U.S. patent application number 11/539789 was filed with the patent office on 2007-11-15 for polynucleotides encoding cytokine receptor.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Francis J. Grant, Wayne R. Kindsvogel, Kevin M. Klucher, Julia E. Novak, SCOTT R. PRESNELL, Theodore E. Whitmore, Wenfeng Xu.
Application Number | 20070264685 11/539789 |
Document ID | / |
Family ID | 29251089 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264685 |
Kind Code |
A1 |
PRESNELL; SCOTT R. ; et
al. |
November 15, 2007 |
POLYNUCLEOTIDES ENCODING CYTOKINE RECEPTOR
Abstract
Novel methods are disclosed for forming a heterodimeric receptor
complex with IL-28R and CRF2-4. The methods may be used for
detecting and treating viral infections in in vitro and in vivo.
Ligand-binding receptor polypeptides can also be used to block
ligand activity in vitro and in vivo. The present invention also
includes methods for producing the protein, uses therefor and
antibodies thereto.
Inventors: |
PRESNELL; SCOTT R.; (Tacoma,
WA) ; Xu; Wenfeng; (Seattle, WA) ; Novak;
Julia E.; (Suquamish, WA) ; Whitmore; Theodore
E.; (Redmond, WA) ; Grant; Francis J.;
(Seattle, WA) ; Kindsvogel; Wayne R.; (Seattle,
WA) ; Klucher; Kevin M.; (Bellevue, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
29251089 |
Appl. No.: |
11/539789 |
Filed: |
October 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10420034 |
Apr 18, 2003 |
|
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11539789 |
Oct 9, 2006 |
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60373813 |
Apr 19, 2002 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/7155
20130101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C07K 14/705 20060101
C07K014/705; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06 |
Claims
1. An isolated polynucleotide that encodes a soluble receptor
polypeptide comprising a sequence of amino acid residues that is at
least 90% identical to the amino acid sequence selected from: (a)
amino acid residue 21 to amino acid residue 163 as shown in SEQ ID
NO:21; (b) amino acid residue 1 to amino acid residue 163 as shown
in SEQ ID NO:21; (c) amino acid residue 21 to amino acid residue
211 as shown in SEQ ID NO:21, (d) amino acid residue 1 to amino
acid residue 211 as shown in SEQ ID NO:21; (e) amino acid residue
21 to amino acid residue 226 as shown in SEQ ID NO: 2; and (f)
amino acid residue 21 to amino acid residue 226 as shown in SEQ ID
NO: 19.
2. The polynucleotide of claim 1, wherein the soluble receptor
polypeptide encoded by the polynucleotide sequence binds a ligand
comprising a polypeptide selected from the group consisting of SEQ
ID NO:52; SEQ ID NO:55; SEQ ID NO:60; and SEQ ID NO:62.
3. The isolated polynucleotide according to claim 1, wherein the
soluble receptor polypeptide encoded by the polynucleotide forms a
heterodimeric receptor complex with the subunit as shown in SEQ ID
NO: 64.
4. The isolated polynucleotide according to claim 1, wherein the
soluble receptor polypeptide comprises a sequence of amino acid
residues selected from: (a) amino acid residue 21 to amino acid
residue 163 as shown in SEQ ID NO:21; (b) amino acid residue 1 to
amino acid residue 163 as shown in SEQ ID NO:21; (c) amino acid
residue 21 to amino acid residue 211 as shown in SEQ ID NO:21, (d)
amino acid residue 1 to amino acid residue 211 as shown in SEQ ID
NO:21; (e) amino acid residue 21 to amino acid residue 226 as shown
in SEQ ID NO: 2; and (f) amino acid residue 21 to amino acid
residue 226 as shown in SEQ ID NO: 19.
5. The polynucleotide of claim 4, wherein the soluble receptor
polypeptide encoded by the polynucleotide sequence binds a ligand
comprising a polypeptide selected from the group consisting of of
SEQ ID NO:52; SEQ ID NO:55; SEQ ID NO:60; and SEQ ID NO:62.
6. The isolated polynucleotide according to claim 4, wherein the
soluble receptor polypeptide encoded by the polynucleotide forms a
heterodimeric receptor complex with the subunit as shown in SEQ ID
NO: 64.
7. The isolated polynucleotide according to claim 4, wherein the
soluble receptor polypeptide further comprises an affinity tag.
8. An expression vector comprising the following operably linked
elements: (a) a transcription promoter; a first DNA segment
encoding the soluble receptor polypeptide of claim 1; and a
transcription terminator; and (b) a second transcription promoter;
a second DNA segment encoding a soluble CRF2-4 polypeptide as shown
in SEQ ID NO:64; and a transcription terminator; and wherein the
first and second DNA segments are contained within a single
expression vector or are contained within independent expression
vectors.
9. The expression vector according to claim 8, further comprising a
secretory signal sequence operably linked to the first and second
DNA segments.
10. A cultured cell comprising the expression vector according to
claim 9, wherein the cell expresses the polypeptides encoded by the
DNA segments.
11. A cultured cell comprising the expression vector according to
claim 9, wherein the first and second DNA segments are located on
independent expression vectors and are co-transfected into the
cell, and cell expresses the polypeptides encoded by the DNA
segments.
12. A method of producing polypeptides that form a heterodimeric
comprising: culturing a cell according to claim 11; and isolating
the polypeptides produced by the cell.
13. An isolated polynucleotide that encodes a first subunit of a
soluble heterodimeric receptor polypeptide wherein the first
subunit comprises a polypeptide comprising the sequence of amino
acid residues that is at least 90% identical to the amino acid
sequence selected from: (a) amino acid residue 21 to amino acid
residue 163 as shown in SEQ ID NO:21; (b) amino acid residue 1 to
amino acid residue 163 as shown in SEQ ID NO:21; (c) amino acid
residue 21 to amino acid residue 211 as shown in SEQ ID NO:21, (d)
amino acid residue 1 to amino acid residue 211 as shown in SEQ ID
NO:21; (e) amino acid residue 21 to amino acid residue 226 as shown
in SEQ ID NO: 2; and (f) amino acid residue 21 to amino acid
residue 226 as shown in SEQ ID NO: 19.
14. The isolated polynucleotide according to claim 13, wherein the
first subunit forms a heterodimeric receptor with a second subunit
comprising the amino acid sequence as shown in SEQ ID NO:64.
15. The isolated polynucleotide according to claim 14, wherein the
heterodimeric receptor specifically binds a ligand comprising a
polypeptide selected from the group consisting of SEQ ID NO:52; SEQ
ID NO:55; SEQ ID NO:60; and SEQ ID NO:62.
16. An isolated polynucleotide that encodes a first subunit of a
soluble heterodimeric receptor polypeptide wherein the first
subunit comprises a polypeptide comprising the sequence of amino
acid residues that is at least 90% identical to the amino acid
sequence selected from: (a) amino acid residue 21 to amino acid
residue 249 as shown in SEQ ID NO: 2; (b) amino acid residue 21 to
amino acid residue 491 as shown in SEQ ID NO: 2; (c) amino acid
residue 21 to amino acid residue 249 as shown in SEQ ID NO: 19; and
(d) amino acid residue 21 to amino acid residue 520 as shown in SEQ
ID NO: 19.
17. The isolated polynucleotide according to claim 16, wherein the
first subunit forms a heterodimeric receptor with a second subunit
comprising the amino acid sequence as shown in SEQ ID NO:64.
18. The isolated polynucleotide according to claim 17, wherein the
heterodimeric receptor specifically binds a ligand comprising a
polypeptide selected from the group consisting of SEQ ID NO:52; SEQ
ID NO:55; SEQ ID NO:60; and SEQ ID NO:62.
19. The isolated polynucleotide according to claim 18, wherein the
binding of the heterodimeric receptor to the ligand induces signal
transduction.
