenia (Ihara,
K et al., Proc. Nat. Acad. Sci. 96: 3132-3136, 1999), and severe
mycobacterial and Salmonella infections result in interleukin-12
receptor-deficient patients (de Jong, R et al., Science 280:
1435-1438, 1998), amongst others. Thus, similarly, defects in
zcytor19 can cause a disease state or susceptibility to disease or
infection. As, zcytor19 is a cytokine receptor in a chromosomal hot
spot for aberrations involved in numerous cancers and is shown to
be expressed in pre-B-cell acute leukemia cells, and other cancers
described herein, the molecules of the present invention could also
be directly involved in cancer formation or metastasis. As the
zcytor19 gene is located at the 1p36.11 region zcytor19,
polynucleotide probes can be used to detect chromosome 1p36.11
loss, trisomy, duplication or translocation associated with human
diseases, such as immune cell cancers, neuroblastoma, bone marrow
cancers, thyroid, parathyroid, prostate, melanoma, or other
cancers, or immune diseases. Moreover, 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 zcytor19 genetic defect.
[0262] 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-zcytor19
antibodies, polynucleotides, and polypeptides can be used for the
detection of zcytor19 polypeptide, mRNA or anti-zcytor19
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,
zcytor19 polynucleotide probes can be used to detect abnormalities
or genotypes associated with chromosome 1p36.11 deletions and
translocations associated with human diseases, other translocations
involved with malignant progression of tumors or other 1p36.11
mutations, which are expected to be involved in chromosome
rearrangements in malignancy; or in other cancers, or in
spontaneous abortion. Similarly, zcytor19 polynucleotide probes can
be used to detect abnormalities or genotypes associated with
chromosome 1p36.11 trisomy and chromosome loss associated with
human diseases. Thus, zcytor19 polynucleotide probes can be used to
detect abnormalities or genotypes associated with these
defects.
[0263] As discussed above, defects in the zcytor19 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 zcytor19 genetic defect. In addition, zcytor19
polynucleotide probes can be used to detect allelic differences
between diseased or non-diseased individuals at the zcytor19
chromosomal locus. As such, the zcytor19 sequences can be used as
diagnostics in forensic DNA profiling.
[0264] 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 zcytor19 polynucleotide probe may
comprise an entire exon or more. Exons are readily determined by
one of skill in the art by comparing zcytor19 sequences (SEQ ID
NO:1 SEQ ID NO:18, or SEQ ID NO:20) with the human genomic DNA for
zcytor19 (Genbank Accession No. AL358412). 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 zcytor19 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 zcytor19
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 zcytor19 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 SEQ ID NO:18, or SEQ ID
NO:20 the complement of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20
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)).
[0265] Mutations associated with the zcytor19 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 zcytor19
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)).
[0266] Mice engineered to express the zcytor19 gene, referred to as
"transgenic mice," and mice that exhibit a complete absence of
zcytor19 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 zcytor19, 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 zcytor19
polypeptide, polypeptide fragment or a mutant thereof may alter
normal cellular processes, resulting in a phenotype that identifies
a tissue in which zcytor19 expression is functionally relevant and
may indicate a therapeutic target for the zcytor19, its agonists or
antagonists. For example, a preferred transgenic mouse to engineer
is one that expresses a "dominant-negative" phenotype, such as one
that over-expresses the zcytor19 polypeptide comprising an
extracellular cytokine binding domain with the transmembrane domain
attached (approximately amino acids 21 (Arg) to 249 (Trp) of SEQ ID
NO:2 or SEQ ID NO:19; or SEQ ID NO:4 attached in frame to a
transmembrane domain). Another preferred transgenic mouse is one
that over-expresses zcytor19 soluble receptors, such as those
disclosed herein. Moreover, such over-expression may result in a
phenotype that shows similarity with human diseases. Similarly,
knockout zcytor19 mice can be used to determine where zcytor19 is
absolutely required in vivo. The phenotype of knockout mice is
predictive of the in vivo effects of a zcytor19 antagonist, such as
those described herein, may have. The mouse or the human zcytor19
cDNA can be used to isolate murine zcytor19 mRNA, cDNA and genomic
DNA, which are subsequently used to generate knockout mice. These
transgenic and knockout mice may be employed to study the zcytor19
gene and the protein encoded thereby in an in vivo system, and can
be used as in vivo models for corresponding human or animal
diseases (such as those in commercially viable animal populations).
The mouse models of the present invention are particularly relevant
as tumor models for the study of cancer biology and progression.
Such models are useful in the development and efficacy of
therapeutic molecules used in human cancers. Because increases in
zcytor19 expression, as well as decreases in zcytor19 expression
are associated with specific human cancers, both transgenic mice
and knockout mice would serve as useful animal models for cancer.
Moreover, in a preferred embodiment, zcytor19 transgenic mouse can
serve as an animal model for specific tumors, particularly
esophagus, liver, ovary, rectum, stomach, and uterus tumors, and
melanoma, B-cell leukemia and other lymphoid cancers. Moreover,
transgenic mice expression of zcytor19 antisense polynucleotides or
ribozymes directed against zcytor19, described herein, can be used
analogously to transgenic mice described above.
[0267] For pharmaceutical use, the soluble receptor polypeptides 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 zcytor19 soluble
receptor polypeptide 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 zcytor19 soluble receptor
polypeptide is an amount sufficient to produce a clinically
significant effect.
[0268] Polynucleotides and polypeptides of the present invention
will additionally find use as educational tools as a laboratory
practicum kits for courses related to genetics and molecular
biology, protein chemistry and antibody production and analysis.
Due to its unique polynucleotide and polypeptide sequence molecules
of zcytor19 can be used as standards or as "unknowns" for testing
purposes. For example, zcytor19 polynucleotides can be used as an
aid, such as, for example, to teach a student how to prepare
expression constructs for bacterial, viral, and/or mammalian
expression, including fusion constructs, wherein zcytor19 is the
gene to be expressed; for determining the restriction endonuclease
cleavage sites of the polynucleotides; determining mRNA and DNA
localization of zcytor19 polynucleotides in tissues (i.e., by
Northern and Southern blotting as well as polymerase chain
reaction); and for identifying related polynucleotides and
polypeptides by nucleic acid hybridization.
[0269] Zcytor19 polypeptides can be used educationally as an aid to
teach preparation of antibodies; identifying proteins by Western
blotting; protein purification; determining the weight of expressed
zcytor19 polypeptides as a ratio to total protein expressed;
identifying peptide cleavage sites; coupling amino and carboxyl
terminal tags; amino acid sequence analysis, as well as, but not
limited to monitoring biological activities of both the native and
tagged protein (i.e., receptor binding, signal transduction,
proliferation, and differentiation) in vitro and in vivo. Zcytor19
polypeptides can also be used to teach analytical skills such as
mass spectrometry, circular dichroism to determine conformation,
especially of the four alpha helices, x-ray crystallography to
determine the three-dimensional structure in atomic detail, nuclear
magnetic resonance spectroscopy to reveal the structure of proteins
in solution. For example, a kit containing the zcytor19 can be
given to the student to analyze. Since the amino acid sequence
would be known by the professor, the specific protein can be given
to the student as a test to determine the skills or develop the
skills of the student, the teacher would then know whether or not
the student has correctly analyzed the polypeptide. Since every
polypeptide is unique, the educational utility of zcytor19 would be
unique unto itself.
[0270] Moreover, since zcytor19 has a tissue-specific expression
and is a polypeptide with a class II cytokine receptor structure
and a distinct chromosomal localization, and expressin pattern,
activity can be measured using proliferation assays; luciferase and
binding assays described herein. Moreover, expression of zcytor19
polynucleotides and polypeptides in lymphoid and other tissues can
be analyzed in order to train students in the use of diagnostic and
tissue-specific identification and methods. Moreover zcytor19
polynucleotides can be used to train students on the use of
chromosomal detection and diagnostic methods, since it's locus is
known. Moreover, students can be specifically trained and educated
about human chromosome 1, and more specifically the locus 1p36.11
wherein the zcytor19 gene is localized. Such assays are well known
in the art, and can be used in an educational setting to teach
students about cytokine receptor proteins and examine different
properties, such as cellular effects on cells, enzyme kinetics,
varying antibody binding affinities, tissue specificity, and the
like, between zcytor19 and other cytokine receptor polypeptides in
the art.
[0271] The antibodies which bind specifically to zcytor19 can be
used as a teaching aid to instruct students how to prepare affinity
chromatography columns to purify zcytor19, cloning and sequencing
the polynucleotide that encodes an antibody and thus as a practicum
for teaching a student how to design humanized antibodies.
Moreover, antibodies which bind specifically to zcytor19 can be
used as a teaching aid for use in detection of B-cell tumor tissue,
esophagus, liver, ovary, rectum, stomach, and uterus tumors, and
melanoma, pre-B-cell lymphoblastic leukemia and other lymphoid
cancers using histological, and in situ methods amongst others
known in the art. The zcytor19 gene, polypeptide or antibody would
then be packaged by reagent companies and sold to universities and
other educational entities so that the students gain skill in art
of molecular biology. Because each gene and protein is unique, each
gene and protein creates unique challenges and learning experiences
for students in a lab practicum. Such educational kits containing
the zcytor19 gene, polypeptide or antibody are considered within
the scope of the present invention.
[0272] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Identification and Isolation of Full-Length Human Zcytor19 cDNA
[0273] Zcytor19 was identified as a predicted full-length cDNA from
human genomic DNA AL358412 (Genbank). The sequence of the predicted
full length zcytor19 polynucleotide is shown in SEQ ID NO:1 and the
corresponding polypeptide is shown in SEQ ID NO:2. A variant
full-length zcytor19 cDNA sequence was identified and is shown in
SEQ ID NO:18 and the corresponding polynucleotides shown in SEQ ID
NO:19. Moreover, a trucnated soluble form of zcytor19 cDNA sequence
was identified and is shown in SEQ ID NO:20 and the corresponding
polynucleotides shown in SEQ ID NO:21.
Example 2
Tissue Distribution in Tissue Panels Using Northern Blot and
PCR
A. Human Zcytor19 Tissue Distribution Using Northern Blot
[0274] Human Multiple Tissue Northern Blots (Human 12-lane MTN Blot
I and II, and Human Immune System MTN Blot II) (Clontech) are
probed to determine the tissue distribution of human zcytor19
expression. A PCR derived probe that hybridizes to SEQ ID NO:1 or
SEQ ID NO:18 is amplified using standard PCR amplification methods.
An exemplary PCR reaction is carried out as follows using primers
designed to hybridize to SEQ ID NO:1, SEQ ID NO:18 or its
complement: 30 cycles of 94.degree. C. for 1 minute, 65.degree. C.
for 1 minute, and 72.degree. C. for 1 minute; followed by 1 cycle
at 72.degree. C. for 7 minutes. The PCR product is visualized by
agarose gel electrophoresis and the PCR product is gel purified as
described herein. The probe is radioactively labeled using, e.g.,
the PRIME IT II.TM. Random Primer Labeling Kit (Stratagene)
according to the manufacturer's instructions. The probe is purified
using, e.g., a NUCTRAP.TM. push column (Stratagene). EXPRESSHYB.TM.
(Clontech) solution is used for the prehybridization and as a
hybridizing solution for the Northern blots. Prehybridization is
carried out, for example, at 68.degree. C. for 2 hours.
Hybridization takes place overnight at about 68.degree. C. with
about 1.5.times.10.sup.6 cpm/ml of labeled probe. The blots are
washed three times at room temperature in 2.times.SSC, 0.05% SDS,
followed by 1 wash for 10 minutes in 2.times.SSC, 0.1% SDS at
50.degree. C. After exposure to X-ray film, a transcript
corresponding to the length of SEQ ID NO:1 SEQ ID NO:18, or SEQ ID
NO:20 or of an mRNA encoding SEQ ID NO:2, SEQ ID NO:19 or SEQ ID
NO:21 is expected to be seen in tissues that specifically express
zcytor19, but not other tissues.
[0275] Northern analysis is also performed using Human Cancer Cell
Line MTN.TM. (Clontech). PCR and probing conditions are as
described above. A strong signal in a cancer line suggests that
zcytor19 expression may be expressed in activated cells and/or may
indicate a cancerous disease state. Moreover, using methods known
in the art, Northern blots or PCR analysis of activated lymphocyte
cells can also show whether zcytor19 is expressed in activated
immune cells. Based on electronic Northern information zcytor19 was
shown to be expressed specifically in pre-B cell acute
lymphoblastic leukemia cells.
B. Tissue Distribution in Tissue Panels Using PCR
[0276] A panel of cDNAs from human tissues was screened for
zcytor19 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 5, below.
The cDNAs came from in-house libraries or marathon cDNAs from
in-house RNA preps, Clontech RNA, or Invitrogen RNA. The marathon
cDNAs were made using the marathon-Ready.TM. kit (Clontech, Palo
Alto, Calif.) and QC tested with clathrin primers ZC21195 (SEQ ID
NO:6) and ZC21196 (SEQ ID NO:7) and then diluted based on the
intensity of the clathrin band. To assure quality of the panel
samples, three tests for quality control (QC) were run: (1) To
assess the RNA quality used for the libraries, the in-house cDNAs
were tested for average insert size by PCR with vector oligos that
were specific for the vector sequences for an individual cDNA
library; (2) Standardization of the concentration of the cDNA in
panel samples was achieved using standard PCR methods to amplify
full length alpha tubulin or G3PDH cDNA using a 5' vector oligo
ZC14,063 (SEQ ID NO:8) and 3' alpha tubulin specific oligo primer
ZC17,574 (SEQ ID NO:9) or 3' G3PDH specific oligo primer ZC17,600
(SEQ ID NO:10); 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 was
set up using oligos ZC37685 (SEQ ID NO:26) and ZC37681 (SEQ ID
NO:27), TaKaRa Ex Taq.TM. (TAKARA Shuzo Co LTD, Biomedicals Group,
Japan), and Rediload dye (Research Genetics, Inc., Huntsville,
Ala.). The amplification was carried out as follows: 1 cycle at
94.degree. C. for 2 minutes, 5 cycles of 94.degree. C. for 30
seconds, 70.degree. C. for 30 seconds, 35 cycles of 94.degree. C.
for 30 seconds, 64.degree. C. for 30 seconds and 72.degree. C. for
30 seconds, followed by 1 cycle at 72.degree. C. for 5 minutes.
About 10 .mu.l of the PCR reaction product was subjected to
standard Agarose gel electrophoresis using a 4% agarose gel. The
correct predicted DNA fragment size was observed in adrenal gland,
bladder, cervix, colon, fetal heart, fetal skin, liver, lung,
melanoma, ovary, salivary gland, small intestine, stomach, brain,
fetal liver, kidney, prostate, spinal cord, thyroid, placenta,
testis, tumor esophagus, tumor liver, tumor ovary, tumor rectum,
tumor stomach, tumor uterus, bone marrow, CD3+ library, HaCAT
library, HPV library and HPVS library. As this primer pair does not
span an intron, there may be risk that some tissues that are
contaminated with genomic DNA or unprocessed mRNA messages would
create a false positive in this assay.
[0277] Therefore, a different primer pair ZC38481 (SEQ ID NO:47)
and ZC38626 (SEQ ID NO:48) that span introns were used using the
methods described above, to re-evaluate the tissue distribution.
The correct predicted DNA fragment size (256 bp) was observed in
colon, fetal heart, fetal liver, kidney, liver, lung, mammary
gland, prostate, salivary gland, small intestine, adipocyte
library, brain library, islet library, and prostate library, RPMI
1788 (B-cell line), spinal cord, placenta library, testis, tumor
esophagus, tumor ovary, tumor rectum, tumor stomach, HaCAT library,
HPV library and HPVS library.
[0278] Mouse tissue panels were also examined using another set of
primer pairs: (1) ZC38706 (SEQ ID NO:49) and ZC38711 (SEQ ID NO:50)
(800 bp product) using the methods described above. This panel
showed a limited tissue distribution for mouse zcytor19: mouse
prostate cell lines, salivary gland library, and skin.