20. An isolated polynucleotide encoding a first subunit of a
heterodimeric receptor comprising wherein the first subunit
comprises a polypeptide selected from: (a) a polypeptide comprising
the amino acid sequence as shown in SEQ ID NO:21 from amino acid
residue 21 to amino acid residue 163; (b) a polypeptide comprising
the amino acid sequence as shown in SEQ ID NO:2 from amino acid
residue 21 to amino acid residue 226; (c) a polypeptide comprising
to the amino acid sequence as shown in SEQ ID NO: 19 from amino
acid residue 21 to amino acid residue 226 wherein the heterodimeric
receptor further comprises an affinity tag.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/420,034, filed Apr. 18, 2003, which is herein incorporated
by reference, and which claims the benefit under 35 U.S.C.
.sctn.119(e)(1) of U.S. Provisional Application Ser. No. 60/373,813
filed Apr. 19, 2002.
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. These and other aspects of the invention
will become evident upon reference to the following detailed
description of the invention.
DESCRIPTION OF THE INVENTION
[0006] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0007] 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.).
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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'.
[0012] 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).
[0013] 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.
[0014] 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).
[0015] 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.
[0016] 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.
[0017] 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.
[0018] "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.
[0019] 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.
[0020] 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".
[0021] "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.
[0022] 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.
[0023] 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.
[0024] 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. 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
.alpha..beta. isoform), and multimers having component subunits
with disparate functions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
and GM-CSF receptors). Some receptor subunits are common to a
plurality of receptors. For example, the AIC2B subunit, which
cannot bind ligand on its own but includes an intracellular signal
transduction domain, is a component of IL-3 and GM-CSF receptors.
Many cytokine receptors can be placed into one of four related
families on the basis of the structure and function. Hematopoietic
receptors, for example, are characterized by the presence of a
domain containing conserved cysteine residues and the WSXWS motif
(SEQ ID NO: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).
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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%.
[0030] All references cited herein are incorporated by reference in
their entirety.
[0031] 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.
[0032] 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.
[0033] 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 Nos. 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.
[0034] 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. Due to its ability to bind IL28A,
IL28B, and IL29 ligands, the Zcytor19 receptor (ZcytoR19) has been
designated IL29RA.
[0035] 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.
[0036] In addition, 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 polypeptide (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.
[0037] A truncated soluble form of the zcytor19 receptor mRNA
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 polypeptide (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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] Analysis of the zcytor19 sequence has revealed 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. 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. Zcytor19 has been
shown to form a heterodimer with CRF2-4.
[0042] CRF2-4 has also been shown to be a binding partner with
zcytor11 (IL-22R) to bind the IL-10, and the binding partner for
zcytor11 to bind 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; 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). Receptors in this
subfamily may also associate to form heterodimers that transduce a
signal. 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.
[0043] 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.
[0044] The receptors of the present invention have been shown to
form complexes with a genus of polynucleotide and polypeptide
molecules that have functional and structural similarity to the
interferons. In this new family, which includes molecules
designated zcyto20 (SEQ ID NOS: 51 and 52), zcyto21 (SEQ ID NOS: 54
and 55), zcyto22 (SEQ ID NOS: 56 and 57), zcyto24 (SEQ ID NOS: 59
and 60), zcyto25 (SEQ ID NOS: 61 and 62), zcyto20, 21, and 22 are
human sequences and zcyto24 and 25 are mouse sequences.
Furthermore, certain biological activities have been shown to be
exhibited by each molecule in the family. These activities include,
for example, antiviral activities and increasing circulating
myeloid cell levels. While not wanting to be bound by theory, these
molecules appear to all signal through zcytor19 receptor via the
same pathway.
[0045] Homology within the ligand family at the nucleotide and
amino acid levels is shown in Table 1, ranging from approximately
72% to 98% at the nucleotide level, and 51% to 97% at the amino
acid level. TABLE-US-00001 TABLE 1 nucleotide sequence identity
zcyto20 zcyto22 zcyto21 zcyto24 zcyto25 rat protein zcyto20 100
98.2 72.9 74.0 72.1 73.4 se- zcyto22 96.0 100 73.0 73.9 71.9 72.9
quence zcyto21 66.5 67.5 100 64.9 62.9 64.6 iden- zcyto24 62.7 63.7
51.7 100 97.2 90.3 tity zcyto25 59.8 60.8 48.8 93.6 100 88.4
[0046] Table 2 is an illustration of the sequence identity between
zcyto20, zcyto21, zcyto22, IFN.alpha., IFN.beta., IFN.gamma., and
IL10 at the amino acid level. TABLE-US-00002 TABLE 2 amino acid
sequence identity Zcyto20 Zcyto22 Zcyto21 IFN.quadrature.
IFN.quadrature. IFN.quadrature. IL10 Zcyto20 100 Zcyto21 81 100
Zcyto22 96 74 100 IFN.quadrature. 17 16 17 100 IFN.quadrature. 14
13 14 31 100 IFN.quadrature. 4 4 4 7 5 100 IL10 13 12 14 7 5 8
100
[0047] Zcyto20 gene encodes a polypeptide of 205 amino acids, as
shown in SEQ ID NO:52. The signal sequence for Zcyto20 can be
predicted as comprising amino acid residue 1 (Met) through amino
acid residue 21 (Ala) of SEQ ID NO: 52. The mature peptide for
Zcyto20 begins at amino acid residue 22 (Val).
[0048] Zcyto21 gene encodes a polypeptide of 200 amino acids, as
shown in SEQ ID NO:55. The signal sequence for Zcyto21 can be
predicted as comprising amino acid residue 1 (Met) through amino
acid residue 19 (Ala) of SEQ ID NO: 55. The mature peptide for
Zcyto21 begins at amino acid residue 20 (Gly). Zcyto21 has been
described in PCT application WO 02/02627.
[0049] Zcyto22 gene encodes a polypeptide of 205 amino acids, as
shown in SEQ ID NO:57. The signal sequence for Zcyto22 can be
predicted as comprising amino acid residue 1 (Met) through amino
acid residue 21 (Ala) of SEQ ID NO: 57. The mature peptide for
Zcyto22 begins at amino acid residue 22 (Val).
[0050] Zcyto24 gene encodes a polypeptide of 202 amino acids, as
shown in SEQ ID NO:60. Zcyto24 secretory signal sequence comprises
amino acid residue 1 (Met) through amino acid residue 28 (Ala) of
SEQ ID NO:60. An alternative site for cleavage of the secretory
signal sequence can be found at amino acid residue 24 (Thr). The
mature polypeptide comprises amino acid residue 29 (Asp) to amino
acid residue 202 (Val).
[0051] Zcyto25 gene encodes a polypeptide of 202 amino acids, as
shown in SEQ ID NO:62. Zcyto25 secretory signal sequence comprises
amino acid residue 1 (Met) through amino acid residue 28 (Ala) of
SEQ ID NO:62. An alternative site for cleavage of the secretory
signal sequence can be found at amino acid residue 24 (Thr). The
mature polypeptide comprises amino acid residue 29 (Asp) to amino
acid residue 202 (Val).
[0052] Evidence that CRF2-4 (SEQ ID NOS: 63 and 64) is the putative
signaling partner for zcytor19 provides further support that the
receptor plays an important role in the immunomodulatory system,
affecting physiologies such as the innate immune system and the
inflammatory response system.