TABLE-US-00005 TABLE 5 Tissue/Cell line #samples Tissue/Cell line
#samples Adrenal gland 1 Bone marrow 3 Bladder 1 Fetal brain 3 Bone
Marrow 1 Islet 2 Brain 1 Prostate 3 Cervix 1 RPMI #1788 (ATCC #
CCL-156) 2 Colon 1 Testis 4 Fetal brain 1 Thyroid 2 Fetal heart 1
WI38 (ATCC # CCL-75 2 Fetal kidney 1 ARIP (ATCC # CRL-1674 - rat) 1
Fetal liver 1 HaCat - human keratinocytes 1 Fetal lung 1 HPV (ATCC
# CRL-2221) 1 Fetal muscle 1 Adrenal gland 1 Fetal skin 1 Prostate
SM 2 Heart 2 CD3+ selected PBMC's Ionomycin + PMA 1 stimulated K562
(ATCC # CCL-243) 1 HPVS (ATCC # CRL-2221) - 1 selected Kidney 1
Heart 1 Liver 1 Pituitary 1 Lung 1 Placenta 2 Lymph node 1 Salivary
gland 1 Melanoma 1 HL60 (ATCC # CCL-240) 3 Pancreas 1 Platelet 1
Pituitary 1 HBL-100 1 Placenta 1 Renal mesangial 1 Prostate 1
T-cell 1 Rectum 1 Neutrophil 1 Salivary Gland 1 MPC 1 Skeletal
muscle 1 Hut-102 (ATCC # TIB-162) 1 Small intestine 1 Endothelial 1
Spinal cord 1 HepG2 (ATCC # HB-8065) 1 Spleen 1 Fibroblast 1
Stomach 1 E. Histo 1 Testis 2 Thymus 1 Thyroid 1 Trachea 1 Uterus 1
Esophagus tumor 1 Gastric tumor 1 Kidney tumor 1 Liver tumor 1 Lung
tumor 1 Ovarian tumor 1 Rectal tumor 1 Uterus tumor 1
Example 3
PCR-Based Chromosomal Manping of the Zcytor19 Gene
[0279] Zcytor19 is mapped to chromosome 1 using the commercially
available "GeneBridge 4 Radiation Hybrid (RH) Mapping
Panel"(Research Genetics, Inc., Huntsville, Ala.). The GeneBridge 4
RH panel contains DNA from each of 93 radiation hybrid clones, plus
two control DNAs (the HFL donor and the A23 recipient). A publicly
available WWW server
(http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allows
mapping relative to the Whitehead Institute/MIT Center for Genome
Research's radiation hybrid map of the human genome (the "WICGR"
radiation hybrid map) which is constructed with the GeneBridge 4 RH
panel.
[0280] For the mapping of Zcytor19 with the GeneBridge 4 RH panel,
20 .mu.l reactions are set up in a 96-well microtiter plate
compatible for PCR (Stratagene, La Jolla, Calif.) and used in a
"RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the
95 PCR reactions consisted of 2 .mu.l 10.times. 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, ZC27,895 (SEQ ID NO:14), 1 .mu.l
antisense primer, ZC27,899 (SEQ ID NO:24), 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 are overlaid
with an equal amount of mineral oil and sealed. The PCR cycler
conditions are 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 54.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 are
separated by electrophoresis on a 2% agarose gel (EM Science,
Gibbstown, N.J.) and visualized by staining with ethidium bromide.
The results show that Zcytor19 maps on the chromosome 1 WICGR
radiation hybrid map in the 1p36.11 chromosomal region.
Example 4
Construction of Mammalian Expression Vectors that Express Zcytor19
Soluble Receptors: Zcytor19CEE Zcytor19CFLG Zcytor19CHIS and
Zcytor19-Fc4
A. Construction of Zcytor19 Mammalian Expression Vector Containing
Zcytor19CEE, Zcytor19CFLG and Zcytor19CHIS
[0281] An expression vector is prepared for the expression of the
soluble, extracellular domain of the zcytor19 polypeptide,
pC4zcytor19CEE, wherein the construct is designed to express a
zcytor19 polypeptide comprised of the predicted initiating
methionine and truncated adjacent to the predicted transmembrane
domain, and with a C-terminal Glu-Glu tag (SEQ ID NO:11).
[0282] A zcytor19 DNA fragment comprising the zcytor19
extracellular or cytokine binding domain of zcytor19 described
herein, is created using PCR, and purified using standard methods.
The excised DNA is subcloned into a plasmid expression vector that
has a signal peptide, e.g., the native zcytor19 signal peptide, and
attaches a Glu-Glu tag (SEQ ID NO:11) to the C-terminus of the
zcytor19 polypeptide-encoding polynucleotide sequence. Such a
mammalian expression vector contains an expression cassette having
a mammalian promoter, multiple restriction sites for insertion of
coding sequences, a stop codon and a mammalian terminator. The
plasmid can also have an E. coli origin of replication, a mammalian
selectable marker expression unit having an SV40 promoter, enhancer
and origin of replication, a DHFR gene and the SV40 terminator.
[0283] Restriction digested zcytor19 insert and previously digested
vector are ligated using standard molecular biological techniques,
and electroporated into competent cells such as DH10B competent
cells (GIBCO BRL, Gaithersburg, Md.) according to manufacturer's
direction and plated onto LB plates containing 50 mg/ml ampicillin,
and incubated overnight. Colonies are screened by restriction
analysis of DNA prepared from individual colonies. The insert
sequence of positive clones is verified by sequence analysis. A
large scale plasmid preparation is done using a QIAGEN.RTM. Maxi
prep kit (Qiagen) according to manufacturer's instructions.
[0284] The same process is used to prepare the zcytor19 soluble
receptors with a C-terminal his tag, composed of 6 His residues in
a row; and a C-terminal FLAGR tag (SEQ ID NO:12), zcytor19CFLAG. To
construct these constructs, the aforementioned vector has either
the CHIS or the FLAG.RTM. tag in place of the glu-glu tag (SEQ ID
NO:11).
B. Mammalian Expression Construction of Soluble Human Zcytor19
Receptor: Zcytor19-Fc4
[0285] An expression vector, zcytor19/Fc4/pzmp20, was prepared to
express a C-terminally Fc4 tagged soluble version of zcytor19
(human zcytor19-Fc4) in BHK cells. A fragment of zcytor19 cDNA that
includes the polynucleotide sequence from extracellular domain of
the zcytor19 receptor was fused in frame to the Fc4 polynucleotide
sequence (SEQ ID NO:13) to generate a zcytor19-Fc4 fusion (SEQ ID
NO:22 and SEQ ID NO:23). The pzmp20 vector is a mammalian
expression vector that contains the Fc4 polynucleotide sequence and
a cloning site that allows rapid construction of C-terminal Fc4
fusions using standard molecular biology techniques.
[0286] A 630 base pair fragment was generated by PCR, containing
the extracellular domain of human zcytor19 with BamH1 and Bgl2
sites coded on the 5' and 3' ends, respectively. This PCR fragment
was generated using primers ZC37967 (SEQ ID NO:24) and ZC37972 (SEQ
ID NO:25) by amplification from human brain cDNA library. The PCR
reaction conditions were as follows: 30 cycles of 94.degree. C. for
20 seconds, and 68.degree. C. for 2 minutes; 1 cycle at 68.degree.
C. for 4 minutes; followed by a 10.degree. C. soak. The fragment
was digested with BamHI and Bgl2 restriction endonucleases and
subsequently purified by 1% gel electrophoresis and band
purification using QiaQuick gel extraction kit (Qiagen). The
resulting purified DNA was ligated for 5 hours at room temperature
into a pzmp20 vector previously digested with Bgl2 containing Fc4
3' of the Bgl2 sites.
[0287] One .mu.l of the ligation mix was electroporated in 37 .mu.l
DH10B electrocompetent E. coli (Gibco) according to the
manufacturer's directions. The transformed cells were diluted in
400 .mu.l of LB media and plated onto LB plates containing 100
.mu.g/ml ampicillin. Clones were analyzed by restriction digests
and positive clones were sent for DNA sequencing to confirm the
sequence of the fusion construct.
Example 5
Transfection and Expression of Zcytor19 Soluble Receptor
Polypeptides
A. Mammalian Expression Human Zcytor19 Soluble Receptor:
Zcytor19/Fc4
[0288] BHK 570 cells (ATCC NO: CRL-10314) were plated in T-75
tissue culture flasks and allowed to grow to approximately 50 to
70% confluence at 37.degree. C., 5% CO.sub.2, in DMEM/FBS media
(DMEM, Gibco/BRL High Glucose, (Gibco BRL, Gaithersburg, Md.), 5%
fetal bovine serum, 1 mM L-glutamine (JRH Biosciences, Lenea,
Kans.), 1 mM sodium pyruvate (Gibco BRL)). The cells were then
transfected with the plasmid zcytor19/Fc4/pzmp20 (Example 4B) using
Lipofectamine.TM. (Gibco BRL), in serum free (SF) media formulation
(DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1%
L-glutamine and 1% sodium pyruvate). Ten .mu.g of the plasmid DNA
zcytor19/Fc4/pzmp20 (Example 4B) was diluted into a 15 ml tube to a
total final volume of 500 .mu.l with SF media. 50 .mu.l of
Lipofectamine was mixed with 450 .mu.l of SF medium. The
Lipofectamine mix was added to the DNA mix and allowed to incubate
approximately 30 minutes at room temperature. Four ml of SF media
was added to the DNA:Lipofectamine mixture. The cells were rinsed
once with 5 ml of SF media, aspirated, and the DNA:Lipofectamine
mixture was added. The cells were incubated at 37.degree. C. for
five hours, and then 5 ml of DMEM/10% FBS media was added. The
flask was incubated at 37.degree. C. overnight after which time the
cells were split into the selection media (DMEM/FBS media from
above with the addition of 1 .mu.M methotrexate (Sigma Chemical
Co., St. Louis, Mo.) in 150 mm plates at 1:2, 1:10, and 1:50.
Approximately 10 days post-transfection, one 150 mm plate of 1
.mu.M methotrexate resistant colonies was trypsinized, the cells
were pooled, and one-half of the cells were replated in 10 .mu.M
methotrexate; to further amplify expression of the zcytor19/Fc4
protein. A conditioned-media sample from this pool of amplified
cells was tested for expression levels using SDS-PAGE and Western
analysis.
[0289] Single clones expressing the soluble receptors can also
isolated, screened and grown up in cell culture media, and purified
using standard techniques. Moreover, CHO cells are also suitable
cells for such purposes.
Example 6
Assessing Zcytor19 Receptor Heterodimerization Using ORIGEN
Assay
[0290] Soluble zcytor19 receptor zcytor19CFLAG (Example 4 and
Example 5), or gp130 (Hibi, M. et al., Cell 63:1149-1157, 1990) are
biotinylated by reaction with a five-fold molar excess of
sulfo-NHS-LC-Biotin (Pierce, Inc., Rockford, Ill.) according to the
manufacturer's protocol. Soluble zcytor19 receptor and another
soluble receptor subunit, for example, soluble class II cytokine
receptors, for example, interferon-gamma, alpha and beta chains and
the interferon-alpha/beta receptor alpha and beta chains, zcytor11
(commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7
(commonly owned U.S. Pat. No. 5,945,511) soluble receptors.
Receptors in this subfamily may associate to form homodimers that
transduce a signal. These soluble receptors are labeled with a five
fold molar excess of Ru-BPY-NHS (Igen, Inc., Gaithersburg, Md.)
according to manufacturer's protocol. The biotinylated and
Ru-BPY-NHS-labeled forms of the soluble zcytor19 receptor can be
respectively designated Bio-zcytor19 receptor and Ru-zcytor19; the
biotinylated and Ru-BPY-NHS-labeled forms of the other soluble
receptor subunit can be similarly designated. Assays can be carried
out using conditioned media from cells expressing a ligand that
binds zcytor19 heterodimeric receptors, or using purified ligands.
Preferred ligands are those that can bind class II heterodimeric
cytokine receptors such as, IL-10, IL-9, IL-TIF, interferons, TSLP
(Levine, S D et al., ibid.; Isaksen, D E et al., ibid.; Ray, R J et
al., ibid.; Friend, S L et al., ibid.), and the like.
[0291] For initial receptor binding characterization a panel of
cytokines or conditioned medium are tested to determine whether
they can mediate homodimerization of zcytor19 receptor and if they
can mediate the heterodimerization of zcytor19 receptor with the
soluble receptor subunits described above. To do this, 50 .mu.l of
conditioned media or TBS-B containing purified cytokine, is
combined with 50 .mu.l of TBS-B (20 mM Tris, 150 mM NaCl, 1 mg/ml
BSA, pH 7.2) containing e.g., 400 ng/ml of Ru-zcytor19 receptor and
Bio-zcytor19, or 400 ng/ml of Ru-zcytor19 receptor and e.g.,
Bio-gp130, or 400 ng/ml of e.g., Ru-classIIsubunit and
Bio-zcytor19. Following incubation for one hour at room
temperature, 30 .mu.g of streptavidin coated, 2.8 mm magnetic beads
(Dynal, Inc., Oslo, Norway) are added and the reaction incubated an
additional hour at room temperature. 200 .mu.l ORIGEN assay buffer
(Igen, Inc., Gaithersburg, Md.) is then added and the extent of
receptor association measured using an M8 ORIGEN analyzer (Igen,
Inc.).
Example 7
Construct for Generating a Zcytor19 Receptor Heterodimer
[0292] A vector expressing a secreted human zcytor19 heterodimer is
constructed. In this construct, the extracellular cytokine-binding
domain of zcytor19 is fused to the heavy chain of IgG gamma 1
(IgGy1) (SEQ ID NO:14 and SEQ ID NO:15), while the extracellular
portion of the heteromeric cytokine receptor subunit (E.g., class
II cytokine receptors, for example, interferon-gamma, alpha and
beta chains and the interferon-alpha/beta receptor alpha and beta
chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4,
DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511) receptors))
is fused to a human kappa light chain (human K light chain) (SEQ ID
NO:16 and SEQ ID NO:17).
A. Construction of IgG Gamma 1 and Human K Light Chain Fusion
Vectors
[0293] The heavy chain of IgGy1 (SEQ ID NO:14) is cloned into the
Zem229R mammalian expression vector (ATCC deposit No. 69447) such
that any desired cytokine receptor extracellular domain having a 5'
EcoRI and 3' NheI site can be cloned in resulting in an N-terminal
extracellular domain-C-terminal IgG.gamma.1 fusion. The IgG.gamma.1
fragment used in this construct is made by using PCR to isolate the
IgG.gamma.1 sequence from a Clontech hFetal Liver cDNA library as a
template. PCR products are purified using methods described herein
and digested with MluI and EcoRI (Boerhinger-Mannheim), ethanol
precipitated and ligated with oligos that comprise an MluI/EcoRI
linker, into Zem229R previously digested with and EcoRI using
standard molecular biology techniques disclosed herein.
[0294] The human K light chain (SEQ ID NO:16) is cloned in the
Zem228R mammalian expression vector (ATCC deposit No. 69446) such
that any desired cytokine receptor extracellular domain having a 5'
EcoRI site and a 3' KpnI site can be cloned in resulting in a
N-terminal cytokine extracellular domain-C-terminal human .kappa.
light chain fusion. As a KpnI site is located within the human
.kappa. light chain sequence (cleaved by the KpnI enzyme after
nucleotide 62 in SEQ ID NO:16), a special primer is designed to
clone the 3' end of the desired extracellular domain of a cytokine
receptor into this KpnI site: The primer is designed so that the
resulting PCR product contains the desired cytokine receptor
extracellular domain with a segment of the human .kappa. light
chain up to the KpnI site (SEQ ID NO:16). This primer preferably
comprises a portion of at least 10 nucleotides of the 3' end of the
desired cytokine receptor extracellular domain fused in frame 5' to
SEQ ID NO: 16. The human .kappa. light chain fragment used in this
construct is made by using PCR to isolate the human .kappa. light
chain sequence from the same Clontech human Fetal Liver cDNA
library used above. PCR products are purified using methods
described herein and digested with MluI and EcoRI
(Boerhinger-Mannheim), ethanol precipitated and ligated with the
MluI/EcoRI linker described above, into Zem228R previously digested
with and EcoRI using standard molecular biology techniques
disclosed herein.
B. Insertion of Zcytor19 Receptor or Heterodimeric Subunit
Extracellular Domains into Fusion Vector Constructs
[0295] Using the construction vectors above, a construct having
zcytor19 fused to IgG.gamma.1 is made. This construction is done by
PCRing the extracellular domain or cytokine-binding domain of
zcytor19 receptor described herein from a prostate cDNA library
(Clontech) or activated lymphocyte cDNA library using standard
methods, and oligos that provide EcoRI and NheI restriction sites.
The resulting PCR product is digested with EcoRI and NheI, gel
purified, as described herein, and ligated into a previously EcoRI
and NheI digested and band-purified Zem229R/IgG.gamma.1 described
above. The resulting vector is sequenced to confirm that the
zcytor19/IgG gamma 1 fusion (zcytor19/Ch1 IgG) is correct.