[0053] Localizing the expression of a receptor for a
ligand/receptor pair may have significance for identifying the
target cell or tissue at which the ligand acts. This is
particularly useful when the receptor/ligand complex involves a
heterodimeric receptor in which one of the subunits is expressed
widely and another of the subunits is expressed in a limited
manner, either spatially or temporally restricted. Using in situ
hybridization expression of zcytor19 has been identified in a skin
carcinoma sample, where the cancerous granular epithelium was
strongly positive, while no positive signal is observed in normal
skin. Other tissues identified as expressing zcytor19 included
fetal liver, where signal was observed in a mixed population of
mononuclear cells in sinusoid spaces; in lung expression was
observed in type II alveolar epithelium; and in macrophage-like
mononuclear cells in the interstitial tissue. Northern analysis of
zcytor19 identified expression of a .about.4.5 kb transcript which
was in greatest in heart, skeletal muscle, pancreas, and prostate
tissue, in addition to in the Burkitt's lymphoma (RAJI) cell line
and SW-480 colorectal carcinoma cell line.
[0054] 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.
[0055] 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 3 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-00003 TABLE 3 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
[0056] The degenerate codons used in SEQ ID NOs:3, 28, 29, 53, and
58 encompassing all possible codons for a given amino acid, are set
forth in Table 4. TABLE-US-00004 TABLE 4 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
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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).
[0061] 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.
[0062] 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. An alternative way to prepare a
full-length gene is to synthesize a specified set of overlapping
oligonucleotides (40 to 100 nucleotides). 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.
[0063] 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.
[0064] 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.
[0065] 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 5 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: Total .times. .times. number .times. .times.
of .times. .times. identical .times. .times. matches [ length
.times. .times. of .times. .times. the .times. .times. longer
.times. .times. sequence .times. .times. plus .times. .times. the
number .times. .times. of .times. .times. gaps .times. .times.
introduced .times. .times. into .times. .times. the .times. .times.
longer sequence .times. .times. in .times. .times. order .times.
.times. to .times. .times. align .times. .times. the .times.
.times. two .times. .times. sequences ] .times. 100 ##EQU1##
TABLE-US-00005 TABLE 5 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
[0066] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0067] 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).
[0068] 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).
[0069] 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.
[0070] 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).
[0071] 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 6) 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-00006 TABLE 6
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
[0072] 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.
[0073] 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).
[0074] 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.
[0075] 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.
[0076] Determination of amino acid residues that are within regions
or domains that are critical to maintaining structural integrity
can be determined. Within these regions one can determine specific
residues that will be more or less tolerant of change and maintain
the overall tertiary structure of the molecule. Methods for
analyzing sequence structure include, but are not limited to,
alignment of multiple sequences with high amino acid or nucleotide
identity and computer analysis using available software (e.g., the
Insight II.RTM. 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.
[0077] 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).
[0078] 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.).
[0079] 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.
[0080] 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.
[0081] 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).
[0082] 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.
[0083] 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.
[0084] Standard methods for identifying functional domains are
well-known to those of skill in the art. For example, studies on
the truncation at either or both termini of interferons have been
summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507
(1995). Moreover, standard techniques for functional analysis of
proteins are described by, for example, Treuter et al., Molec. Gen.
Genet. 240:113 (1993); Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2-5A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987); Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985); Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995); and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0085] 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).
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).
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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).
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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, pFastBac1.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).
[0098] The recombinant virus is used to infect host cells,
typically a cell line derived from the fall armyworm, Spodoptera
frugiperda. See, in general, Glick and Pasternak, Molecular
Biotechnology: Principles and Applications of Recombinant DNA, ASM
Press, Washington, D.C., 1994. Another suitable cell line is the
High FiveO.TM. cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
are used to grow and maintain the cells. Suitable media are Sf900
II.TM. (Life Technologies) or ESF 921.TM. (Expression Systems) for
the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences, Lenexa, Kans.)
or Express FiveO.TM. (Life Technologies) for the T. ni cells.
Procedures used are generally described in available laboratory
manuals (King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et
al., ibid.; Richardson, C. D., ibid.). Subsequent purification of
the zcytor19 polypeptide from the supernatant can be achieved using
methods described herein.
[0099] 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. 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.
[0100] 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.).
[0101] 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).
[0102] 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.
[0103] 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.
[0104] 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.
[0105] A secretion trap method employing zcytor19 soluble receptor
polypeptide was used to isolate a zcytor19 ligand (Aldrich, et al,
Cell 87: 1161-1169, 1996), as explained in the Examples. Other
methods for identifying natural ligand for zcytor19 include
mutagenizing a cytokine-dependent cell line expressing zcytor19 and
culturing it under conditions that select for autocrine growth. See
WIPO publication WO 95/21930.
[0106] 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. 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 mp1 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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).
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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).
[0119] 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.
[0120] 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, Calif.--A Cancer
Journal for Clinicians 44:43 (1994).
[0121] 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.
[0122] 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-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-19, IL-20, IL-21, IL-22, IL-28A, IL-28B, and IL-29),
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-.hoarfrost., TNF-.hoarfrost., and the like.
[0123] 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-.alpha., are believed to produce cytotoxic effects
by inducing DNA fragmentation.
[0124] 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. Other suitable toxins
are known to those of skill in the art.
[0125] 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).
[0126] 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. Nat'l 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).
[0127] 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.193MPt,
.sup.197Hg, and the like.
[0128] 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.
[0129] 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.
[0130] 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).
[0131] The approaches described above can also be used to prepare
multispecific antibody compositions that comprise an
immunoconjugate.
[0132] Antibodies disclosed herein include antibodies that bind the
zcytor19/CRF2-4 heterodimeric complex, including the heterodimeric
soluble receptor.
[0133] Anti-zcytor19 antibodies, and multispecific antibody
compositions can be used to modulate the immune system by
preventing the binding of zcytor19 ligands (for example, zcyto20,
zcyto21, zcyto22, zcyto24, and zcyto25) 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.
[0134] 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.
[0135] 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).
[0136] 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.
[0137] 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.
[0138] 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.
[0139] Additionally, the invention provides methods employing
anti-zcytor19 antibodies, or multispecific antibody compositions,
for selectively blocking or neutralizing the viral infection
associated with the liver. As shown in Example 24, while normal and
diseased liver specimens show expression of zcytoR19 mRNA, there is
specific expression of the receptor in liver specimens that are
positive for Hepatitis Virus C, and Hepatitis B.
[0140] When liver disease is inflammatory and continuing for at
least six months, it is generally considered chronic hepatitis.
Hepatitis C virus (HCV) patients actively infected will be positive
for HCV-RNA in their blood, which is detectable by reverse
transcritptase/polymerase chain reaction (RT-PCR) assays. The
methods of the present invention will slow the progression of the
liver disease, and can be measured, for example, as improved serum
alanine transaminase (ALT) levels, improved levels of aspartate
aminotrasnferase (AST), decreased portal inflammation as determined
by biopsy, or decrease in hepatocytic necrosis. Histological
improvement can be measured using the Histological Activity Index
(Davis et al., New Eng. J. Of Med. 321:1501-1506, 1989; Knodell et
al., Hepatology 1:431-435, 1981). Other means for measuring
improvement are known in the art, and will be determined by the
clinician, and can include, for example, evaluation of HCV
antibodies (Kuo, et al. Science, 244:362-364, 1989).
[0141] The present invention also provides methods for treatment of
renal or urological neoplasms, multiple myelomas, lymphomas,
leukemias, light chain neuropathy, or amyloidosis.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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).
[0149] 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.
[0150] 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)).
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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)).
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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 zcytor19 receptor 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.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] In an alternative approach, a receptor extracellular domain
can be expressed as a fusion with immunoglobulin heavy chain
constant regions, typically an Fc 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.
[0166] 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.
[0167] 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 antibodies, or peptide
binding fragments can be used to detect zcytor19 receptor
expressing cancers. In a preferred embodiment zcytor19
polynucleotides, polypeptides, anti-zcytor19 antibodies, or peptide
binding fragments can be used to detect leukemias, more preferably
B-cell leukemias, and most preferably pre-B-cell acute
lymphoblastic leukemia.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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).