[0296] A separate construct having a heterodimeric cytokine
receptor subunit extracellular domain fused to .kappa. light is
also constructed as above. The cytokine receptor/human .kappa.
light chain construction is performed as above by PCRing from,
e.g., a lymphocyte cDNA library (Clontech) using standard methods,
and oligos that provide EcoRI and KpnI restriction sites. The
resulting PCR product is digested with EcoRI and KpnI and then
ligating this product into a previously EcoRI and KpnI digested and
band-purified Zem228R/human K light chain vector described above.
The resulting vector is sequenced to confirm that the cytokine
receptor subunit/human .kappa. light chain fusion is correct.
D. Co-Expression of the Zcytor19 and Heterodimeric Cytokine
Receptor Subunit Extracellular Domain
[0297] Approximately 15 .mu.g of each of vectors above, are
co-transfected into mammalian cells, e.g., BHK-570 cells (ATCC No.
CRL-10314) using LipofectaminePlus.TM. reagent (Gibco/BRL), as per
manufacturer's instructions. The transfected cells are selected for
10 days in DMEM+5% FBS (Gibco/BRL) containing 1 .mu.M of
methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5 mg/ml G418
(Gibco/BRL) for 10 days. The resulting pool of transfectants is
selected again in 10 .mu.m of MTX and 0.5 mg/ml G418 for 10
days.
[0298] The resulting pool of doubly selected cells is used to
generate protein. Three Factories (Nunc, Denmark) of this pool are
used to generate 10 L of serum free conditioned medium. This
conditioned media is passed over a 1 ml protein-A column and eluted
in about 10, 750 microliter fractions. The fractions having the
highest protein concentration are pooled and dialyzed (10 kD MW
cutoff) against PBS. Finally the dialyzed material is submitted for
amino acid analysis (AAA) using routine methods.
Example 8
Reconstitution of Zcytor19 Receptor in Vitro
[0299] To identify components involved in the zcytor19-signaling
complex, receptor reconstitution studies are performed as follows.
For example, BHK 570 cells (ATCC No. CRL-10314) transfected, using
standard methods described herein, with a luciferase reporter
mammalian expression vector plasmid serve as a bioassay cell line
to measure signal transduction response from a transfected zcytor19
receptor complex to the luciferase reporter in the presence of
zcytor19 Ligand. BHK cells would be used in the event that BHK
cells do not endogenously express the zcytor19 receptor. Other cell
lines can be used. An exemplary luciferase reporter mammalian
expression vector is the KZ134 plasmid which is constructed with
complementary oligonucleotides that contain STAT transcription
factor binding elements from 4 genes. A modified c-fos Sis
inducible element (m67SIE, or hSIE) (Sadowski, H. et al., Science
261:1739-1744, 1993), the p21 SIE1 from the p21 WAF1 gene (Chin, Y.
et al., Science 272:719-722, 1996), the mammary gland response
element of the .beta.-casein gene (Schmitt-Ney, M. et al., Mol.
Cell. Biol. 11:3745-3755, 1991), and a STAT inducible element of
the Fcg RI gene, (Seidel, H. et al., Proc. Natl. Acad. Sci.
92:3041-3045, 1995). These oligonucleotides contain Asp718-XhoI
compatible ends and are ligated, using standard methods, into a
recipient firefly luciferase reporter vector with a c-Fos promoter
(Poulsen, L. K. et al., J. Biol. Chem. 273:6229-6232, 1998)
digested with the same enzymes and containing a neomycin selectable
marker. The KZ134 plasmid is used to stably transfect BHK, or BaF3
cells, using standard transfection and selection methods, to make a
BHK/KZ134 or BaF3/KZ134 cell line respectively.
[0300] The bioassay cell line is transfected with zcytor19 receptor
alone, or co-transfected with zcytor19 receptor along with one of a
variety of other known receptor subunits. Receptor complexes
include but are not limited to zcytor19 receptor only, various
combinations of zcytor19 receptor with class II cytokine receptors,
for example, interferon-gamma, alpha and beta chains and the
interferon-alpha/beta receptor alpha and beta chains, zcytor11
(commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7
(commonly owned U.S. Pat. No. 5,945,511) receptors. Each
independent receptor complex cell line is then assayed in the
presence of cytokine-conditioned media or purified cytokines and
luciferase activity measured using routine methods. The
untransfected bioassay cell line serves as a control for the
background luciferase activity, and is thus used as a baseline to
compare signaling by the various receptor complex combinations. The
conditioned medium or cytokine that binds the zcytor19 receptor in
the presence of the correct receptor complex, is expected to give a
luciferase readout of approximately 5 fold over background or
greater.
[0301] As an alternative, a similar assay can be performed wherein
the a Baf3/zcytor19 cell line isco-transfected as described herein
and proliferation is measured, using a known assay such as a
standard Alamar Blue proliferation assay.
Example 9
COS Cell Transfection and Secretion Trap
[0302] A secretion trap assay can be used to identify the zcytor19
receptor ligand. Since zcytor19 is a Class II cytokine receptor,
the binding of zcytor19sR/Fc4 fusion protein with known or orphan
cytokines was tested. The pZP7 expression vectors containing cDNAs
of cytokines (including human IL-TIF, interferon alpha, interferon
beta, interferon gamma, IL-10, amongst others) are transfected into
COS cells, and the binding of zcytor19sR/Fc4 to transfected COS
cells are carried out using the secretion trap assay described
below. Positive binding in this assay shows potential zcytor19
receptor-ligand pairs.
A. COS Cell Transfections
[0303] The COS cell transfection was performed as follows: Mix 0.75
.mu.g cytokine DNA in 50 .mu.l serum free DMEM media (55 mg sodium
pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mg insulin, 1
.mu.g selenium and 5 mg fetuin in 500 ml DMEM (Gibco BRL)), with 5
.mu.l Lipofectamine.TM. and 45 .mu.l serum free DMEM media.
Incubate at room temperature for 30 minutes and then add 400 .mu.l
serum free DMEM media. Add this 500 .mu.l mixture onto
1.5.times.10.sup.5 COS cells/well plated on 12-well tissue culture
plate and incubate for 5 hours at 37.degree. C. Add 500 .mu.l 20%
FBS DMEM media (100 ml FBS, 55 mg sodium pyruvate and 146 mg
L-glutamine in 500 ml DMEM) and incubate overnight.
B. Secretion Trap Assay
[0304] The secretion trap was performed as follows: Media was
rinsed off cells with PBS and then fixed for 15 minutes with 1.8%
Formaldehyde in PBS. Cells were then washed 2 times with TNT (0.1M
Tris-HCL, 0.15M NaCl, and 0.05% Tween-20 in H.sub.2O), and
permeabilized with 0.1% Triton-X in PBS for 15 minutes, and washed
3 times with TNT. Cells were blocked for 1 hour with TNB (0.1M
Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NEN Renaissance
TSA-Direct Kit) in H.sub.2O. The cells were incubated for 1 hour
with 1 .mu.g/ml, 0.5 .mu.g/ml, or 0.25 .mu.g/ml zcytor19-Fc4
soluble receptor fusion protein (Example 10) in TNB. Cells were
then washed 3 times with TNT and were incubated for another hour
with 1:1000 diluted goat-anti-human Ig-HRP (Fcy specific) (Jackson
Immuno Research) in TNB. Again cells were washed with TNT.
[0305] Positive binding was detected with fluorescein tyramide
reagent diluted 1:50 in dilution buffer (NEN kit) and incubated for
4.5 minutes, and washed with TNT. Cells were preserved with
Vectashield Mounting Media (Vector Labs Burlingame, Calif.) diluted
1:5 in TNT. Cells were visualized using a FITC filter on
fluorescent microscope.
[0306] Since zcytor19 is a Class II cytokine receptor, the binding
of zcytor19sR/Fc4 fusion protein with known or orphan cytokines is
tested. The pZP7 expression vectors containing cDNAs of cytokines
(including human IL-TIF, interferon alpha, interferon beta,
interferon gamma, IL-10, amongst others are transfected into COS
cells, and the binding of zcytor19sR/Fc4 to transfected COS cells
are carried out using the secretion trap assay described above.
Positive binding in this assay shows potential zcytor19
receptor-ligand pairs.
Example 10
Expression of Human Zcytor19 in E. coli
A. Construction of Zcytor19-MBP Fusion Expression Vector
pTAP170/Zcytor19
[0307] An expression plasmid containing a polynucleotide encoding
part of the human zcytor19 fused N-terminally to maltose binding
protein (MBP) was constructed via homologous recombination. A
fragment of human zcytor19 cDNA (SEQ ID NO:1) was isolated using
PCR. Two primers were used in the production of the human zcytor19
fragment in a PCR reaction: (1) Primer ZC39204 (SEQ ID NO:30),
containing 40 bp of the vector flanking sequence and 24 bp
corresponding to the amino terminus of the human zcytor19, and (2)
primer ZC39205 (SEQ ID NO:31), containing 40 bp of the 3' end
corresponding to the flanking vector sequence and 24 bp
corresponding to the carboxyl terminus of the human zcytor19. The
PCR reaction conditions were as follows: 1 cycle of 94 C for 1
minute. Then 20 cycles of 94.degree. C. for 30 seconds, 60.degree.
C. for 30 seconds, and 68.degree. C. for 1.5 minutes; followed by
4.degree. C. soak, run in duplicate. Five .mu.l of each 100 .mu.l
PCR reaction were run on a 1.0% agarose gel with 1.times.TBE buffer
for analysis, and the expected band of approximately 700 bp
fragment was seen. The remaining 95 .mu.l of PCR reaction was
combined with the second PCR tube precipitated with 400 .mu.l of
absolute ethanol and resuspended in 10 .mu.l of water to be used
for recombining into the SmaI cut recipient vector pTAP170 to
produce the construct encoding the MBP-human zcytor19 fusion, as
described below.
[0308] Plasmid pTAP170 was derived from the plasmids pRS316 and
pMAL-c2. The plasmid pRS316 is a Saccharomyces cerevisiae shuttle
vector (Hieter P. and Sikorski, R., Genetics 122:19-27, 1989).
pMAL-C2 (NEB) is an E. coli expression plasmid. It carries the tac
promoter driving MalE (gene encoding MBP) followed by a His tag, a
thrombin cleavage site, a cloning site, and the rrnB terminator.
The vector pTAP170 was constructed using yeast homologous
recombination. 100 ng of EcoR1 cut pMAL-c2 was recombined with 1
.mu.g PvuI cut pRS316, 1 .mu.g linker, and 1 .mu.g ScaI/EcoR1 cut
pRS316. The linker consisted of oligos zc19,372 (100 pmole):
zc19,351 (1 pmole): zc19,352 (1 pmole), and zc19,371 (100 pmole)
combined in a PCR reaction. Conditions were as follows: 10 cycles
of 94.degree. C. for 30 seconds, 50.degree. C. for 30 seconds, and
72.degree. C. for 30 seconds; followed by 4.degree. C. soak. PCR
products were concentrated via 100% ethanol precipitation.
[0309] One hundred microliters of competent yeast cells (S.
cerevisiae) were combined with 10 .mu.l of a mixture containing
approximately 1 .mu.g of the human zcytor19 insert, and 100 ng of
SmaI digested pTAP170 vector, and transferred to a 0.2 cm
electroporation cuvette. The yeast/DNA mixture was electropulsed at
0.75 kV (5 kV/cm), infinite ohms, 25 .mu.F. To each cuvette was
added 600 .mu.l of 1.2 M sorbitol. The yeast was then plated in two
300 .mu.l aliquots onto two -URA D plates and incubated at
30.degree. C.
[0310] After about 48 hours, the Ura+ yeast transformants from a
single plate were resuspended in 1 ml H.sub.2O and spun briefly to
pellet the yeast cells. The cell pellet was resuspended in 1 ml of
lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH
8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture was
added to an Eppendorf tube containing 300 .mu.l acid washed glass
beads and 500 .mu.l phenol-chloroform, vortexed for 1 minute
intervals two or three times, followed by a 5 minute spin in a
Eppendorf centrifuge at maximum speed. Three hundred microliters of
the aqueous phase was transferred to a fresh tube, and the DNA
precipitated with 600 .mu.l ethanol (EtOH), followed by
centrifugation for 10 minutes at 4.degree. C. The DNA pellet was
resuspended in 100 .mu.l H.sub.2O.
[0311] Transformation of electrocompetent E. coli cells (MC1061,
Casadaban et. al. J. Mol. Biol. 138, 179-207) was done with 1 .mu.l
yeast DNA prep and 40 .mu.l of MC1061 cells. The cells were
electropulsed at 2.0 kV, 25 .mu.F and 400 ohms. Following
electroporation, 0.6 ml SOC (2% Bactol Tryptone (Difco, Detroit,
Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl2, 10 mM MgSO4, 20 mM glucose) was added to the cells. After
incubation for one hour at 37.degree. C., the cells were plated in
one aliquot on LB Kan plates (LB broth (Lennox), 1.8% Bacto.TM.
Agar (Difco), 30 mg/L kanamycin).
[0312] Individual clones harboring the correct expression construct
for human zcytor19 were identified by expression. Cells were grown
in Superbroth II (Becton Dickinson) with 30 .mu.g/ml of kanamycin
overnight. 50 .mu.l of the overnight culture was used to inoculate
2 ml of fresh Superbroth II +30 .mu.g/ml kanamycin. Cultures were
grown at 37.degree. C., shaking for 2 hours. 1 ml of the culture
was induced with 1 mM IPTG. 2-4 hours later the 250 .mu.l of each
culture was mixed 250 .mu.l Thomer buffer with 5% .beta.ME and dye
(8M urea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS).
Samples were boiled for 5-10 minutes. 20 .mu.l were loaded per lane
on a 4%-12% PAGE gel (NOVEX). Gels were run in 1.times.MES buffer.
The positive clones were designated pTAP317 and subjected to
sequence analysis. The polynucleotide sequence of MBP-zcytor19
fusion within pTAP317 is shown in SEQ ID NO:32, and the
corresponding polypeptide sequence of the MBP-zcytor19 fusion is
shown in SEQ ID NO:33.
B. Bacterial Expression of Human Zcytor19.
[0313] Ten microliters of sequencing DNA was digested with Not1
(NEB) in the following reaction to remove the CEN-ARS: 10 .mu.l
DNA, 3 .mu.l buffer3 (NEB), 15 .mu.l water, and 2PI Notl (10
U/.mu.l NEB) at 37.degree. C. for one hour. Then 7 .mu.l of the
digest was mixed with 2 .mu.l of 5.times. buffer and T4DNA ligase
(1 u/.mu.l BRL). Reaction was incubated at room temperature for one
hour. One microliter of the reaction was transformed into the
E.coli strain W3110 (ATCC). The cells were electropulsed at 2.0 kV,
25 .mu.F and 400 ohms. Following electroporation, 0.6 ml SOC (2%
Bacto.TM. Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract
(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM
glucose) was added to the cells. After a one hour incubation at
37.degree. C., the cells were plated in one aliquot on LB Kan
plates (LB broth (Lennox), 1.8% Bacto.TM. Agar (Difco), 30 mg/L
Kanamycin). Individual clones were analyzed by diagnostic digests
for the absence of yeast marker and replication sequence.
[0314] A positive clone was used to inoculate an overnight starter
culture of Superbroth II (Becton Dickinson) with 30 .mu.g/ml of
kanamycin. The starter culture was used to inoculate 4 2L-baffled
flasks each filled with 500 ml of Superbroth II+Kan. Cultures shook
at 37.degree. C. at 250 rpm until the OD.sub.600 reached 4.1. At
this point, the cultures were induced with 1 mMIPTG. Cultures grew
for two more hours at 37.degree. C., 250 rpm at which point 2 ml
was saved for analysis and the rest was harvested via
centrifugation. Pellet was saved at -80.degree. C. until
transferred to protein purification.
Example 11
Purification Scheme for Zcytor19-FC4 Fusion
[0315] All procedures performed at 4 C, unless otherwise noted. The
conditioned media was concentrated first 20 times by using an
Amicon/Millipore Spiral cartridge, 10 kD MWCO. (at ambient
temperature) The concentrated media was then applied to an
appropriately sized POROS 50 A (coupled protein A) column at an
optimal capture flow rate. The column was washed with 10 column
volumes (CV) of equilibration buffer, then rapidly eluted with 3 CV
of 0.1 M Glycine pH 3. The collected fractions had a predetermined
volume of 2M TRIS pH 8.0 added prior to the elution to neutralize
the pH to about 7.2.
[0316] Brilliant Blue (Sigma) stained NuPAGE gels were ran to
analyze the elution. Fractions of interested were pooled and
concentrated using a 30 kD MWCO centrifugal concentrator to a
nominal volume. The concentrated Protein A pool was injected onto
an appropriately sized Phamicia Sephacryl 200 column to remove
aggregates and to buffer exchange the protein into PBS pH
7.3.Brilliant Blue (Sigma) stained NuPAGE gels were again used to
analyze the elution. Fractions were pooled. Western and Brilliant
Blue (Sigma) stained NuPAGE gels were ran to confirm purity and
content. For further analysis, the protein was submitted for AAA,
and N-terminal sequencing. AAA analysis and N-terminal sequencing
verified the zcytor19-Fc polyepptide; the N-terminal amino acid
sequence was as expected SRPRL APPQX VTLLS QNFSV (SEQ ID
NO:34).
Example 12
Human Zcytor19 Expression Based on RT-PCR Analysis of Multiple
Tissue and Blood Fraction First-Strand cDNA Panels
[0317] Gene expression of zcytor19 was examined using commercially
available normalized multiple tissue first-strand cDNA panels
(OriGene Technologies, Inc. Rockville, Md.; BD Biosciences
Clontech, Palo Alto, Calif.). These included OriGene's Human Tissue
Rapid-Scan.TM. Panel (containing 24 different tissues) and the
following BD Biosciences Clontech Multiple Tissue cDNA (MTC.TM.)
Panels: Human MTC Panel I (containing 8 different adult tissues),
Human MTC Panel II (containing 8 different adult tissues), Human
Fetal MTC Panel (containing 8 different fetal tissues), Human Tumor
MTC Panel (containing carcinomas from 7 different organs), Human
Blood Fractions MTC Panel (containing 9 different blood fractions),
and Human Immune System MTC Panel (containing 6 different organs
and peripheral blood leukocyte).
[0318] PCR reactions were set up using zcytor19 specific oligo
primers ZC40285 (SEQ ID NO:35) and ZC40286 (SEQ ID NO:36) which
yield a 426 bp product, Qiagen HotStarTaq DNA Polymerase (Qiagen,
Inc., Valencia, Calif.) and RediLoad.TM. dye (Research Genetics,
Inc., Huntville, Ala.). The PCR cycler conditions were as follows:
an initial 1 cycle 15 minute denaturation at 95.degree. C., 35
cycles of a 45 second denaturation at 95.degree. C., 1 minute
annealing at 63.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.
[0319] A DNA fragment of the correct size was observed in the
following human adult tissues: adrenal gland, bone marrow, colon,
heart, liver, lung, lymph node, muscle, ovary, pancreas, placenta,
prostate, salivary gland, small intestine, spleen, stomach, testis,
thyroid, and tonsil. A DNA fragment of the correct size was
observed in the following human fetal tissues: heart, liver, lung,
kidney, skeletal muscle, spleen, and thymus. A DNA fragment of the
correct size was observed in the following human blood fractions:
peripheral blood leukocyte, mononuclear cells (B-cells, T-cells,
and monocytes), resting CD8+ cells (T-suppressor/cytotoxic),
resting CD19+ cells (B-cells), activated CD19+ cells, activated
mononuclear cells, and activated CD4+ cells. A DNA fragment of the
correct size was observed in the following tumor tissues: breast
carcinoma, colon adenocarcinoma, lung carcinoma, ovarian carcinoma,
pancreatic adenocarcinoma, and prostatic adenocarcinoma.
[0320] Because zcytor19 is expressed in these specific tumor
tissues, zcytor19 polynucleotides, polypeptides and antibodies can
be used as a tumor marker as disclosed herein. Moreove, an antibody
to zcytor19 could have anti-tumor activity, as well as
toxin-conjugates, cytokine conjugates or other conjugates of an
antibody, or the zcytor19 receptor ligand itself. The antagonist of
zcytor19 ligand, such as anti-zcytor19 antibodies or soluble
receptors can also act as anti-tumor reagents.
Example 13
Generation and Analysis of Zcytor19 KO Mice
A. Identification of BAC Clones Positive for Mouse Zcytor19
Gene
[0321] One BAC clone positive for mouse zcytor19 gene was
identified using Incyte Genomic's (St. Louis, Mo.) Easy-to-Screen
DNA Pools, BAC Mouse ES (Release I) following Manufacturer's
instructions. Oligonucleotides were designed to generate a PCR
fragment containing partial exon 6, complete intron 6 and partial
exon 7 sequences.
[0322] PCR reactions were carried out in 25 .mu.l using 1.75 units
of Advantage 2 polymerase (Clontech). Either 2 .mu.l or 10 .mu.l of
BAC library DNA was used as template in buffer containing 67 mM
Tris pH 8.8, 16.6 mM (H.sub.4).sub.2SO.sub.4, 6.7 mM MgCl.sub.2
[0323] 5 mM 2-Mercaptoethanol, 100 .mu.g/ml gelatin, 10% Dimethyl
Sulfoxide, 1 mM deoxynucleotides, 140 nM forward primer ZC39128
(SEQ ID NO:37) and 140 nM reverse primer ZC39129 (SEQ ID NO:38).
PCR conditions were as follows 95.degree. C. for 1 min,; 30 cycles
of 95.degree. C. for 15 seconds, 55.degree. C. for 30 seconds, and
68.degree. C. for 30 seconds; and 68.degree. C. for 2 minutes;
followed by a 4.degree. C. hold. PCR products were analyzed by
agarose gel electrophoresis. Positive PCR products were found to be
1,149 bp.
[0324] Four additional BAC clones positive for mouse zcytor19 gene
were identified using Incyte's BAC Mouse Filter Set (Release II)
following Manufacturer's instructions. Oligonucleotides were
designed to generate a PCR fragment containing partial exon 6, and
partial exon 7 sequences from mouse cDNA template.
[0325] PCR reactions were carried out in 25 .mu.l using 1.75 units
of Advantage 2 polymerase (Clontech). 2 .mu.l of Neonatal Mouse
skin cDNA library (JAK 062700B) was used as template in buffer
containing 67 mM Tris pH 8.8, 16.6 mM (NH.sub.4).sub.2SO.sub.4, 6.7
mM MgCl.sub.2
[0326] 5 mM 2-Mercaptoethanol, 100 .mu.g/ml gelatin, 10% Dimethyl
Sulfoxide, 1 mM deoxynucleotides, 140 nM forward primer ZC39128
(SEQ ID NO:37) and 140 nM reverse primer ZC39129 (SEQ ID NO:38).
PCR conditions were as described above. PCR products were separated
by agarose gel electrophoresis and purified using Qiaquick (Qiagen)
gel extraction kit. The isolated, approximately 400 bp, DNA
fragment was labeled using Prime-It II (Stratagene) Random Primer
labeling kit and purified using MicroSpin S-200HR columns
(AmershamPharmacia).
[0327] The labeled probe was used to screen Incyte's 7 filter BAC
library set. Hybridizations were carried out at 55.degree. C.
overnight using ExpressHyb (Clontech). Filters were then washed 3
times for 30 minutes at 50.degree. C. with 0.1.times.SSC, 0.1% SDS,
autoradiographed overnight and compared to manufacturer's grid
patterns to identify positive clones.
B. Characterization of Zcytor19 Mouse Positive BACs.
[0328] Five zcytor19 mouse positive BAC clones from 129/SvJ
Embryonic Stem Cell libraries (Release I and II) were obtained from
Incyte Genomics. BAC clones were grown within Escherichia coli host
strain DH10B in liquid media and extracted using BAC large plasmid
purification kit MKB-500 (Incyte Genomics) according to
manufacturer's instructions. 4 of 5 BACs were found to contain at
least 2,000 bp of 5' untranslated region, exon1, and exon 5 as
determined by PCR. 100 ng of each BAC DNA was used as template
using the following conditions: PCR reactions were carried out in
25 .mu.l using 1.75 units of Advantage 2 polymerase (Clontech) in
buffer containing 67 mM Tris pH 8.8, 16.6 mM
(H.sub.4).sub.2SO.sub.4, 6.7 mM MgCl.sub.2, 5 mM 2-Mercaptoethanol,
100 .mu.g/ml gelatin, 10% Dimethyl Sulfoxide, 1 mM
deoxynucleotides, 140 nM forward and 140 nM reverse primer. PCR
conditions were as follows 95.degree. C. for 1 min,; 30 cycles of
95.degree. C. for 15 seconds, 55.degree. C. for 30 seconds, and
68.degree. C. for 30 seconds; and 68.degree. C. for 2 minutes;
followed by a 4.degree. C. hold. PCR products were analyzed by
agarose gel electrophoresis. Using forward primer ZC40784 (SEQ ID
NO:39) and reverse primer ZC40785 (SEQ ID NO:40) partial 5' UTR was
amplified and found to be 957 bp. Using forward primer ZC40786 (SEQ
ID NO:41) and reverse primer ZC40787 (SEQ ID NO:42) partial 5' UTR,
complete exon 1 and partial intron 1 was amplified and found to be
approximately 950 bp. Using forward primer ZC39128 (SEQ ID NO:37)
and forward primer ZC39129 (SEQ ID NO:38) containing partial exon
6, complete intron 6 and partial exon 7 sequence was amplified and
found to be 1,149 bp.
[0329] Four of the 5 BAC clones were found to contain at least
3,796 bp of 5' UTR and at 6,922 bp of 3' UTR by Southern Blot
analysis. Oligonucleotides ZC40784 (SEQ ID NO:39) and ZC39129 (SEQ
ID NO:38) were end labeled using T4 polynucleotide kinase (Roche)
and used to probe Southern Blots containing 5 BAC candidates
digested with restriction endonucleases EcoRI (Life Technologies)
and XbaI (New England Biolabs). Results indicated 4 of 5 BACs
contained at least 3,796 bp of 5' UTR and 5 of 5 BACs contained at
least 6,922 bp of 3' UTR.
C. Determination of Zcytor19 Mouse Intron 6 Sequence.
[0330] Oligonucleotides were designed to generate a PCR fragment
containing partial exon 6, complete intron 6 and partial exon 7
sequences.
[0331] PCR reactions were carried out in 25 .mu.l using 1.75 units
of Advantage 2 polymerase (Clontech). 100 ng of 129/Sv mouse
genomic DNA was used as template in buffer containing 67 mM Tris pH
8.8, 16.6 mM (NH.sub.4).sub.2SO.sub.4, 6.7 mM MgCl.sub.2, 5 mM
2-Mercaptoethanol, 100 .mu.g/ml gelatin, 10% Dimethyl Sulfoxide, 1
mM deoxynucleotides, 140 nM forward primer ZC39128 (SEQ ID NO:37)
and 140 nM reverse primer ZC39129 (SEQ ID NO:38). PCR conditions
were as described above. PCR products were analyzed by agarose gel
electrophoresis and found to be 1,149 bp. PCR products were then
purified using Qiaquick (Qiagen) PCR purification kit.
Determination of intron 6 sequence was made by sequence analysis
using oligos ZC39128 (SEQ ID NO:37) and ZC 39129 (SEQ ID
NO:38).
D. Determination of Zcytor19 Mouse Intron 5 Sequence
[0332] Oligonucleotides were designed to generate a PCR fragment
containing partial exon5, complete intron5 and partial exon6. PCR
reactions were carried out in 25 .mu.l using 1.75 units of
Advantage 2 polymerase (Clontech). 100 ng of 129/Sv mouse genomic
DNA was used as template in buffer containing 67 mM Tris pH 8.8,
16.6 mM (NH4).sub.2SO.sub.4, 6.7 mM MgCl.sub.2, 5 mM
2-Mercaptoethanol, 100 .mu.g/ml gelatin, 10% Dimethyl Sulfoxide, 1
mM deoxynucleotides, 140 nM forward primer ZC39408 (SEQ ID NO:43)
and 140 nM reverse primer ZC39409 (SEQ ID NO:44). PCR conditions
were as follows 95.degree. C. for 1 min,; 30 cycles of 95.degree.
C. for 15 seconds, 55.degree. C. seconds, and 68.degree. C. for 30
seconds; and 68.degree. C. for 2 minutes; followed by a 4.degree.
C. hold. PCR products were analyzed by agarose gel electrophoresis
and found to be 356 bp. PCR products were then purified using
Qiaquick (Qiagen) PCR purification kit. Determination of intron 6
sequence was made by sequence analysis using oligos ZC39408 (SEQ ID
NO:43) and ZC 39409 (SEQ ID NO:44).
E. Design of Oligonucleotides for Generating of KO Constructs of
the Mouse Zcytor19 Gene
[0333] To investigate biological function of zytor19 gene, a
knockout mouse model is being generated by homologous recombination
technology in embryonic stem (ES) cells. In this model, the coding
exon 1, 2 and 3 are deleted to create a null mutation of the
zcytor19 gene. This deletion removes the translation initiation
codon, the signal domain and part of the extracellular domain of
the zcytor19 protein, thus inactivating the zcytor19 gene.
[0334] ET cloning technique will be used to generate the KO vector
(Stewart et al, Nucl. Acids Res. 27:6, 1999) First, Kanomycin
resistance cassette is used to replace introns1, 2 and 3 of
zcytor19 mouse gene. A forward knockout oligonucleotide (SEQ ID
NO:45) was designed to be 121 nucleotides in length, having 52 bp
of homology to the 5'UTR of zcytor19m a 42 bp linker having SfiI,
FseI, BamHI and HindIII restriction sites and 27 bp of homology to
the 5' end of the Kanomycin resistance cassette. A reverse knockout
oligonucleotide (SEQ ID NO:46) was designed to be 125 nucleotides
in length, having 50 bp of homology to intron 3 of zcytor19 mouse,
a 48 bp linker having SfiI, AscI, BamHI and HindlII restriction
sites and 27 bp of homology to the 3' end of the Kanomycin
resistance cassette. The above oligonucleotides can be used to
synthesize a PCR fragment 1073 bp in length containing the entire
Kanomycin resistance cassette with the first 52 bp having homology
to the 5' UTR of zcytor19 mouse and the last 50 bp having homology
to intron 3.
[0335] The fragment will then be used to construct a Knockout
vector through ET Cloning, in which cytor19 mouse positive BAC cell
hosts are made competent through treatment with glycerol then
transfected with the plasmid pBADalpha/beta/gamma(Amp). Resistance
to chloramphenical and ampicillin selects for transformed cell.
Cells are then re-transformed with the Kanomycin PCR fragment
containing homology arms. The Beta and gamma recombination proteins
of pBADalpha/beta/gamma(Amp) are induced by the addition of
arabinose to the growth media through the activation of the Red
alpha gene. Recombinant BACs are selected for by resistance to
kanomycin and ampicillin then screened by PCR. Once a recombinant
BAC is identified a fragment is subcloned containing at least 1,800
bp of sequence upstream of kanomycin resistance cassette insertion
and at least 6,000 bp of sequence downstream into a pGEM7 derived
vector. The Kanomycin resistance cassette is then replaced by
standard ligation cloning with a IRES/LacZ/Neo-MCl cassette. The
IRES is an internal ribosome entry sequence derived from
encephalomyocarditis virus. It is fused in-frame to the reporter
lacZ gene, linked to a polyA signal. Downstream of the IRES/LacZ
reporter gene, MCl promoter drives the expression of a G418
resistance selectable marker Neo gene. The selectable maker
cassette contains termination codons in all three reading frames.
Thus, the drug resistance gene Neo is used for selection of
homologous recombination events in embryonic stem (ES) cells.
IRES/LacZ reporter gene will be used to monitor the expression of
the replaced gene after homologous recombination Homologous
recombination of the knockout vector and the target locus in ES
cells leads to the replacement of a total 17,980 bp, including
complete exons 1, 2 and 3, of the wild type locus with the
IRES/LacZ/Neo-MCl cassette, which is about 5,200 bp in length.
F. Generation of zCytor19 KO Mice
[0336] The KO vector, described above, is linearized by PmeI
digestion, and electroporated into ES cells. Homologous
recombination events are identified by PCR screening strategy, and
confirmed by Southern Blot Analysis, using a standard KO protocol.
See, A. L. Joyner, Gene Targeting. A Practical Approach. IRL Press
1993.
[0337] Once homologous recombination events are identified, ES
cells will be expanded, and injected into blastocysts to generate
chimeras. Chimeric males will be used to breed to C57black females
to achieve germ line transmission of the null mutation, according
to standard procedures. See Hogan, B. et al., Manipulating the
Mouse Embryo. A Laboratory Manual Cold Spring Harbor Laboratory
Press, 1994.
[0338] Heterozygous KO animals will be bred to test biological
functions of the zcytor19 gene. Of offspring produced, 1/4 should
be wild type, 1/2 should be heterozygous, and 1/4 should be
homozygous. Homozygous will be analyzed in details as described
below.
G. Microscopic Evaluation of Tissues from Zcytor19 Homozygous
Animals.