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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).
[0181] 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. 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, N.Y., 1989; and Hurrell, J. G.
R., Ed., Monoclonal Hybridoma Antibodies: Techniques and
Applications, CRC Press, Inc., Boca Raton, Fla., 1982.
[0182] 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.
[0183] 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. Antibodies in the present
invention include, but are not limited to, antibodies that bind the
zcytor19/CRF2-4 heterodimer, as well as the heterodimeric soluble
receptor complex.
[0184] 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). 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.
[0185] 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.-1 or greater, preferably 10.sup.7 M.sup.-1 or
greater, more preferably 10.sup.8 M.sup.-1 or greater, and most
preferably 10.sup.9 M.sup.-1 or greater. The binding affinity of an
antibody can be readily determined by one of ordinary skill in the
art, for example, by Scatchard analysis (Scatchard, G., Ann. NY
Acad. Sci. 51: 660-672, 1949).
[0186] 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. 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.
[0187] 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.
[0188] 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. 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.
[0189] 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.
[0190] 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.
[0191] 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.
Similarly, in another embodiment, zcytor19 binding
polypeptide-cytokine or antibody-cytokine fusion proteins can be
used for enhancing in vivo killing of target tissues, if the
binding polypeptide-cytokine or anti-zcytor19 antibody targets the
hyperproliferative cell (See, generally, Hornick 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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).
[0198] 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.
[0199] 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 wherein aberrations are prevalent in neuroblastoma, one of
skill in the art would appreciate 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.):A19, 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, wherein 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 polynucleotides of the present invention have use
in detecting such aberrations that are clearly associated with
human disease, as described herein.
[0200] 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 preferably
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.
[0201] 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 (SCID) (Noguchi, M et al., Cell 73:
147-157, 1993), c-Mp1 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.
[0202] 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)).
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Identification and Isolation of Full-Length Human Zcytor19 cDNA
[0208] 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 truncated 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
[0209] 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.
[0210] 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
[0211] 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.
[0212] 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.
[0213] 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-00007 TABLE 7 Tissue/Cell line #samples Adrenal gland 1
Bladder 1 Bone Marrow 1 Brain 1 Cervix 1 Colon 1 Fetal brain 1
Fetal heart 1 Fetal kidney 1 Fetal liver 1 Fetal lung 1 Fetal
muscle 1 Fetal skin 1 Heart 2 K562 (ATCC # CCL-243) 1 Kidney 1
Liver 1 Lung 1 Lymph node 1 Melanoma 1 Pancreas 1 Pituitary 1
Placenta 1 Prostate 1 Rectum 1 Salivary Gland 1 Skeletal muscle 1
Small intestine 1 Spinal cord 1 Spleen 1 Stomach 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 Bone marrow 3 Fetal brain 3 Islet 2 Prostate
3 RPMI #1788 (ATCC # CCL-156) 2 Testis 4 Thyroid 2 WI38 (ATCC #
CCL-75 2 ARIP (ATCC # CRL-1674 - rat) 1 HaCat - human keratinocytes
1 HPV (ATCC # CRL-2221) 1 Adrenal gland 1 Prostate SM 2 CD3+
selected PBMC's Ionomycin + 1 PMA stimulated HPVS (ATCC # CRL-2221)
- selected 1 Heart 1 Pituitary 1 Placenta 2 Salivary gland 1 HL60
(ATCC # CCL-240) 3 Platelet 1 HBL-100 1 Renal mesangial 1 T-cell 1
Neutrophil 1 MPC 1 Hut-102 (ATCC # TIB-162) 1 Endothelial 1 HepG2
(ATCC # HB-8065) 1 Fibroblast 1 E. Histo 1
Example 3
PCR-Based Chromosomal Mapping of the Zcytor19 Gene
[0214] 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.
[0215] 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
[0216] 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).
[0217] A zcytor19 DNA fragment comprising a 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.
[0218] 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.
[0219] 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 FLAG.RTM. 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
[0220] 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.
[0221] A 630 base pair fragment was generated by PCR, containing
the extracellular domain of human zcytor19 with BamHI 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.
[0222] 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
[0223] 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 15ml 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.
[0224] 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
[0225] 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 (SEQ ID NO:64),
DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511) soluble
receptors. Receptors in this subfamily may associate to form
heterodimers 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 zcyto20 (SEQ ID NO:52),
zcyto21 (SEQ ID NO:55), zcyto22 (SEQ ID NO:57), zcyto24 (SEQ ID
NO:60), zcyto25 (SEQ ID NO:62), and ligands 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.
[0226] For initial soluble receptor binding characterization, the
cytokines mentioned above, 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-CRF2-4, or 400 ng/ml of e.g., Ru--CRF2-4 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
[0227] 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
(IgG.gamma.1) (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, CRF2-4) 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 .kappa. Light Chain Fusion
Vectors
[0228] The heavy chain of IgG.gamma.1 (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.
[0229] The human .kappa. 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 K .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
[0230] 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.
[0231] A separate construct having a heterodimeric cytokine
receptor subunit extracellular domain, i.e., CRF2-4 (SEQ ID NO: 64)
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 .kappa. light chain
vector described above. The resulting vector is sequenced to
confirm that the cytokine receptor subunit/human .kappa. light
chain fusion is correct.
D. Co-Expression of the Zcytor19 and Heterodimeric Cytokine
Receptor Subunit Extracellular Domain
[0232] 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.
[0233] 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
[0234] 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.
[0235] 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.
[0236] As an alternative, a similar assay can be performed wherein
the a Baf3/zcytor19 cell line is co-transfected as described herein
and proliferation is measured, using a known assay such as a
standard Alamar Blue proliferation assay.
Example 9
[0237] A: COS Cell Transfection and Secretion Trap
[0238] Biotinylated zcyto21 (SEQ ID NO:55) was tested for binding
to known or orphan cytokine receptors. The pZP7 expression vectors
containing cDNAs of cytokine receptors (including human
IFN.alpha.R1, IFN.beta.R1, IFN.alpha.R2, IFN.beta.R2, IL-10R,
CRF2-4, ZcytoR7, DIRS1, Zcytor19, and Tissue Factor) were
transfected into COS cells, and the binding of biotinylated zcyto20
to transfected COS cells was carried out using the secretion trap
assay described below. Positive binding in this assay showed
receptor-ligand pairs.
[0239] COS Cell Transfections
[0240] The COS cell transfections were performed as follows: COS
cells were plated (1.times.10.sup.5 cells/well) on fibronectin
coated, 12-well, tissue culture plates (Becton Dickinson, Bedford,
Mass.) and incubated at 37.degree. C. overnight. Cytokine receptor
DNA (0.75 .mu.g) was mixed with 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), then
mixed with 5 .mu.l Lipofectamine.TM. (Invitrogen, Carlsbad, Calif.)
in 45 .mu.l serum free DMEM media, and incubated at room
temperature for 30 minutes. An additional 400 .mu.l serum free DMEM
media was added. The cells were rinsed with serum free DMEM, and
500 .mu.l of the DNA mixture was added. The cells were incubated
for 5 hours at 37.degree. C., at which time an additional 500 .mu.l
20% FBS DMEM media (100 ml FBS, 55 mg sodium pyruvate and 146 mg
L-glutamine in 500 ml DMEM) was added and the cells were incubated
overnight.
[0241] Secretion Trap Assay
[0242] The secretion trap was performed as follows: Media was
aspirated and cells were rinsed twice with 1% BSA in PBS. 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, NEN Life
Science Products, Boston, Mass.) in H.sub.2O. The cells were
incubated for 1 hour with 3 .mu.g/ml biotinylated zcyto21 protein
(Example 27) in TNB. Cells were then washed 3 times with 1% BSA in
PBS and were incubated for another hour with 1:300 diluted
Streptavidin-HRP (NEN kit) in TNB. Again cells were washed 3 times
with 1% BSA in PBS, and then fixed for 15 minutes with 1.8%
Formaldehyde in PBS. Cells were then washed 3 times with TNT (0.1M
Tris-HCL, 0.15M NaCl, and 0.05% Tween-20 in H.sub.2O).
[0243] Positive binding was detected with fluorescein tyramide
reagent diluted 1:50 in dilution buffer (NEN kit), 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.
[0244] Positive binding was detected on cells transfected with
human zcytor19 cDNA and incubated with biotinylated zcyto21. None
of the other transfected receptors bound zcyto21, and zcytor19 did
not bind a control biotinylated protein. These data indicate that
zcytor19 is a receptor for zcyto21.
[0245] Further experiments have shown positive binding between both
human and mouse Zcytor19 with biotinylated zcyto21. Positive
binding was also detected on cells transfected with human zcytor19
cDNA and incubated with biotinylated zcyto20, and zcyto24.
Example 10
Expression of Human Zcytor19 in E. coli
A. Construction of Zcytor19-MBP Fusion Expression Vector
pTAP170/Zcytor19
[0246] 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 Sma1 cut recipient vector pTAP170 to
produce the construct encoding the MBP-human zcytor19 fusion, as
described below.
[0247] 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 Pvul cut pRS316, 1 .mu.g linker, and 1 .mu.g Sca1/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.
[0248] 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.
[0249] 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.
[0250] 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% BactoI 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).
[0251] 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.
[0252] 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 2 .mu.l Not1 (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.
[0253] 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 2 L-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
[0254] 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.
[0255] 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
[0256] 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).
[0257] 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.
[0258] 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.
[0259] Because zcytor19 is expressed in these specific tumor
tissues, zcytor19 polynucleotides, polypeptides and antibodies can
be used as a tumor marker as disclosed herein. Moreover, 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
[0260] 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.
[0261] 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 (NH.sub.4).sub.2SO.sub.4, 6.7 mM
MgCl.sub.2
[0262] 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.
[0263] 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.
[0264] 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
[0265] 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).
[0266] 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.
[0267] 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
(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 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.
[0268] 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.
[0269] Oligonucleotides were designed to generate a PCR fragment
containing partial exon 6, complete intron 6 and partial exon 7
sequences.
[0270] 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 gelation, 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
[0271] 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 (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 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. 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 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
[0272] 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.
[0273] 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 HindIII 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.
[0274] 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-MC1 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, MC1 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-MC1 cassette, which is about 5,200 bp in length.
F. Generation of ZCytor19 KO Mice
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] Homozygous animals missing zcytor19 gene are to be
sacrificed for flow cytometric analysis of peripheral blood,
thymus, lymph node, bone marrow, and spleen.
[0281] 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.
[0282] 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 1 mg/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).
[0283] 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
[0284] Specific human tissues were isolated and screened for
zcytor19 expression by in situ hybridization. Various human tissues
prepared, sectioned and subjected to in situ hybridization included
normal and carcinoma colon, cervical carcinoma, endometrial
carcinoma, normal and carcinoma ovary, normal and neoplasmic skin,
fetal liver, lung, heart and MFH (muscle sarcoma). The tissues were
fixed in 10% buffered formalin and blocked in paraffin using
standard techniques. Tissues were sectioned at 4 to 8 microns.
Tissues were prepared using a standard protocol. Briefly, tissue
sections were deparaffinized with Histo-Clear.RTM. (National
Diagnostics, Atlanta, Ga.) and then dehydrated with ethanol. Next
they were digested with Proteinase K (50 .mu.g/ml) (Boehringer
Diagnostics, Indianapolis, Ind.) at 37.degree. C. for 2 to 7
minutes. This step was followed by acetylation and re-hydration of
the tissues.
[0285] One in situ probe was designed against the human zcytor19
(variant x1) sequence (INC7128744, as shown in SEQ ID NO: 25),
containing the 3'UTR of zcytor19 using standard methods. T7 RNA
polymerase was used to generate an antisense probe. The probe was
labeled using an In Vitro transcription System (Riboprobe.RTM. in
vitro Transcription System, Promega, Madison, Wis.) as per
manufacturer's instruction, except that the probes digoxigenin was
used instead of radiolabeled rCTP and that the water was adjusted
to accomodate the reduced volume of the rNTP's. In situ
hybridization was performed with a digoxigenin-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 TSA.TM. (Tyramide Signal
Amplification; PerkinElmer Life Sciences Inc., Boston, Mass.) and
visualized with VECTOR Red substrate kit (Vector Laboratories,
Burlingame, Calif.) as per manufacturer's instructions. The slides
were then counter-stained with hematoxylin.
[0286] Signals were observed in several tissues tested: In colon
carcinoma tissues, weak signal was observed in carcinoma cells and
a few immune infiltrations. However, there was no positive signal
observed in the normal colon and intestine, including cells in
lamina propria, epithelium, immune nodules and peripheral ganglia
nerve cells. In cervical carcinoma tissues, there is weak signal in
carcinoma cells and some cells in the immune nodules. In
endometrial carcinoma tissues, weak signals present in the
carcinoma cells. In normal uterus tissues, no positive signal was
observed. In ovarian carcinoma samples, some carcinoma cells are
weakly positive. In normal ovary samples, some endothelium of
capillaries and epithelium of large follicles may be weakly
positive. In the skin carcinoma sample, the cancerous granular
epithelium is strongly positive, while no positive signal is
observed in the normal skin. In fetal liver, signal is observed in
a mixed population of mononuclear cells in sinusoid spaces. In
lung, zcytor19 appears to be positive in type II alveolar
epithelium. Occasionally bronchial epithelium may also be weakly
positive. Macrophage-like mononuclear cells in the interstitial
tissue are also positive. In heart, myocytes are negative while
some circulating mononuclear cells are positive for zcytor19. In
one of the samples, endothelium of the vessels may be weakly
positive. Other tissues tested including a MFH (muscle sarcoma)
sample and a Kaposi's sarcoma skin sample. There is no conclusive
positive signal in these tissues.
[0287] 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. 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.
[0288] 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.
[0289] 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.).
[0290] 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.
[0291] Because zcytor19 is expressed in these specific tumor
tissues, zcytor19 polynucleotides, polypeptides and antibodies can
be used as a tumor marker as disclosed herein. Moreover, 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
[0292] 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
[0293] BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell
line derived from murine bone marrow (Palacios and Steinmetz, Cell
41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:
4133-4135, 1986), was maintained in complete media (RPMI medium
(JRH Bioscience Inc., Lenexa, Kans.) supplemented with 10%
heat-inactivated fetal calf serum, 2 ng/ml murine IL-3 (mIL-3) (R
& D, Minneapolis, Minn.), 2 mM L-glutaMax-1.TM. (Gibco BRL), 1
mM Sodium Pyruvate (Gibco BRL). 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 .mu.lasmid DNA, or 30 .quadrature.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 lFad/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
[0294] 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.
[0295] 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 .quadrature.l of 10.times. cDNA PCR
Reaction Buffer (Clonetech), 3 .quadrature.l DNA from the RT
reaction, 0.5 .quadrature.l Advantage2 Polymerase (Clonetech), made
to a final volume of 50.quadrature.1 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.
Example 16
[0296] Polyclonal Antibodies:
[0297] Polyclonal antibodies are prepared by immunizing 2 female
New Zealand white rabbits with the purified recombinant protein
huzcytor19/MBP-6H. The rabbits are each given an initial
intraperitoneal (ip) injection of 200 .mu.g of purified protein in
Complete Freund's Adjuvant followed by booster ip injections of 100
.mu.g peptide in Incomplete Freund's Adjuvant every three weeks.