[0339] Since zcytor19 is expressed in following tissues, we will
examine these tissues carefully: colon, ovary placenta, pituitary,
lymph node, small intestine, salivary gland, rectum, prostate,
testis, brain, lung, kidney, thyroid, spinal cord, bone marrow, and
cervix.
[0340] Spleen, thymus, and mesenteric lymph nodes are collected and
prepared for histologic examination from transgenic animals
expressing zcytor19. Other tissues which are routinely harvested
included the following: Liver, heart, lung, spleen, thymus,
mesenteric lymph nodes, kidney, skin, mammary gland, pancreas,
stomach, small and large intestine, brain, salivary gland, trachea,
esophagus, adrenal, pituitary, reproductive tract, accessory male
sex glands, skeletal muscle including peripheral nerve, and femur
with bone marrow. The tissues are harvested from homozygous animals
as well as wild type controls. Samples are fixed in 10% buffered
formalin, routinely processed, embedded in paraffin, sectioned at 5
microns, and stained with hematoxylin and eosin. The slides are
examined for histological, and pathological changes, such as
inflammatory reactions, and hypo-proliferation of certain cell
types.
H. Flow Cytometric Analysis of Tissues from Homozygous Mouse
Mutants Missing Zcytor19.
[0341] Homozygous animals missing zcytor19 gene are to be
sacrificed for flow cytometric analysis of peripheral blood,
thymus, lymph node, bone marrow, and spleen.
[0342] Cell suspensions are made from spleen, thymus and lymph
nodes by teasing the organ apart with forceps in ice cold culture
media (500 ml RPMI 1640 Medium (JRH Biosciences. Lenexa, Kans.); 5
ml 100.times. L-glutamine (Gibco BRL. Grand Island, N.Y.); 5 ml
100.times. Na Pyruvate (Gibco BRL); 5 ml 100.times. Penicillin,
Streptomycin, Neomycin (PSN) (Gibco BRL) and then gently pressing
the cells through a cell strainer (Falcon, VWR Seattle, Wash.).
Peripheral blood (200 ml) is collected in heparinized tubes and
diluted to 10 mls with HBSS containing 10 U Heparin/ml.
Erythrocytes are removed from spleen and peripheral blood
preparations by hypotonic lysis. Bone marrow cell suspensions are
made by flushing marrow from femurs with ice-cold culture media.
Cells are counted and tested for viability using Trypan Blue (GIBCO
BRL, Gaithersburg, Md.). Cells are resuspended in ice cold staining
media (HBSS, 1% fetal bovine serum, 0.1% sodium azide) at a
concentration of ten million per milliliter. Blocking of Fc
receptor and non-specific binding of antibodies to the cells was
achieved by adding 10% normal goat sera and Fc Block (PharMingen,
La Jolla, Calif.) to the cell suspension.
[0343] Cell suspensions are mixed with equal volumes of
fluorochrome labeled monoclonal antibodies (PharMingen), incubated
on ice for 60 minutes and then washed twice with ice cold wash
buffer (PBS, 1% fetal bovine serum, 0.1% sodium azide) prior to
resuspending in 400 ml wash buffer containing lmg/ml 7-AAD
(Molecular Probes, Eugene, Oreg.) as a viability marker in some
samples. Flow data was acquired on a FACSCalibur flow cytometer (BD
Immunocytometry Systems, San Jose, Calif.). Both acquisition and
analysis were performed using CellQuest software (BD
Immunocytometry Systems).
[0344] The cell populations in all lymphoid organs will be analyzed
to detect abnormalities in specific lineages of T cell, B cell, or
other lymphocytes, and cellularity in these organs.
Example 14
Identification of Cells Expressing Zcytor19 Using in Situ
Hybridization
[0345] Human tissues from cervical carcinoma, normal and carcinoma
colon, duodenum, endometrial carcinoma, normal and carcinoma ovary,
uterus, heart, liver, lung, muscle sarcoma, and normal and
carcinoma skin were screened for zcytor19 expression by in situ
hybridization. The tissues were fixed in 10% buffered formalin and
blocked in paraffin using standard techniques. Tissues were
sectioned at 5 microns. Tissues were prepared using a standard
protocol ("Development of non-isotopic in situ hybridization" at
http://dir.niehs.nih.gov/dirlep/ish.html). Briefly, tissue sections
were deparaffinized with HistoClear (National Diagnostics, Atlanta,
Ga.) and then dehydrated with ethanol. Next they were digested with
Proteinase K (50 .mu.g/ml) (Boehringer Diagnostics, Indianapolis,
Ind.) at 23.degree. C. for 4-15 minutes. This step was followed by
acetylation and re-hydration of the tissues.
[0346] One in situ probe was designed against the human zcytor19
sequence. Plasmid DNA 100933 was digested with restriction enzyme
HindIII, which covers 0.7 kb from the end of 3'UTR. The T-7 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.
[0347] In situ hybridization was performed with a digoxigenin- or
biotin-labeled zcytor19 probe (above). The probe was added to the
slides at a concentration of 1 to 5 pmol/ml for 12 to 16 hours at
60.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.).
[0348] Positive signal were observed in most of carcinoma samples.
In cervical carcinoma, carcinoma epithelial cells were positive.
There were also some signals in a subset of lymphocytes in the
lymphoid follicles. Similarly, both carcinoma and some immune cells
were positive in the colon carcinoma samples, while normal colon
samples were negative. Weak staining was also in the endometrial
carcinoma and ovarian carcinoma, while normal ovary and uterus were
negative. There was weak staining in the cancer area of the muscle
sarcoma sample. Keratinocytes were positive in the skin carcinoma
and Kaposi's sarcoma samples, while no staining was observed in the
normal skin. In heart and liver, a subset of cells possibly
circulating WBC, were positive for zcytor19. It appears endothelial
cells in some vessels may also be positive. In lung, type II
pneumocytes and macrophage-like cells were positive. Bronchial
epithelium and endothelium were also positive in some lung
specimens. In summary, zcytor19 appears to be up-regulated in
carcinoma cells. There is low level of zcytor19 mRNA in a subset of
lymphocytes and endothelial cells.
[0349] Because zcytor19 is expressed in these specific tumor
tissues, zcytor19 polynucleotides, polypeptides and antibodies can
be used as a tumor marker as disclosed herein. Moreove, an antibody
to zcytor19 could have anti-tumor activity, as well as
toxin-conjugates, cytokine conjugates or other conjugates of an
antibody, or the zcytor19 receptor ligand itself. The antagonist of
zcytor19 ligand, such as anti-zcytor19 antibodies or soluble
receptors can also act as anti-tumor reagents.
Example 15
Construction of BaF3 Cells Expressing the Zcytor19 Receptor (BaF3
Zcytor19 Cells) with Puromycin Resistant and Zeomycin Resistant
Vectors.
[0350] Two types of BaF3 cells expressing the full-length zcytor19
receptor were constructed using 30 .mu.g of zcytor19 expression
vectors, one resistant to puromycin, one resistant to zeomycin
described below. The BaF3 cells expressing the zcytor19 receptor
mRNA with puromycin resistance were designated as BaF3/zcytor19-p.
The BaF3 cells expressing the zcytor19 receptor mRNA with zeomycin
resistance were designated as BaF3/zcytor19-z.
A. Construction of BaF3 Cells Expressing the Zcytor19 Receptor
[0351] 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, 2ng/ml murine IL-3 (mIL-3) (R
& D, Minneapolis, Minn.), 2 mM L-glutaMax-1.TM. (Gibco BRL), 1
mM Sodium Pyruvate (Gibco BRL). Prior to electroporation,
pZP-5N/CRF2-4 was prepared and purified using a Qiagen Maxi Prep
kit (Qiagen) as per manufacturer's instructions. BaF3 cells for
electroporation were washed twice in PBS (Gibco BRL) and then
resuspended in RPMI media at a cell density of 10.sup.7 cells/ml.
One ml of resuspended BaF3 cells was mixed with 30 .mu.g of the
pZP-7p/zcytor19 plasmid DNA, or 30 .hoarfrost.g of the
pZP-7z/zcytor19 plasmid DNA, and transferred to separate disposable
electroporation chambers (GIBCO BRL). The cells were given two
serial shocks (800 lFad/300 V.; 1180 IFad/300 V.) delivered by an
electroporation apparatus (CELL-PORATOR.TM.; GIBCO BRL), with a 1
minute rest between the shocks. 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 Puromycin (Clonetech) selection (2
.quadrature.g/ml) for the cells transfected with pZP-7p/zcytor19,
or Zeocin selection (1:150-1:333) for the cells transfected with
pZP-7z/zcytor19, and placed in a T-162 flask to isolate the
antibiotic-resistant pools. Pools of the transfected BaF3 cells,
hereinafter called BaF3/zcytor19-puro and BaF3/zcytor19-zeo cells,
were assayed for expression of zcytor19 by RT-PCR.
B. Confirmation of zcytor19 Expression by RT-PCR.
[0352] The BaF3/zcytor19-puro and BaF3/zcytor19-zeo cells were
harvested for RNA, which was then put into a reverse transcriptase
reaction, and subsequently tested by PCR for the presence of
zcytor19.
[0353] Flasks of cells were grown to confluence, then 10 ml were
removed and spun down to obtain a cell pellet. RNA was purified
from the pellet using the RNeasy Total RNA Purification kit, with
the additional RNase-free DNase set (Qiagen), following the
manufacturer's protocol. Reverse transcription was then done on the
samples using the StrataScript RT-PCR kit (Stratagene), following
the manufacturer's protocol through the completion of the RT
reaction. PCR was then done by mixing 0.2 pmol each of primers
ZC40279 and ZC37863, 0.2 mM of dNTP mix (Roche) containing equal
amounts of each nucleotide, 5 .mu.l of 10.times. cDNA PCR Reaction
Buffer (Clonetech), 3 .mu.l DNA from the RT reaction, 0.5 .mu.l
Advantage2 Polymerase (Clonetech), made to a final volume of 50
.mu.l with water. The reaction ran for 95.degree. C., 5 min, then
30 cycles of 95.degree. C. 30 sec, 60.degree. C. 30 sec, 72.degree.
C. 1 min, then 72.degree. C. 7 min and a 4.degree. C. soak, on a
Perkin Elmer GeneAmp PCR System 2400. The samples were mixed with 3
ml loading dye, and 25 ml was run on a 1% OmniPur Agarose (Merck)
gel. Zcytor19 bands were detected on the gel for both
BaF3/zcytor19-puro and BaF3/zcytor19-zeo, indicating that those
cells are expressing the gene.
[0354] 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
50 1 1476 DNA Homo sapiens CDS (1)...(1473) 1 atg gcg ggg ccc gag
cgc tgg ggc ccc ctg ctc ctg tgc ctg ctg cag 48 Met Ala Gly Pro Glu
Arg Trp Gly Pro Leu Leu Leu Cys Leu Leu Gln 1 5 10 15 gcc gct cca
ggg agg ccc cgt ctg gcc cct ccc cag aat gtg acg ctg 96 Ala Ala Pro
Gly Arg Pro Arg Leu Ala Pro Pro Gln Asn Val Thr Leu 20 25 30 ctc
tcc cag aac ttc agc gtg tac ctg aca tgg ctc cca ggg ctt ggc 144 Leu
Ser Gln Asn Phe Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly 35 40
45 aac ccc cag gat gtg acc tat ttt gtg gcc tat cag agc tct ccc acc
192 Asn Pro Gln Asp Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr
50 55 60 cgt aga cgg tgg cgc gaa gtg gaa gag tgt gcg gga acc aag
gag ctg 240 Arg Arg Arg Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys
Glu Leu 65 70 75 80 cta tgt tct atg atg tgc ctg aag aaa cag gac ctg
tac aac aag ttc 288 Leu Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu
Tyr Asn Lys Phe 85 90 95 aag gga cgc gtg cgg acg gtt tct ccc agc
tcc aag tcc ccc tgg gtg 336 Lys Gly Arg Val Arg Thr Val Ser Pro Ser
Ser Lys Ser Pro Trp Val 100 105 110 gag tcc gaa tac ctg gat tac ctt
ttt gaa gtg gag ccg gcc cca cct 384 Glu Ser Glu Tyr Leu Asp Tyr Leu
Phe Glu Val Glu Pro Ala Pro Pro 115 120 125 gtc ctg gtg ctc acc cag
acg gag gag atc ctg agt gcc aat gcc acg 432 Val Leu Val Leu Thr Gln
Thr Glu Glu Ile Leu Ser Ala Asn Ala Thr 130 135 140 tac cag ctg ccc
ccc tgc atg ccc cca ctg ttt ctg aag tat gag gtg 480 Tyr Gln Leu Pro
Pro Cys Met Pro Pro Leu Phe Leu Lys Tyr Glu Val 145 150 155 160 gca
ttt tgg ggg ggg ggg gcc gga acc aag acc cta ttt cca gtc act 528 Ala
Phe Trp Gly Gly Gly Ala Gly Thr Lys Thr Leu Phe Pro Val Thr 165 170
175 ccc cat ggc cag cca gtc cag atc act ctc cag cca gct gcc agc gaa
576 Pro His Gly Gln Pro Val Gln Ile Thr Leu Gln Pro Ala Ala Ser Glu
180 185 190 cac cac tgc ctc agt gcc aga acc atc tac acg ttc agt gtc
ccg aaa 624 His His Cys Leu Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val
Pro Lys 195 200 205 tac agc aag ttc tct aag ccc acc tgc ttc ttg ctg
gag gtc cca gaa 672 Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu
Glu Val Pro Glu 210 215 220 gcc aac tgg gct ttc ctg gtg ctg cca tcg
ctt ctg ata ctg ctg tta 720 Ala Asn Trp Ala Phe Leu Val Leu Pro Ser
Leu Leu Ile Leu Leu Leu 225 230 235 240 gta att gcc gca ggg ggt gtg
atc tgg aag acc ctc atg ggg aac ccc 768 Val Ile Ala Ala Gly Gly Val
Ile Trp Lys Thr Leu Met Gly Asn Pro 245 250 255 tgg ttt cag cgg gca
aag atg cca cgg gcc ctg gaa ctg acc aga ggg 816 Trp Phe Gln Arg Ala
Lys Met Pro Arg Ala Leu Glu Leu Thr Arg Gly 260 265 270 gtc agg ccg
acg cct cga gtc agg gcc cca gcc acc caa cag aca aga 864 Val Arg Pro
Thr Pro Arg Val Arg Ala Pro Ala Thr Gln Gln Thr Arg 275 280 285 tgg
aag aag gac ctt gca gag gac gaa gag gag gag gat gag gag gac 912 Trp
Lys Lys Asp Leu Ala Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp 290 295
300 aca gaa gat ggc gtc agc ttc cag ccc tac att gaa cca cct tct ttc
960 Thr Glu Asp Gly Val Ser Phe Gln Pro Tyr Ile Glu Pro Pro Ser Phe
305 310 315 320 ctg ggg caa gag cac cag gct cca ggg cac tcg gag gct
ggt ggg gtg 1008 Leu Gly Gln Glu His Gln Ala Pro Gly His Ser Glu
Ala Gly Gly Val 325 330 335 gac tca ggg agg ccc agg gct cct ctg gtc
cca agc gaa ggc tcc tct 1056 Asp Ser Gly Arg Pro Arg Ala Pro Leu
Val Pro Ser Glu Gly Ser Ser 340 345 350 gct tgg gat tct tca gac aga
agc tgg gcc agc act gtg gac tcc tcc 1104 Ala Trp Asp Ser Ser Asp
Arg Ser Trp Ala Ser Thr Val Asp Ser Ser 355 360 365 tgg gac agg gct
ggg tcc tct ggc tat ttg gct gag aag ggg cca ggc 1152 Trp Asp Arg
Ala Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly 370 375 380 caa
ggg ccg ggt ggg gat ggg cac caa gaa tct ctc cca cca cct gaa 1200
Gln Gly Pro Gly Gly Asp Gly His Gln Glu Ser Leu Pro Pro Pro Glu 385
390 395 400 ttc tcc aag gac tcg ggt ttc ctg gaa gag ctc cca gaa gat
aac ctc 1248 Phe Ser Lys Asp Ser Gly Phe Leu Glu Glu Leu Pro Glu
Asp Asn Leu 405 410 415 tcc tcc tgg gcc acc tgg ggc acc tta cca ccg
gag ccg aat ctg gtc 1296 Ser Ser Trp Ala Thr Trp Gly Thr Leu Pro