Seven to ten days after the administration of the second booster
injection (3 total injections), the animals are bled and the serum
is collected. The animals are then boosted and bled every three
weeks.
[0298] The huzcyotr19/MBP-6H specific rabbit serum is pre-adsorbed
of anti-MBP antibodies using a CNBr-SEPHAROSE 4B protein column
(Pharmacia LKB, Peapack, N.J.) that is prepared using 10 mg of
purified recombinant MBP per gram of CNBr-SEPHAROSE. The
huzcytor19-specific polyclonal antibodies are affinity purified
from the rabbit serum using a CNBr-SEPHAROSE 4B protein column that
is prepared using 10 mg of the specific antigen purified
recombinant protein huzcytor19/MBP-6H followed by 20.times.
dialysis in PBS overnight. Huzcytor19-specific antibodies are
characterized by ELISA using 500 ng/ml of the purified recombinant
proteins huzcytor19/MBP-6H or huzcytor19-Fc4 as antibody targets.
The lower limit of detection (LLD) of the rabbit
anti-huzcytor19/MBP-6H affinity purified antibody on its specific
purified recombinant antigen huzcytor19/MBP-6H and on purified
recombinant huzcytor19-Fc4 is determined.
Example 17
[0299] Signal Transduction Reporter Assay
[0300] A signal transduction reporter assay can be used to
determine the functional interaction of zcyto20, zcyto21, zcyto22,
zcyto24, and zcyto25 with zcytor19. Human embryonal kidney (HEK)
cells are transfected with a reporter plasmid containing an
interferon-stimulated response element (ISRE) driving transcription
of a luciferase reporter gene in the presence or absence of pZP7
expression vectors containing cDNAs for class II cytokine receptors
(including human DIRS1, IFNaR1, IFNaR2 and Zcytor19 (SEQ ID
NO:23)). Luciferase activity following stimulation of transfected
cells with class II ligands (including zcyto20 (SEQ ID NO:52),
zcyto21 (SEQ ID NO:55), zcyto22 (SEQ ID NO:57), zcyto10, huIL10 and
huIFNa-2a) reflects the interaction of the ligand with transfected
and native cytokine receptors on the cell surface. The results and
methods are described below.
[0301] Cell Transfections
[0302] 293 HEK cells were transfected as follows: 700,000 293
cells/well (6 well plates) were plated approximately 18 h prior to
transfection in 2 milliliters DMEM+10% fetal bovine serum. Per
well, 1 microgram pISRE-Luciferase DNA (Stratagene), 1 microgram
cytokine receptor DNA and 1 microgram pIRES2-EGFP DNA (Clontech,)
were added to 9 microliters Fugene 6 reagent (Roche Biochemicals)
in a total of 100 microliters DMEM. Two micrograms pIRES2-EGFP DNA
was used when cytokine receptor DNA was not included. This
transfection mix was added 30 minutes later to the pre-plated 293
cells. Twenty-four hours later the transfected cells were removed
from the plate using trypsin-EDTA and replated at approximately
25,000 cells/well in 96 well microtiter plates. Approximately 18 h
prior to ligand stimulation, media was changed to DMEM+0.5%
FBS.
[0303] Signal Transduction Reporter Assays
[0304] The signal transduction reporter assays were done as
follows: Following an 18 h incubation at 37.degree. C. in DMEM+0.5%
FBS, transfected cells were stimulated with dilutions (in DMEM+0.5%
FBS) of the following class II ligands; zcyto20, zcyto21, zcyto22,
zcyto10, huIL10 and huIFNa-2a. Following a 4-hour incubation at
37.degree. C., the cells were lysed, and the relative light units
(RLU) were measured on a luminometer after addition of a luciferase
substrate. The results obtained are shown as the fold induction of
the RLU of the experimental samples over the medium alone control
(RLU of experimental samples/RLU of medium alone=fold induction).
Table 14 shows that zcyto20, zcyto21 and zcyto22 induce ISRE
signaling in 293 cells transfected with ISRE-luciferase giving a 15
to 17-fold induction in luciferase activity over medium alone. The
addition of zcytor19 DNA to the transfection mix results in a 6 to
8-fold further induction in ISRE signaling by zcyto20, zcyto21 and
zcyto22 giving a 104 to 125-fold total induction. None of the other
transfected class II cytokine receptor DNAs resulted in increased
ISRE signaling. These results indicate that zcyto20, zcyto21 and
zcyto22 functionally interact with the zcytor19 cytokine receptor.
Table 8 also shows that huIFNa-2a can induce ISRE signaling in
ISRE-luciferase transfected 293 cells giving a 205-fold induction
of luciferase activity compared to medium alone. However, the
addition of zcytor19 DNA to the transfection leads to an 11-fold
reduction in ISRE-signaling (compared to ISRE-luciferase DNA
alone), suggesting that zcytor19 over-expression negatively effects
interferon signaling, in contrast to the positive effects of
zcytor19 over-expression on zcyto20, zcyto21 and zcyto22 signaling.
TABLE-US-00008 TABLE 8 Interferon Stimulated Response Element
(ISRE) Signaling of Transfected 293 Cells Following Class II
Cytokine Stimulation (Fold Induction) Ligand ISRE-Luc.
ISRE-Luc./Zcytor19 Zcyto20 (125 ng/ml) 15 125 Zcyto21 (125 ng/ml)
17 108 Zcyto22 (125 ng/ml) 17 104 HuIFNa-2a (100 ng/ml) 205 18
Zcyto10 (125 ng/ml) 1.3 1 HuIL10 (100 ng/ml) 1 0.5
Example 18
Identification of IL10Rb (CRF2-4) as a Receptor subunit for
zcytor19
[0305] A: IL10Rb Neutralizing Antibody Inhibits ISRE Signaling:
[0306] A signal transduction reporter assay was used to determine
the functional interaction of zcyto20, zcyto21, and zcyto22 with
zcytor19 and IL10Rb (CRF2-4). Human embryonal kidney (HEK) cells or
human embryonal kidney (HEK) cells stably overexpressing human
zcytoR19 were transfected with a reporter plasmid containing an
interferon-stimulated response element (ISRE) driving transcription
of a luciferase reporter. Luciferase activity following stimulation
of transfected cells with class II ligands (including zcyto20,
zcyto21, zcyto22 and huIFNa-2a) in the presence or absence of a
neutralizing antibody to IL10Rb (CRF2-4) reflects the interaction
of the ligand with cytokine receptors on the cell surface. The
results and methods are described below.
Cell Transfections:
[0307] To produce 293 HEK cells stably overexpressing human
zcytoR19, 293 cells were transfected as follows: 300,000 293
cells/well (6 well plates) were plated approximately 6 h prior to
transfection in 2 milliliters DMEM+10% fetal bovine serum. Per
well, 2 micrograms of a pZP7 expression vector containing the cDNA
of human zcytoR19 (SEQ ID NO:23) was added to 6 microliters Fugene
6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM.
This transfection mix was added 30 minutes later to the pre-plated
293 cells. Forty-eight hours later the transfected cells were
placed under 2 microgram/milliliter puromicin selection. Puromicin
resistant cells were carried as a population of cells.
[0308] The 293 HEK cells (wild type or overexpressing human
zcytoR19) were transfected as follows: 700,000 293 cells/well (6
well plates) were plated approximately 18 h prior to transfection
in 2 milliliters DMEM+10% fetal bovine serum. Per well, 1 microgram
pISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFP DNA
(Clontech) were added to 6 microliters Fugene 6 reagent (Roche
Biochemicals) in a total of 100 microliters DMEM. This transfection
mix was added 30 minutes later to the pre-plated 293 cells.
Twenty-four hours later the transfected cells were removed from the
plate using trypsin-EDTA and replated at approximately 25,000
cells/well in 96 well microtiter plates. Approximately 18 h prior
to ligand stimulation, media was changed to DMEM+0.5% FBS.
Signal Transduction Reporter Assays:
[0309] The signal transduction reporter assays were done as
follows: Following an 18 h incubation at 37 degrees in DMEM+0.5%
FBS, transfected cells were pretreated with a neutralizing
polyclonal goat antibody to IL10Rb (2.5 micrograms/ml for zcyto21;
8 micrograms/ml for zcyto20 and zcyto22, R&D Systems) or PBS
for 1 hour at 37 C. Human embryonal kidney (HEK) cells stably
overexpressing human zcytoR19 were also pretreated with a
non-neutralizing polyclonal goat antibody to IFNAR1 (8
micrograms/ml, R&D Systems) as an antibody control for
experiments involving zcyto20 and zcyto22. Pretreated cells were
stimulated with dilutions (in DMEM+0.5% FBS) of the following class
II ligands; zcyto20, zcyto21, or zcyto22. As a control, huIFNa-2a
was run in each experiment. Following a 4-hour incubation at 37
degrees, the cells were lysed, and the relative light units (RLU)
were measured on a luminometer after addition of a luciferase
substrate. The results obtained are shown as the fold induction of
the RLU of the experimental samples over the medium alone control
(RLU of experimental samples/RLU of medium alone=fold
induction).
[0310] Tables 9 and 10 show that induction of ISRE signaling by
zcyto20 is inhibited by pretreatment of wild type 293 cells or 293
cells overexpressing human zcytoR19 with a neutralizing antibody to
IL10Rb. No or little inhibition is seen of huIFNa-2a induction of
ISRE signaling. These results indicate that zcyto20 requires
interaction with IL10Rb (CRF2-4) for maximal induction of ISRE
signaling and that the receptor for zcyto20 is the heterdimeric
combination of zcytoR19 and IL10Rb (CRF2-4). TABLE-US-00009 TABLE 9
IL10Rb Inhibition of ISRE Signaling of Transfected wild-type 293
Cells Following Class II Cytokine Stimulation (Fold Induction)
Zcyto20 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentration
neutralizing neutralizing (ng/ml) Zcyto20 Antibody HuIFNa-2a
Antibody 100 8.4 0.8 152 102 10 4 0.9 160 117 1 1 0.9 90 69 0.1 1 1
12 6 0.01 1 0.8 1.2 1 0 1 1 1 1
[0311] TABLE-US-00010 TABLE 10 IL10Rb Inhibition of ISRE Signaling
of Transfected zcytoR19-overexpressing 293 Cells Following Class II
Cytokine Stimulation (Fold Induction) Zcyto20 + HuIFNa-2a +
Cytokine IL10Rb IL10Rb Concentration neutralizing neutralizing
(ng/ml) Zcyto20 Antibody HuIFNa-2a Antibody 100 91 60 16 16 10 97
23 14 15 1 68 1.3 8 8.4 0.1 6 1.1 1.5 1.9 0.01 1.1 1.2 1.2 1.3 0 1
1 1 1
[0312] Tables 11 and 12 show that ISRE signaling by zcyto21 is
inhibited by pretreatment of wild type 293 cells or 293 cells
overexpressing human zcytoR19 with a neutralizing antibody to
IL10Rb. No inhibition is seen of huIFNa-2a induction of ISRE
signaling. These results indicate that zcyto21 requires interaction
with IL10Rb (CRF2-4) for maximal induction of ISRE signaling and
that the receptor for zcyto21 is the heterdimeric combination of
zcytoR19 and IL10Rb (CRF2-4). TABLE-US-00011 TABLE 11 IL10Rb
Inhibition of ISRE Signaling of Transfected wild-type 293 Cells
Following Class II Cytokine Stimulation (Fold Induction) Zcyto21 +
HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentration neutralizing
neutralizing (ng/ml) Zcyto21 Antibody HuIFNa-2a Antibody 100 4.1
1.8 31 30 10 3.2 1.4 32 31 1 1.5 1.3 16.3 15 0.1 1.1 1.3 1.4 2 0.01
1.2 1.3 1.1 1.2 0.001 1.2 1.3 0.9 2.1 0 1 1 1 1
[0313] TABLE-US-00012 TABLE 12 IL10Rb Inhibition of ISR) Signaling
of Transfected zcytoR19-overexpressing 293 Cells Following Class II
Cytokine Stimulation (Fold Induction) Zcyto21 + HuIFNa-2a +
Cytokine IL10Rb IL10Rb Concentration neutralizing neutralizing
(ng/ml) Zcyto21 Antibody HuIFNa-2a Antibody 100 45 31 9 7.7 10 48
28 9 8.5 1 35 5.8 4.3 4.3 0.1 3.5 1 1.4 1.3 0.01 1.5 1.1 0.9 1.2
0.001 1.1 1 1.2 1 0 1 1 1 1
[0314] Tables 13 and 14 show that induction of ISRE signaling by
zcyto22 is inhibited by pretreatment of wild type 293 cells or 293
cells overexpressing human zcytoR19 with a neutralizing antibody to
IL10Rb. No or little inhibition is seen of huIFNa-2a induction of
ISRE signaling. These results indicate that zcyto22 requires
interaction with IL10Rb (CRF2-4) for maximal induction of ISRE
signaling and that the receptor for zcyto22 is the heterdimeric
combination of zcytoR19 and IL10Rb (CRF2-4). TABLE-US-00013 TABLE
13 IL10Rb Inhibition of ISRE Signaling of Transfected wild-type 293
Cells Following Class II Cytokine Stimulation (Fold Induction)
Zcyto22 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentration
neutralizing neutralizing (ng/ml) Zcyto22 Antibody HuIFNa-2a
Antibody 100 11 1.2 152 102 10 8 1 160 117 1 1.8 0.8 90 69 0.1 1.2
0.8 12 6 0.01 0.9 0.9 1.2 1 0 1 1 1 1
[0315] TABLE-US-00014 TABLE 14 IL10Rb Inhibition of ISRE Signaling
of Transfected zcytoR19-overexpressing 293 Cells Following Class II
Cytokine Stimulation (Fold Induction) Zcyto22 + HuIFNa-2a +
Cytokine IL10Rb IL10Rb Concentration neutralizing neutralizing
(ng/ml) Zcyto22 Antibody HuIFNa-2a Antibody 100 82 76 16 16 10 97
39 14 15 1 69 2.3 8 8.4 0.1 8.4 1.1 1.5 1.9 0.01 1 1.3 1.2 1.3 0 1
1 1 1
[0316] B: A: Anti-IL10Rb Antibody Blocks Antiviral Activity
[0317] An antiviral assay was performed to determine the ability of
anti-IL10Rb antibody to block the antiviral activity of zcyto20.
The assay was carried out using 293 HEK cells (wild type or
overexpressing human zcytoR19). On the first day, antibodies
(anti-human IL10R beta, anti-human Leptin receptor, R&D
Systems) were diluted into cell media at 5 micrograms/ml and then
plated with 50,000 cells per well into a 96-well plate. Following a
one-hour incubation at 37.degree. C., zcyto20-CEE (from example 3)
(200 ng/ml for wild-type 293 cells, 0.5 ng/ml for 293 cells
overexpressing human zcytoR19) or human interferon-a-2a (1 ng/ml
for wild-type 293 cells, 100 ng/ml for 293 cells overexpressing
human zcytoR19) were added to the wells and incubated overnight at
37.degree. C. The next day, the medium was removed and replaced
with medium containing encephalomyocarditis virus (EMCV) at a
multiplicity of infection of 0.1. The cells were then incubated at
37.degree. C. overnight. Subsequently, 25 uL of 5 mg/ml
Methylthiazoletetrazolium (MTT) (Sigma) were added to each well,
incubated 2 hours at 37 degrees, and wells were then extracted with
100 uL extraction buffer (12.5% SDS, 45% DMF). Following overnight
incubation at 37.degree. C., the optical density at 570 nM was
measured on a Spectromax plate reader (Molecular Devices, Calif.).