Pro Glu Pro Asn Leu Val 420 425 430 cct ggg gga ccc cca gtt tct ctt
cag aca ctg acc ttc tgc tgg gaa 1344 Pro Gly Gly Pro Pro Val Ser
Leu Gln Thr Leu Thr Phe Cys Trp Glu 435 440 445 agc agc cct gag gag
gaa gag gag gcg agg gaa tca gaa att gag gac 1392 Ser Ser Pro Glu
Glu Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp 450 455 460 agc gat
gcg ggc agc tgg ggg gct gag agc acc cag agg acc gag gac 1440 Ser
Asp Ala Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu Asp 465 470
475 480 agg ggc cgg aca ttg ggg cat tac atg gcc agg tga 1476 Arg
Gly Arg Thr Leu Gly His Tyr Met Ala Arg 485 490 2 491 PRT Homo
sapiens 2 Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu
Leu Gln 1 5 10 15 Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gln
Asn Val Thr Leu 20 25 30 Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr
Trp Leu Pro Gly Leu Gly 35 40 45 Asn Pro Gln Asp Val Thr Tyr Phe
Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60 Arg Arg Arg Trp Arg Glu
Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70 75 80 Leu Cys Ser Met
Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95 Lys Gly
Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105 110
Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 115
120 125 Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala
Thr 130 135 140 Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Phe Leu Lys
Tyr Glu Val 145 150 155 160 Ala Phe Trp Gly Gly Gly Ala Gly Thr Lys
Thr Leu Phe Pro Val Thr 165 170 175 Pro His Gly Gln Pro Val Gln Ile
Thr Leu Gln Pro Ala Ala Ser Glu 180 185 190 His His Cys Leu Ser Ala
Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys 195 200 205 Tyr Ser Lys Phe
Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu 210 215 220 Ala Asn
Trp Ala Phe Leu Val Leu Pro Ser Leu Leu Ile Leu Leu Leu 225 230 235
240 Val Ile Ala Ala Gly Gly Val Ile Trp Lys Thr Leu Met Gly Asn Pro
245 250 255 Trp Phe Gln Arg Ala Lys Met Pro Arg Ala Leu Glu Leu Thr
Arg Gly 260 265 270 Val Arg Pro Thr Pro Arg Val Arg Ala Pro Ala Thr
Gln Gln Thr Arg 275 280 285 Trp Lys Lys Asp Leu Ala Glu Asp Glu Glu
Glu Glu Asp Glu Glu Asp 290 295 300 Thr Glu Asp Gly Val Ser Phe Gln
Pro Tyr Ile Glu Pro Pro Ser Phe 305 310 315 320 Leu Gly Gln Glu His
Gln Ala Pro Gly His Ser Glu Ala Gly Gly Val 325 330 335 Asp Ser Gly
Arg Pro Arg Ala Pro Leu Val Pro Ser Glu Gly Ser Ser 340 345 350 Ala
Trp Asp Ser Ser Asp Arg Ser Trp Ala Ser Thr Val Asp Ser Ser 355 360
365 Trp Asp Arg Ala Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly
370 375 380 Gln Gly Pro Gly Gly Asp Gly His Gln Glu Ser Leu Pro Pro
Pro Glu 385 390 395 400 Phe Ser Lys Asp Ser Gly Phe Leu Glu Glu Leu
Pro Glu Asp Asn Leu 405 410 415 Ser Ser Trp Ala Thr Trp Gly Thr Leu
Pro Pro Glu Pro Asn Leu Val 420 425 430 Pro Gly Gly Pro Pro Val Ser
Leu Gln Thr Leu Thr Phe Cys Trp Glu 435 440 445 Ser Ser Pro Glu Glu
Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp 450 455 460 Ser Asp Ala
Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu Asp 465 470 475 480
Arg Gly Arg Thr Leu Gly His Tyr Met Ala Arg 485 490 3 1473 DNA
Artificial Sequence Degenerate polynucleotide seuquence of SEQ ID
NO2 misc_feature (1)...(1473) n = A,T,C or G 3 atggcnggnc
cngarmgntg gggnccnytn ytnytntgyy tnytncargc ngcnccnggn 60
mgnccnmgny tngcnccncc ncaraaygtn acnytnytnw sncaraaytt ywsngtntay
120 ytnacntggy tnccnggnyt nggnaayccn cargaygtna cntayttygt
ngcntaycar 180 wsnwsnccna cnmgnmgnmg ntggmgngar gtngargart
gygcnggnac naargarytn 240 ytntgywsna tgatgtgyyt naaraarcar
gayytntaya ayaarttyaa rggnmgngtn 300 mgnacngtnw snccnwsnws
naarwsnccn tgggtngarw sngartayyt ngaytayytn 360 ttygargtng
arccngcncc nccngtnytn gtnytnacnc aracngarga rathytnwsn 420
gcnaaygcna cntaycaryt nccnccntgy atgccnccny tnttyytnaa rtaygargtn
480 gcnttytggg gnggnggngc nggnacnaar acnytnttyc cngtnacncc
ncayggncar 540 ccngtncara thacnytnca rccngcngcn wsngarcayc
aytgyytnws ngcnmgnacn 600 athtayacnt tywsngtncc naartaywsn
aarttywsna arccnacntg yttyytnytn 660 gargtnccng argcnaaytg
ggcnttyytn gtnytnccnw snytnytnat hytnytnytn 720 gtnathgcng
cnggnggngt nathtggaar acnytnatgg gnaayccntg gttycarmgn 780
gcnaaratgc cnmgngcnyt ngarytnacn mgnggngtnm gnccnacncc nmgngtnmgn
840 gcnccngcna cncarcarac nmgntggaar aargayytng cngargayga
rgargargar 900 gaygargarg ayacngarga yggngtnwsn ttycarccnt
ayathgarcc nccnwsntty 960 ytnggncarg arcaycargc nccnggncay
wsngargcng gnggngtnga ywsnggnmgn 1020 ccnmgngcnc cnytngtncc
nwsngarggn wsnwsngcnt gggaywsnws ngaymgnwsn 1080 tgggcnwsna
cngtngayws nwsntgggay mgngcnggnw snwsnggnta yytngcngar 1140
aarggnccng gncarggncc nggnggngay ggncaycarg arwsnytncc nccnccngar
1200 ttywsnaarg aywsnggntt yytngargar ytnccngarg ayaayytnws
nwsntgggcn 1260 acntggggna cnytnccncc ngarccnaay ytngtnccng
gnggnccncc ngtnwsnytn 1320 caracnytna cnttytgytg ggarwsnwsn
ccngargarg argargargc nmgngarwsn 1380 garathgarg aywsngaygc
nggnwsntgg ggngcngarw snacncarmg nacngargay 1440 mgnggnmgna
cnytnggnca ytayatggcn mgn 1473 4 203 PRT Homo sapiens 4 Arg Pro Arg
Leu Ala Pro Pro Gln Asn Val Thr Leu Leu Ser Gln Asn 1 5 10 15 Phe
Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly Asn Pro Gln Asp 20 25
30 Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr Arg Arg Arg Trp
35 40 45 Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu Leu Cys
Ser Met 50 55 60 Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe
Lys Gly Arg Val 65 70 75 80 Arg Thr Val Ser Pro Ser Ser Lys Ser Pro
Trp Val Glu Ser Glu Tyr 85 90 95 Leu Asp Tyr Leu Phe Glu Val Glu
Pro Ala Pro Pro Val Leu Val Leu 100 105 110 Thr Gln Thr Glu Glu Ile
Leu Ser Ala Asn Ala Thr Tyr Gln Leu Pro 115 120 125 Pro Cys Met Pro
Pro Leu Phe Leu Lys Tyr Glu Val Ala Phe Trp Gly 130 135 140 Gly Gly
Ala Gly Thr Lys Thr Leu Phe Pro Val Thr Pro His Gly Gln 145 150 155
160 Pro Val Gln Ile Thr Leu Gln Pro Ala Ala Ser Glu His His Cys Leu
165 170 175 Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys Tyr Ser
Lys Phe 180 185 190 Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro 195
200 5 5 PRT Artificial Sequence WSXWS motif VARIANT (1)...(5) Xaa =
Any Amino Acid 5 Trp Ser Xaa Trp Ser 1 5 6 23 DNA Artificial
Sequence Oligonucleotide primer ZC21195 6 gaggagacca taacccccga cag
23 7 23 DNA Artificial Sequence Oligonucleotide primer ZC21196 7
catagctccc accacacgat ttt 23 8 25 DNA Artificial Sequence
Oligonucleotide primer ZC14063 8 caccagacat aatagctgac agact 25 9
21 DNA Artificial Sequence Oligonucleotide primer ZC17574 9
ggtrttgctc agcatgcaca c 21 10 24 DNA Artificial Sequence
Oligonucleotide primer ZC17600 10 catgtaggcc atgaggtcca ccac 24 11
6 PRT Artificial Sequence Glu-Glu peptide tag 11 Glu Tyr Met Pro
Met Glu 1 5 12 8 PRT Artificial Sequence FLAG peptide tag 12 Asp
Tyr Lys Asp Asp Asp Asp Lys 1 5 13 699 DNA Homo sapiens 13
gagcccagat cttcagacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgag
60 ggggcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat
gatctcccgg 120 acccctgagg tcacatgcgt ggtggtggac gtgagccacg
aagaccctga ggtcaagttc 180 aactggtacg tggacggcgt ggaggtgcat
aatgccaaga caaagccgcg ggaggagcag 240 tacaacagca cgtaccgtgt
ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300 ggcaaggagt
acaagtgcaa ggtctccaac aaagccctcc catcctccat cgagaaaacc 360
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg
420 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt
ctatcccagc 480 gacatcgccg tggagtggga gagcaatggg cagccggaga
acaactacaa gaccacgcct 540 cccgtgctgg actccgacgg ctccttcttc
ctctacagca agctcaccgt ggacaagagc 600 aggtggcagc aggggaacgt
cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660 tacacgcaga
agagcctctc cctgtctccg ggtaaataa 699 14 990 DNA Homo sapiens CDS
(1)...(990) 14 gct agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc
tcc tcc aag 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 agc acc tct ggg ggc aca gcg gcc ctg ggc tgc
ctg gtc aag gac tac 96 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 ttc ccc gaa ccg gtg acg gtg tcg tgg
aac tca ggc gcc ctg acc agc 144 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 ggc gtg cac acc ttc ccg gct
gtc cta cag tcc tca gga ctc tac tcc 192 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 ctc agc agc gtg gtg
acc gtg ccc tcc agc agc ttg ggc acc cag acc 240 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 tac atc tgc
aac gtg aat cac aag ccc agc aac acc aag gtg gac aag 288 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 aaa
gtt gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc 336 Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110 cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca
384 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc
aca tgc 432 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc
aag ttc aac tgg 480 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 tac gtg gac ggc gtg gag gtg cat aat
gcc aag aca aag ccg cgg gag 528 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 gag cag tac aac agc acg tac
cgt gtg gtc agc gtc ctc acc gtc ctg 576 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 cac cag gac tgg ctg
aat ggc aag gag tac aag tgc aag gtc tcc aac 624 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 aaa gcc ctc
cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg 672 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 cag
ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag 720 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230
235 240 ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc
tat 768 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 ccc agc gac atc
gcc gtg gag tgg gag agc aat ggg cag ccg gag aac 816 Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 aac tac
aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 864 Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285
ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac 912
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290
295 300 gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac
acg 960 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 305 310 315 320 cag aag agc ctc tcc ctg tct ccg ggt aaa 990 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 15 330 PRT Homo sapiens
15 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130
135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 325 330 16 321 DNA Homo sapiens CDS (1)...(321) 16 act
gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cag 48 Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10
15 ttg aaa tct ggt acc gcc tct gtt gtg tgc ctg ctg aat aac ttc tat
96 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30 ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc
caa tcg 144 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser 35 40 45 ggt aac tcc cag gag agt gtc aca gag cag gac agc
aag gac agc acc 192 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr 50 55 60 tac agc ctc agc agc acc ctg acg ctg agc
aaa gca gac tac gag aaa 240 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 65 70 75 80 cac aaa gtc tac gcc tgc gaa gtc
acc cat cag ggc ctg agc tcg ccc 288 His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 85 90 95 gtc aca aag agc ttc aac
agg gga gag tgt tag 321 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys *
100 105 17 106 PRT Homo sapiens 17 Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65
70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 18
1563 DNA Homo sapiens CDS (1)...(1563) 18 atg gcg ggg ccc gag cgc
tgg ggc ccc ctg ctc ctg tgc ctg ctg cag 48 Met Ala Gly Pro Glu Arg
Trp Gly Pro Leu Leu Leu Cys Leu Leu Gln 1 5 10 15 gcc gct cca ggg
agg ccc cgt ctg gcc cct ccc cag aat gtg acg ctg 96 Ala Ala Pro Gly
Arg Pro Arg Leu Ala Pro Pro Gln Asn Val Thr Leu 20 25 30 ctc tcc
cag aac ttc agc gtg tac ctg aca tgg ctc cca ggg ctt ggc 144 Leu Ser
Gln Asn Phe Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly 35 40 45
aac ccc cag gat gtg acc tat ttt gtg gcc tat cag agc tct ccc acc 192
Asn Pro Gln Asp Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr 50
55 60 cgt aga cgg tgg cgc gaa gtg gaa gag tgt gcg gga acc aag gag
ctg 240 Arg Arg Arg Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu
Leu 65 70 75 80 cta tgt tct atg atg tgc ctg aag aaa cag gac ctg tac
aac aag ttc 288 Leu Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr
Asn Lys Phe 85 90 95 aag gga cgc gtg cgg acg gtt tct ccc agc tcc
aag tcc ccc tgg gtg 336 Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser
Lys Ser Pro Trp Val 100 105 110 gag tcc gaa tac ctg gat tac ctt ttt
gaa gtg gag ccg gcc cca cct 384 Glu Ser Glu Tyr Leu Asp Tyr Leu Phe
Glu Val Glu Pro Ala Pro Pro 115 120 125 gtc ctg gtg ctc acc cag acg
gag gag atc ctg agt gcc aat gcc acg 432 Val Leu Val Leu Thr Gln Thr
Glu Glu Ile Leu Ser Ala Asn Ala Thr 130 135 140 tac cag ctg ccc ccc
tgc atg ccc cca ctg gat ctg aag tat gag gtg 480 Tyr Gln Leu Pro Pro
Cys Met Pro Pro Leu Asp Leu Lys Tyr Glu Val 145 150 155 160 gca ttc
tgg aag gag ggg gcc gga aac aag acc cta ttt cca gtc act 528 Ala Phe
Trp Lys Glu Gly Ala Gly Asn Lys Thr Leu Phe Pro Val Thr 165 170 175
ccc cat ggc cag cca gtc cag atc act ctc cag cca gct gcc agc gaa 576
Pro His Gly Gln Pro Val Gln Ile Thr Leu Gln Pro Ala Ala Ser Glu 180
185 190 cac cac tgc ctc agt gcc aga acc atc tac acg ttc agt gtc ccg
aaa 624 His His Cys Leu Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val Pro
Lys 195 200 205 tac agc aag ttc tct aag ccc acc tgc ttc ttg ctg gag
gtc cca gaa 672 Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu Glu
Val Pro Glu 210 215 220 gcc aac tgg gct ttc ctg gtg ctg cca tcg ctt
ctg ata ctg ctg tta 720 Ala Asn Trp Ala Phe Leu Val Leu Pro Ser Leu
Leu Ile Leu Leu Leu 225 230 235 240 gta att gcc gca ggg ggt gtg atc
tgg aag acc ctc atg ggg aac ccc 768 Val Ile Ala Ala Gly Gly Val Ile
Trp Lys Thr Leu Met Gly Asn Pro 245 250 255 tgg ttt cag cgg gca aag
atg cca cgg gcc ctg gac ttt tct gga cac 816 Trp Phe Gln Arg Ala Lys
Met Pro Arg Ala Leu Asp Phe Ser Gly His 260 265 270 aca cac cct gtg
gca acc ttt cag ccc agc aga cca gag tcc gtg aat 864 Thr His Pro Val
Ala Thr Phe Gln Pro Ser Arg Pro Glu Ser Val Asn 275 280 285 gac ttg
ttc ctc tgt ccc caa aag gaa ctg acc aga ggg gtc agg ccg 912 Asp Leu
Phe Leu Cys Pro Gln Lys Glu Leu Thr Arg Gly Val Arg Pro 290 295 300
acg cct cga gtc agg gcc cca gcc acc caa cag aca aga tgg aag aag 960
Thr Pro Arg Val Arg Ala Pro Ala Thr Gln Gln Thr Arg Trp Lys Lys 305
310 315 320 gac ctt gca gag gac gaa gag gag gag gat gag gag gac aca
gaa gat 1008 Asp Leu Ala Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp
Thr Glu Asp 325 330 335 ggc gtc agc ttc cag ccc tac att gaa cca cct
tct ttc ctg ggg caa 1056 Gly Val Ser Phe Gln Pro Tyr Ile Glu Pro
Pro Ser Phe Leu Gly Gln 340 345 350 gag cac cag gct cca ggg cac tcg
gag gct ggt ggg gtg gac tca ggg 1104 Glu His Gln Ala Pro Gly His
Ser Glu Ala Gly Gly Val Asp Ser Gly 355 360 365 agg ccc agg gct cct
ctg gtc cca agc gaa ggc tcc tct gct tgg gat 1152 Arg Pro Arg Ala
Pro Leu Val Pro Ser Glu Gly Ser Ser Ala Trp Asp 370 375 380 tct tca
gac aga agc tgg gcc agc act gtg gac tcc tcc tgg gac agg 1200 Ser
Ser Asp Arg Ser Trp Ala Ser Thr Val Asp Ser Ser Trp Asp Arg 385 390
395 400 gct ggg tcc tct ggc tat ttg gct gag aag ggg cca ggc caa ggg
ccg 1248 Ala Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly Gln
Gly Pro 405 410 415 ggt ggg gat ggg cac caa gaa tct ctc cca cca cct
gaa ttc tcc aag 1296 Gly Gly Asp Gly His Gln Glu Ser Leu Pro Pro
Pro Glu Phe Ser Lys 420 425 430 gac tcg ggt ttc ctg gaa gag ctc cca
gaa gat aac ctc tcc tcc tgg 1344 Asp Ser Gly Phe Leu Glu Glu Leu
Pro Glu Asp Asn Leu Ser Ser Trp 435 440 445 gcc acc tgg ggc acc tta
cca ccg gag ccg aat ctg gtc cct ggg gga 1392 Ala Thr Trp Gly Thr
Leu Pro Pro Glu Pro Asn Leu Val Pro Gly Gly 450 455 460 ccc cca gtt
tct ctt cag aca ctg acc ttc tgc tgg gaa agc agc cct 1440 Pro Pro
Val Ser Leu Gln Thr Leu Thr Phe Cys Trp Glu Ser Ser Pro 465 470 475
480 gag gag gaa gag gag gcg agg gaa tca gaa att gag gac agc gat gcg
1488 Glu Glu Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp Ser Asp
Ala 485 490 495 ggc agc tgg ggg gct gag agc acc cag agg acc gag gac
agg ggc cgg 1536 Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu
Asp Arg Gly Arg 500 505 510 aca ttg ggg cat tac atg gcc agg tga
1563 Thr Leu Gly His Tyr Met Ala Arg * 515 520 19 520 PRT Homo
sapiens 19 Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu
Leu Gln 1 5 10 15 Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gln
Asn Val Thr Leu 20 25 30 Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr
Trp Leu Pro Gly Leu Gly 35 40 45 Asn Pro Gln Asp Val Thr Tyr Phe
Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60 Arg Arg Arg Trp Arg Glu
Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70 75 80 Leu Cys Ser Met
Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95 Lys Gly
Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105 110
Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 115
120 125 Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala
Thr 130 135 140 Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu Lys
Tyr Glu Val 145 150 155 160 Ala Phe Trp Lys Glu Gly Ala Gly Asn Lys
Thr Leu Phe Pro Val Thr 165 170 175 Pro His Gly Gln Pro Val Gln Ile
Thr Leu Gln Pro Ala Ala Ser Glu 180 185 190 His His Cys Leu Ser Ala
Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys 195 200 205 Tyr Ser Lys Phe
Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu 210 215 220 Ala Asn
Trp Ala Phe Leu Val Leu Pro Ser Leu Leu Ile Leu Leu Leu 225 230 235
240 Val Ile Ala Ala Gly Gly Val Ile Trp Lys Thr Leu Met Gly Asn Pro
245 250 255 Trp Phe Gln Arg Ala Lys Met Pro Arg Ala Leu Asp Phe Ser
Gly His 260 265 270 Thr His Pro Val Ala Thr Phe Gln Pro Ser Arg Pro
Glu Ser Val Asn 275 280 285 Asp Leu Phe Leu Cys Pro Gln Lys Glu Leu
Thr Arg Gly Val Arg Pro 290 295 300 Thr Pro Arg Val Arg Ala Pro Ala
Thr Gln Gln Thr Arg Trp Lys Lys 305 310 315 320 Asp Leu Ala Glu Asp
Glu Glu Glu Glu Asp Glu Glu Asp Thr Glu Asp 325 330 335 Gly Val Ser
Phe Gln Pro Tyr Ile Glu Pro Pro Ser Phe Leu Gly Gln 340 345 350 Glu
His Gln Ala Pro Gly His Ser Glu Ala Gly Gly Val Asp Ser Gly 355 360
365 Arg Pro Arg Ala Pro Leu Val Pro Ser Glu Gly Ser Ser Ala Trp Asp
370 375 380 Ser Ser Asp Arg Ser Trp Ala Ser Thr Val Asp Ser Ser Trp
Asp Arg 385 390 395 400 Ala Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly
Pro Gly Gln Gly Pro 405 410 415 Gly Gly Asp Gly His Gln Glu Ser Leu
Pro Pro Pro Glu Phe Ser Lys 420 425 430 Asp Ser Gly Phe Leu Glu Glu
Leu Pro Glu Asp Asn Leu Ser Ser Trp 435 440 445 Ala Thr Trp Gly Thr
Leu Pro Pro Glu Pro Asn Leu Val Pro Gly Gly 450 455 460 Pro Pro Val
Ser Leu Gln Thr Leu Thr Phe Cys Trp Glu Ser Ser Pro 465 470 475 480
Glu Glu Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp Ser Asp Ala 485
490 495 Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu Asp Arg Gly
Arg 500 505 510 Thr Leu Gly His Tyr Met Ala Arg 515 520 20 674 DNA
Homo sapiens CDS (1)...(633) 20 atg gcg ggg ccc gag cgc tgg ggc ccc
ctg ctc ctg tgc ctg ctg cag 48 Met Ala Gly Pro Glu Arg Trp Gly Pro
Leu Leu Leu Cys Leu Leu Gln 1 5 10 15 gcc gct cca ggg agg ccc cgt
ctg gcc cct ccc cag aat gtg acg ctg 96 Ala Ala Pro Gly Arg Pro Arg
Leu Ala Pro Pro Gln Asn Val Thr Leu 20 25 30 ctc tcc cag aac ttc
agc gtg tac ctg aca tgg ctc cca ggg ctt ggc 144 Leu Ser Gln Asn Phe
Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly 35 40 45 aac ccc cag
gat gtg acc tat ttt gtg gcc tat cag agc tct ccc acc 192 Asn Pro Gln
Asp Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60 cgt
aga cgg tgg cgc gaa gtg gaa gag tgt gcg gga acc aag gag ctg 240 Arg
Arg Arg Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70
75 80 cta tgt tct atg atg tgc ctg aag aaa cag gac ctg tac aac aag
ttc 288 Leu Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys
Phe 85 90 95 aag gga cgc gtg cgg acg gtt tct ccc agc tcc aag tcc
ccc tgg gtg 336 Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser
Pro Trp Val 100 105 110 gag tcc gaa tac ctg gat tac ctt ttt gaa gtg
gag ccg gcc cca cct 384 Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val
Glu Pro Ala Pro Pro 115 120 125 gtc ctg gtg ctc acc cag acg gag gag
atc ctg agt gcc aat gcc acg 432 Val Leu Val Leu Thr Gln Thr Glu Glu
Ile Leu Ser Ala Asn Ala Thr 130 135 140 tac cag ctg ccc ccc tgc atg
ccc cca ctg gat ctg aag tat gag gtg 480 Tyr Gln Leu Pro Pro Cys Met
Pro Pro Leu Asp Leu Lys Tyr Glu Val 145 150 155 160 gca ttc tgg aag
gag ggg gcc gga aac aag gtg gga agc tcc ttt cct 528 Ala Phe Trp Lys
Glu Gly Ala Gly Asn Lys Val Gly Ser Ser Phe Pro 165 170 175 gcc ccc
agg cta ggc ccg ctc ctc cac ccc ttc tta ctc agg ttc ttc 576 Ala Pro
Arg Leu Gly Pro Leu Leu His Pro Phe Leu Leu Arg Phe Phe 180 185 190
tca ccc tcc cag cct gct cct gca ccc ctc ctc cag gaa gtc ttc cct 624
Ser Pro Ser Gln Pro Ala Pro Ala Pro Leu Leu
Gln Glu Val Phe Pro 195 200 205 gta cac tcc tgacttctgg cagtcagccc
taataaaatc tgatcaaagt 673 Val His Ser 210 a 674 21 211 PRT Homo
sapiens 21 Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu
Leu Gln 1 5 10 15 Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gln
Asn Val Thr Leu 20 25 30 Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr
Trp Leu Pro Gly Leu Gly 35 40 45 Asn Pro Gln Asp Val Thr Tyr Phe
Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60 Arg Arg Arg Trp Arg Glu
Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70 75 80 Leu Cys Ser Met
Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95 Lys Gly
Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105 110
Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 115
120 125 Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala
Thr 130 135 140 Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu Lys
Tyr Glu Val 145 150 155 160 Ala Phe Trp Lys Glu Gly Ala Gly Asn Lys
Val Gly Ser Ser Phe Pro 165 170 175 Ala Pro Arg Leu Gly Pro Leu Leu
His Pro Phe Leu Leu Arg Phe Phe 180 185 190 Ser Pro Ser Gln Pro Ala
Pro Ala Pro Leu Leu Gln Glu Val Phe Pro 195 200 205 Val His Ser 210
22 1422 DNA Artificial Sequence Zcytor17-Fc4 fusion protein CDS
(1)...(1422) 22 atg gat gca atg aag aga ggg ctc tgc tgt gtg ctg ctg
ctg tgt ggc 48 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu
Leu Cys Gly 1 5 10 15 gcc gtc ttc gtt tcg ctc agc cag gaa atc cat
gcc gag ttg aga cgc 96 Ala Val Phe Val Ser Leu Ser Gln Glu Ile His
Ala Glu Leu Arg Arg 20 25 30 ttc cgt aga tcc agg ccc cgt ctg gcc
cct ccc cag aat gtg acg ctg 144 Phe Arg Arg Ser Arg Pro Arg Leu Ala
Pro Pro Gln Asn Val Thr Leu 35 40 45 ctc tcc cag aac ttc agc gtg
tac ctg aca tgg ctc cca ggg ctt ggc 192 Leu Ser Gln Asn Phe Ser Val
Tyr Leu Thr Trp Leu Pro Gly Leu Gly 50 55 60 aac ccc cag gat gtg
acc tat ttt gtg gcc tat cag agc tct ccc acc 240 Asn Pro Gln Asp Val
Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr 65 70 75 80 cgt aga cgg
tgg cgc gaa gtg gaa gag tgt gcg gga acc aag gag ctg 288 Arg Arg Arg
Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 85 90 95 cta
tgt tct atg atg tgc ctg aag aaa cag gac ctg tac aac aag ttc 336 Leu
Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 100 105
110 aag gga cgc gtg cgg acg gtt tct ccc agc tcc aag tcc ccc tgg gtg
384 Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val
115 120 125 gag tcc gaa tac ctg gat tac ctt ttt gaa gtg gag ccg gcc
cca cct 432 Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala
Pro Pro 130 135 140 gtc ctg gtg ctc acc cag acg gag gag atc ctg agt
gcc aat gcc acg 480 Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser
Ala Asn Ala Thr 145 150 155 160 tac cag ctg ccc ccc tgc atg ccc cca
ctg gat ctg aag tat gag gtg 528 Tyr Gln Leu Pro Pro Cys Met Pro Pro
Leu Asp Leu Lys Tyr Glu Val 165 170 175 gca ttc tgg aag gag ggg gcc
gga aac aag acc cta ttt cca gtc act 576 Ala Phe Trp Lys Glu Gly Ala
Gly Asn Lys Thr Leu Phe Pro Val Thr 180 185 190 ccc cat ggc cag cca
gtc cag atc act ctc cag cca gct gcc agc gaa 624 Pro His Gly Gln Pro
Val Gln Ile Thr Leu Gln Pro Ala Ala Ser Glu 195 200 205 cac cac tgc
ctc agt gcc aga acc atc tac acg ttc agt gtc ccg aaa 672 His His Cys
Leu Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys 210 215 220 tac
agc aag ttc tct aag ccc acc tgc ttc ttg ctg gag gtc cca gaa 720 Tyr
Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu 225 230
235 240 gcc aac tgg aga tct tca gac aaa act cac aca tgc cca ccg tgc
cca 768 Ala Asn Trp Arg Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys
Pro 245 250 255 gca cct gaa gcc gag ggg gca ccg tca gtc ttc ctc ttc
ccc cca aaa 816 Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe
Pro Pro Lys 260 265 270 ccc aag gac acc ctc atg atc tcc cgg acc cct
gag gtc aca tgc gtg 864 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 275 280 285 gtg gtg gac gtg agc cac gaa gac cct
gag gtc aag ttc aac tgg tac 912 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr 290 295 300 gtg gac ggc gtg gag gtg cat
aat gcc aag aca aag ccg cgg gag gag 960 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 305 310 315 320 cag tac aac agc
acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac 1008 Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 325 330 335 cag
gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa 1056
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 340
345 350 gcc ctc cca tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg
cag 1104 Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln 355 360 365 ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc
cgg gat gag ctg 1152 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu 370 375 380 acc aag aac cag gtc agc ctg acc tgc
ctg gtc aaa ggc ttc tat ccc 1200 Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro 385 390 395 400 agc gac atc gcc gtg
gag tgg gag agc aat ggg cag ccg gag aac aac 1248 Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 405 410 415 tac aag
acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc 1296 Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 420 425
430 tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc
1344 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val 435 440 445 ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac
tac acg cag 1392 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln 450 455 460 aag agc ctc tcc ctg tct ccg ggt aaa taa
1422 Lys Ser Leu Ser Leu Ser Pro Gly Lys * 465 470 23 473 PRT
Artificial Sequence Zcytor17-Fc4 fusion protein 23 Met Asp Ala Met
Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val
Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg 20 25 30
Phe Arg Arg Ser Arg Pro Arg Leu Ala Pro Pro Gln Asn Val Thr Leu 35
40 45 Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu
Gly 50 55 60 Asn Pro Gln Asp Val Thr Tyr Phe Val Ala Tyr Gln Ser
Ser Pro Thr 65 70 75 80 Arg Arg Arg Trp Arg Glu Val Glu Glu Cys Ala
Gly Thr Lys Glu Leu 85 90 95 Leu Cys Ser Met Met Cys Leu Lys Lys
Gln Asp Leu Tyr Asn Lys Phe 100 105 110 Lys Gly Arg Val Arg Thr Val
Ser Pro Ser Ser Lys Ser Pro Trp Val 115 120 125 Glu Ser Glu Tyr Leu
Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 130 135 140 Val Leu Val
Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala Thr 145 150 155 160
Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu Lys Tyr Glu Val 165
170 175 Ala Phe Trp Lys Glu Gly Ala Gly Asn Lys Thr Leu Phe Pro Val
Thr 180 185 190 Pro His Gly Gln Pro Val Gln Ile Thr Leu Gln Pro Ala
Ala Ser Glu 195 200 205 His His Cys Leu Ser Ala Arg Thr Ile Tyr Thr
Phe Ser Val Pro Lys 210 215 220 Tyr Ser Lys Phe Ser Lys Pro Thr Cys
Phe Leu Leu Glu Val Pro Glu 225 230 235 240 Ala Asn Trp Arg Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro 245 250 255 Ala Pro Glu Ala
Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys 260 265 270 Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 275 280 285
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 290
295 300 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 305 310 315 320 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His 325 330 335 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 340 345 350 Ala Leu Pro Ser Ser Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln 355 360 365 Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 370 375 380 Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 385 390 395 400 Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 405 410
415 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
420 425 430 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val 435 440 445 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln 450 455 460 Lys Ser Leu Ser Leu Ser Pro Gly Lys 465
470 24 28 DNA Artificial Sequence Oligonucleotide primer ZC37967 24
gcggatccag gccccgtctg gcccctcc 28 25 30 DNA Artificial Sequence
Oligonucleotide primer ZC37972 25 gcagatctcc agttggcttc tgggacctcc
30 26 23 DNA Artificial Sequence Oligonucleotide primer ZC37685 26
ccagccctac attgaaccac ctt 23 27 22 DNA Artificial Sequence
Oligonucleotide primer ZC37681 27 cctcgcctcc tcttcctcct ca 22 28
1560 DNA Artificial Sequence Degenerate Polynucleotide seuquence of
SEQ ID NO19 misc_feature (1)...(1560) n = A,T,C or G 28 atggcnggnc
cngarmgntg gggnccnytn ytnytntgyy tnytncargc ngcnccnggn 60
mgnccnmgny tngcnccncc ncaraaygtn acnytnytnw sncaraaytt ywsngtntay
120 ytnacntggy tnccnggnyt nggnaayccn cargaygtna cntayttygt
ngcntaycar 180 wsnwsnccna cnmgnmgnmg ntggmgngar gtngargart
gygcnggnac naargarytn 240 ytntgywsna tgatgtgyyt naaraarcar
gayytntaya ayaarttyaa rggnmgngtn 300 mgnacngtnw snccnwsnws
naarwsnccn tgggtngarw sngartayyt ngaytayytn 360 ttygargtng
arccngcncc nccngtnytn gtnytnacnc aracngarga rathytnwsn 420
gcnaaygcna cntaycaryt nccnccntgy atgccnccny tngayytnaa rtaygargtn
480 gcnttytgga argarggngc nggnaayaar acnytnttyc cngtnacncc
ncayggncar 540 ccngtncara thacnytnca rccngcngcn wsngarcayc
aytgyytnws ngcnmgnacn 600 athtayacnt tywsngtncc naartaywsn
aarttywsna arccnacntg yttyytnytn 660 gargtnccng argcnaaytg
ggcnttyytn gtnytnccnw snytnytnat hytnytnytn 720 gtnathgcng
cnggnggngt nathtggaar acnytnatgg gnaayccntg gttycarmgn 780
gcnaaratgc cnmgngcnyt ngayttywsn ggncayacnc ayccngtngc nacnttycar
840 ccnwsnmgnc cngarwsngt naaygayytn ttyytntgyc cncaraarga
rytnacnmgn 900 ggngtnmgnc cnacnccnmg ngtnmgngcn ccngcnacnc
arcaracnmg ntggaaraar 960 gayytngcng argaygarga rgargargay
gargargaya cngargaygg ngtnwsntty 1020 carccntaya thgarccncc
nwsnttyytn ggncargarc aycargcncc nggncaywsn 1080 gargcnggng
gngtngayws nggnmgnccn mgngcnccny tngtnccnws ngarggnwsn 1140
wsngcntggg aywsnwsnga ymgnwsntgg gcnwsnacng tngaywsnws ntgggaymgn
1200 gcnggnwsnw snggntayyt ngcngaraar ggnccnggnc arggnccngg
nggngaygg 1260 caycargarw snytnccncc nccngartty wsnaargayw
snggnttyyt ngargaryn 1320 ccngargaya ayytnwsnws ntgggcnacn
tggggnacny tnccnccnga rccnaaytn 1380 gtnccnggng gnccnccngt
nwsnytncar acnytnacnt tytgytggga rwsnwsccn 1440 gargargarg
argargcnmg ngarwsngar athgargayw sngaygcngg nwsntgggn 1500
gcngarwsna cncarmgnac ngargaymgn ggnmgnacny tnggncayta yatgcnmgn
1560 29 633 DNA Artificial Sequence Degenerate polynucleotide
sequence of SEQ ID NO21 misc_feature (1)...(633) n = A,T,C or G 29
atggcnggnc cngarmgntg gggnccnytn ytnytntgyy tnytncargc ngcnccnggn
60 mgnccnmgny tngcnccncc ncaraaygtn acnytnytnw sncaraaytt
ywsngtntay 120 ytnacntggy tnccnggnyt nggnaayccn cargaygtna
cntayttygt ngcntaycar 180 wsnwsnccna cnmgnmgnmg ntggmgngar
gtngargart gygcnggnac naargarytn 240 ytntgywsna tgatgtgyyt
naaraarcar gayytntaya ayaarttyaa rggnmgngtn 300 mgnacngtnw
snccnwsnws naarwsnccn tgggtngarw sngartayyt ngaytayytn 360
ttygargtng arccngcncc nccngtnytn gtnytnacnc aracngarga rathytnwsn
420 gcnaaygcna cntaycaryt nccnccntgy atgccnccny tngayytnaa
rtaygargtn 480 gcnttytgga argarggngc nggnaayaar gtnggnwsnw
snttyccngc nccnmgnytn 540 ggnccnytny tncayccntt yytnytnmgn
ttyttywsnc cnwsncarcc ngcnccngcn 600 ccnytnytnc argargtntt
yccngtncay wsn 633 30 64 DNA Artificial Sequence Oligonucleotide
Primer ZC39204 30 tcaccacgcg aattcggtac cgctggttcc gcgtggatcc
aggccccgtc tggcccctcc 60 ccag 64 31 64 DNA Artificial Sequence
Oligonucleotide Primer ZC39205 31 tctgtatcag gctgaaaatc ttatctcatc
cgccaaaaca ccagttggct tctgggacct 60 ccag 64 32 1922 DNA Artificial
Sequence MBP-human zcytoR19 fusion protein polynucleotide sequence
CDS (123)...(1922) 32 ttgacaatta atcatcggct cgtataatgt gtggaattgt
gagcggataa caatttcaca 60 caggaaacag ccagtccgtt taggtgtttt
cacgagcact tcaccaacaa ggaccataga 120 tt atg aaa act gaa gaa ggt aaa
ctg gta atc tgg att aac ggc gat 167 Met Lys Thr Glu Glu Gly Lys Leu
Val Ile Trp Ile Asn Gly Asp 1 5 10 15 aaa ggc tat aac ggt ctc gct
gaa gtc ggt aag aaa ttc gag aaa gat 215 Lys Gly Tyr Asn Gly Leu Ala
Glu Val Gly Lys Lys Phe Glu Lys Asp 20 25 30 acc gga att aaa gtc
acc gtt gag cat ccg gat aaa ctg gaa gag aaa 263 Thr Gly Ile Lys Val
Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys 35 40 45 ttc cca cag
gtt gcg gca act ggc gat ggc cct gac att atc ttc tgg 311 Phe Pro Gln
Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp 50 55 60 gca
cac gac cgc ttt ggt ggc tac gct caa tct ggc ctg ttg gct gaa 359 Ala
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu 65 70
75 atc acc ccg gac aaa gcg ttc cag gac aag ctg tat ccg ttt acc tgg
407 Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp
80 85 90 95 gat gcc gta cgt tac aac ggc aag ctg att gct tac ccg atc
gct gtt 455 Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile
Ala Val 100 105 110 gaa gcg tta tcg ctg att tat aac aaa gat ctg ctg
ccg aac ccg cca 503 Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu
Pro Asn Pro Pro 115 120 125 aaa acc tgg gaa gag atc ccg gcg ctg gat
aaa gaa ctg aaa gcg aaa 551 Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp
Lys Glu Leu Lys Ala Lys 130 135 140 ggt aag agc gcg ctg atg ttc aac
ctg caa gaa ccg tac ttc acc tgg 599 Gly Lys Ser Ala Leu Met Phe Asn
Leu Gln Glu Pro Tyr Phe Thr Trp 145 150 155 ccg ctg att gct gct gac
ggg ggt tat gcg ttc aag tat gaa aac ggc 647 Pro Leu Ile Ala Ala Asp
Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly 160 165 170 175 aag tac gac
att aaa gac gtg ggc gtg gat aac gct ggc gcg aaa gcg 695 Lys Tyr Asp
Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala 180 185 190 ggt
ctg acc ttc ctg gtt gac ctg att aaa aac aaa cac atg aat gca 743 Gly
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala 195 200
205 gac acc gat tac tcc atc gca gaa gct gcc ttt aat aaa ggc gaa aca
791 Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr
210 215 220 gcg atg acc atc aac ggc ccg tgg gca tgg tcc aac atc gac
acc agc 839 Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn
Ile Asp Thr Ser 225 230 235 aaa gtg aat tat ggt gta acg gta ctg ccg
acc ttc aag ggt caa cca 887 Lys Val Asn Tyr Gly Val Thr Val Leu Pro
Thr Phe Lys Gly Gln Pro 240 245 250 255 tcc aaa ccg ttc gtt ggc gtg
ctg agc gca ggt att aac gcc gcc agt 935 Ser Lys Pro Phe Val Gly Val
Leu Ser Ala Gly Ile Asn Ala Ala Ser 260 265 270 ccg aac aaa gag ctg
gca aaa gag ttc ctc gaa aac tat ctg ctg act 983 Pro Asn Lys Glu Leu
Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr 275 280 285 gat gaa ggt
ctg gaa gcg gtt aat aaa gac aaa ccg ctg ggt gcc gta 1031 Asp Glu
Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val 290 295 300
gcg ctg aag tct tac gag gaa gag ttg gcg aaa gat cca cgt att gcc
1079 Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile
Ala 305 310 315 gcc acc atg gaa aac gcc cag aaa ggt gaa atc atg ccg
aac atc ccg 1127 Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met
Pro Asn Ile Pro 320 325 330 335 cag atg tcc gct ttc tgg tat gcc gtg
cgt act gcg gtg atc aac gcc 1175 Gln Met Ser Ala Phe Trp Tyr Ala
Val Arg Thr Ala Val Ile Asn Ala 340 345 350 gcc agc ggt cgt cag act
gtc gat gaa gcc ctg aaa gac gcg cag act 1223 Ala Ser Gly Arg Gln
Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr 355 360 365 aat tcg agc
tcc cac cat cac cat cac cac gcg aat tcg gta ccg ctg 1271 Asn Ser
Ser Ser His His His His His His Ala Asn Ser Val Pro Leu 370 375 380
gtt ccg cgt gga tcc agg ccc cgt ctg gcc cct ccc cag aat gtg acg
1319 Val Pro Arg Gly Ser Arg Pro Arg Leu Ala Pro Pro Gln Asn Val
Thr 385 390 395 ctg ctc tcc cag aac ttc agc gtg tac ctg aca tgg ctc
cca ggg ctt 1367 Leu Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr Trp
Leu Pro Gly Leu 400 405 410 415 ggc aac ccc cag gat gtg acc tat ttt
gtg gcc tat cag agc tct ccc 1415 Gly Asn Pro Gln Asp Val Thr Tyr
Phe Val Ala Tyr Gln Ser Ser Pro 420 425 430 acc cgt aga cgg tgg cgc
gaa gtg gaa gag tgt gcg gga acc aag gag 1463 Thr Arg Arg Arg Trp
Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu 435 440 445 ctg cta tgt
tct atg atg tgc ctg aag aaa cag gac ctg tac aac aag 1511 Leu Leu
Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys 450 455 460
ttc aag gga cgc gtg cgg acg gtt tct ccc agc tcc aag tcc ccc tgg
1559 Phe Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro
Trp 465 470 475 gtg gag tcc gaa tac ctg gat tac ctt ttt gaa gtg gag
ccg gcc cca 1607 Val Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val
Glu Pro Ala Pro 480 485 490 495 cct gtc ctg gtg ctc acc cag acg gag
gag atc ctg agt gcc aat gcc 1655 Pro Val Leu Val Leu Thr Gln Thr
Glu Glu Ile Leu Ser Ala Asn Ala 500 505 510 acg tac cag ctg ccc ccc
tgc atg ccc cca ctg gat ctg aag tat gag 1703 Thr Tyr Gln Leu Pro
Pro Cys Met Pro Pro Leu Asp Leu Lys Tyr Glu 515 520 525 gtg gca ttc
tgg aag gag ggg gcc gga aac aag acc cta ttt cca gtc 1751 Val Ala
Phe Trp Lys Glu Gly Ala Gly Asn Lys Thr Leu Phe Pro Val 530 535 540
act ccc cat ggc cag cca gtc cag atc act ctc cag cca gct gcc agc
1799 Thr Pro His Gly Gln Pro Val Gln Ile Thr Leu Gln Pro Ala Ala
Ser 545 550 555 gaa cac cac tgc ctc agt gcc aga acc atc tac acg ttc
agt gtc ccg 1847 Glu His His Cys Leu Ser Ala Arg Thr Ile Tyr Thr
Phe Ser Val Pro 560 565 570 575 aaa tac agc aag ttc tct aag ccc acc
tgc ttc ttg ctg gag gtc cca 1895 Lys Tyr Ser Lys Phe Ser Lys Pro
Thr Cys Phe Leu Leu Glu Val Pro 580 585 590 gaa gcc aac tgg tgt ttt
ggc gga tga 1922 Glu Ala Asn Trp Cys Phe Gly Gly * 595 33 599 PRT
Artificial Sequence MBP-human zcytoR19 fusion protein polypeptide
sequence 33 Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly
Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe
Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp
Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp
Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly
Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys
Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val
Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115
120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys
Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe
Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe
Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val
Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp
Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser
Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr
Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235
240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala
Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr
Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys
Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu
Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala
Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala
Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser
Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360
365 Ser Ser Ser His His His His His His Ala Asn Ser Val Pro Leu Val
370 375 380 Pro Arg Gly Ser Arg Pro Arg Leu Ala Pro Pro Gln Asn Val
Thr Leu 385 390 395 400 Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr Trp
Leu Pro Gly Leu Gly 405 410 415 Asn Pro Gln Asp Val Thr Tyr Phe Val
Ala Tyr Gln Ser Ser Pro Thr 420 425 430 Arg Arg Arg Trp Arg Glu Val
Glu Glu Cys Ala Gly Thr Lys Glu Leu 435 440 445 Leu Cys Ser Met Met
Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 450 455 460 Lys Gly Arg
Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 465 470 475 480
Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 485
490 495 Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala
Thr 500 505 510 Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu Lys
Tyr Glu Val 515 520 525 Ala Phe Trp Lys Glu Gly Ala Gly Asn Lys Thr
Leu Phe Pro Val Thr 530 535 540 Pro His Gly Gln Pro Val Gln Ile Thr
Leu Gln Pro Ala Ala Ser Glu 545 550 555 560 His His Cys Leu Ser Ala
Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys 565 570 575 Tyr Ser Lys Phe
Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu 580 585 590 Ala Asn
Trp Cys Phe Gly Gly 595 34 20 PRT Homo sapiens VARIANT (1)...(20)
Xaa = Any Amino Acid 34 Ser Arg Pro Arg Leu Ala Pro Pro Gln Xaa Val
Thr Leu Leu Ser Gln 1 5 10 15 Asn Phe Ser Val 20 35 24 DNA
Artificial Sequence Oligonucleotide primer ZC40285 35 gccccagcca
cccaacagac aaga 24 36 24 DNA Artificial Sequence Oligonucleotide
primer ZC40286 36 ccaggtggcc caggaggaga ggtt 24 37 24 DNA
Artificial Sequence Oligonucleotide primer ZC39128 37 ggcatggaag
ataatgaaag gaaa 24 38 24 DNA Artificial Sequence Oligonucleotide
primer ZC39129 38 gccgtcactc ccaactgggg atgt 24 39 25 DNA
Artificial Sequence Oligonucleotide primer ZC40784 39 ggatagtgtt
ttgagtttct gtgga 25 40 25 DNA Artificial Sequence Oligonucleotide
primer ZC40785 40 accaggagtt caaggttaac cttgg 25 41 24 DNA
Artificial Sequence Oligonucleotide primer ZC40786 41 gggaattcct
gcagaaactc agta 24 42 24 DNA Artificial Sequence Oligonucleotide
primer ZC40787 42 cccttcctgc tcctttgact gcgt 24 43 24 DNA
Artificial Sequence Oligonucleotide primer ZC39408 43 gcccagctgc
atcttcctag aggc 24 44 25 DNA Artificial Sequence Oligonucleotide
primer ZC39409 44 gggcattgcc aggacagctc ttttg 25 45 121 DNA
Artificial Sequence forward zcytor19 knockout oligonucleotide 45
cacctgccgc ccaggggcct tgcggcgggc ggcggggacc ccagggaccg aaggccatag
60 cggccggccc ctaggatccg aattctagaa gctttgtgtc tcaaaatctc
tgatgttaca 120 t 121 46 125 DNA Artificial Sequence reverse
zcytor19 knockout oligonucleotide 46 ggctggtccc ctgcaagagt
agcaagcgct tcttcagcat ccggacttac ggcctcgctg 60 gccggcgcgc
ctaggaattc tctagaggat ccaagctttt agaaaaactc atcgagcatc 120 aaatg
125 47 22 DNA Artificial Sequence Oligonucleotide primer ZC38481 47
cctccttcca gaatgccacc tc 22 48 25 DNA Artificial Sequence
Oligonucleotide primer ZC38626 48 ctgctatgtt ctatgatgtg cctga 25 49
22 DNA Artificial Sequence Oligonucleotide primer ZC38706 49
ggaagataat gaaaggaaac cc 22 50 21 DNA Artificial Sequence
Oligonucleotide primer ZC38711 50 tatgaggagt cccctgtgct g 21
* * * * *
References