Decreased optical density (570 nm) indicates decreased cell
survival (loss of antiviral activity). The optical densities (570
nm) for the different experimental conditions are shown in Table 15
below. The results indicate that blocking human IL10 receptor beta
specifically neutralizes the antiviral activity of zcyto20 without
effecting interferon-a-2a activity. This indicates that human IL10
receptor beta is part of the receptor complex (including human
zcytoR19) involved in zcyto20 antiviral activity. TABLE-US-00015
TABLE 15 Optical Density (570 nm) of ECMV-Infected Cytokine-Treated
Cells HuzcytoR19- HuzcytoR19- Wild-type 293 Wild-type 293 Cells:
overexpressing 293 overexpressing 293 Cytokine Cells: Anti-IL10Rb
Anti-LeptinR Cells: Anti-IL10Rb Cells: Anti-LeptinR Zcyto20-CEE
0.94 1.88 0.95 2.24 HuIFNa-2a 2.58 2.4 2.18 2.05
[0318] C: Zcyto20 Zcyto21, and Zcyto22 Signaling is Enhanced by
Coexpression of ZcytoR19 and IL10Rb:
[0319] A signal transduction reporter assay was used to determine
the functional interaction of zcyto20, zcyto21 and zcyto22 with
zcytor19 and IL10Rb (CRF2-4). Hamster kidney (BHK) cells were
transfected with a reporter plasmid containing an
interferon-stimulated response element (ISRE) driving transcription
of a luciferase reporter gene in the presence or absence of pZP7
expression vectors containing cDNAs for class II cytokine receptors
Zcytor19 and IL10Rb (CRF2-4). Luciferase activity following
stimulation of transfected cells with class II ligands (including
zcyto20, zcyto21 and zcyto22) reflects the interaction of the
ligand with transfected and native cytokine receptors on the cell
surface. The results and methods are described below.
Cell Transfections
[0320] BHK-570 cells were transfected as follows: 200,000 BHK
cells/well (6 well plates) were plated approximately 5 h prior to
transfection in 2 milliliters DMEM+5% fetal bovine serum. Per well,
1 microgram pISRE-Luciferase DNA (Stratagene), 1 microgram cytokine
receptor DNA and 1 microgram pIRES2-EGFP DNA (Clontech) were added
to 9 microliters Fugene 6 reagent (Roche Biochemicals) in a total
of 100 microliters DMEM. Two micrograms pIRES2-EGFP DNA was used
when cytokine receptor DNA was not included. This transfection mix
was added 30 minutes later to the pre-plated BHK cells. Twenty-four
hours later the transfected cells were removed from the plate using
trypsin-EDTA and replated at approximately 25,000 cells/well in 96
well microtiter plates. Approximately 18 h prior to ligand
stimulation, media was changed to DMEM+0.5% FBS.
Signal Transduction Reporter Assays
[0321] The signal transduction reporter assays were done as
follows: Following an 18 h incubation at 37.degree. C. in DMEM+0.5%
FBS, transfected cells were stimulated with dilutions (in DMEM+0.5%
FBS) of zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 ligands.
Following a 4-hour incubation at 37 degrees, the cells were lysed,
and the relative light units (RLU) were measured on a luminometer
after addition of a luciferase substrate. The results obtained are
shown as the fold induction of the RLU of the experimental samples
over the medium alone control (RLU of experimental samples/RLU of
medium alone=fold induction). Table 16 shows that zcyto20, zcyto21
and zcyto22 induce ISRE signaling in BHK cells transfected with
ISRE-luciferase and zcytoR19 in a dose-dependent manner. The
addition of IL10Rb (CRF2-4) DNA to the transfection mix results in
a half-maximal induction of signaling at a 10-100 fold lower
cytokine dose. No response was seen with ISRE transfection alone.
These results show that the ability of zcyto20, zcyto21 and zcyto22
to signal through the interferon stimulated response element is
enhanced by coexpression of zcytoR19 and IL10Rb (CRF2-4) indicating
that the receptor for zcyto20, zcyto21 and zcyto22 is the
heterdimeric combination of zcytoR19 and IL10Rb (CRF2-4).
TABLE-US-00016 TABLE 16 Interferon Stimulated Response Element
(ISRE) Signaling of Transfected BHK Cells Following Class II
Cytokine Stimulation (Fold Induction) zcyto20/cells zcyto21/cells
zcyto22/cells Class II transfected transfected transfected Ligand
zcyto20/cells with zcytoR19 zcyto21/cells with zcytoR19
zcyto22/cells with zcytoR19 Conc. transfected with and IL10Rb
transfected with and IL10Rb transfected with and IL10Rb (ng/ml)
zcytoR19 alone (CRF2-4) zcytoR19 alone (CRF2-4) zcytoR19 alone
(CRF2-4) 1000 2.25 2.1 3.3 2.2 1.8 2.2 100 2.2 2.6 2.6 2.5 2 2.2 10
2.1 2.4 2.4 2.6 1.9 2.7 1 1.3 2.5 2 2.5 1.5 2.7 0.1 1.25 2.1 1.4
2.2 1.1 2.4 0.01 1.2 1.6 1.4 1.6 1.2 1.7 0.001 1.4 1.5 1.3 1.3 1.2
1.3 0 1 1 1 1 1 1
Example 19
[0322] Binding of Ligands to Soluble Receptors
[0323] The binding of the ligands (zcyto20, zcyto21, zcyto22,
zcyto24, and zcyto25) to soluble receptors can be assayed using an
iodo-bead labeling method. For example, .sup.125I labeled
zcyto21-CEE is labeled (1.2.times.10.sup.7 CPM/ml; 1.5 ng/ul; and
8.6.times.10.sup.6 CPM/ug).
[0324] Fifty nanograms of the 125I labeled zcyto21-cEE (See Example
3) (399,600 CPM) is combined with 1000 ng of cold zcytor19/Fc4
homodimer receptor, 1000 ng cold zcytor19/CRF2-4 heterodimer
receptor, or 1000 ng of a control Class II cytokine receptor/Fc4
receptor as a control with about 10,000 ng of cold zcyto21 as a
competitor. Samples are incubated for 2 hours at 4.degree. C.,
after which 30 ul protein-G (Zymed San Francisco, Calif.) is added
to each sample. Samples are incubated for 1 hour at 4.degree. C.,
and washed 3 times with PBS. Radioactivity of the washed protein-G
is measured in a gamma counter (Packard Instruments, Downers Grove,
Ill.).
Example 20
[0325] Flow Cytometry Staining of Human Monocytes with Zcyto20 and
Zcyto21-Biotin
[0326] Peripheral blood leukocytes (PBLs) were isolated by Ficoll
Hypaque (Amersham, Sweden) separation from heparinized human blood.
The PBLs were cultured at 37.degree. C. in standard media at a
density of 1.times.10.sup.e6 cells per milliliter in 6-well tissue
culture plates. Following overnight incubation, the PBLs were
harvested and stained with biotinylated zcyto20-cee and zcyto21-cee
(See Example 18) at a concentration of 10 ug/ml. Staining was
detected with Phycoerythrin-labeled streptavidin (Pharmingen,
Calif., USA) that was prepared at a dilution of 1:1000. Following
staining the PBLs were fixed in 2% Paraformaldehyde, and read on a
Facscaliber (Becton Dickinson, San Diego, Calif.). The data was
analyzed using Cellquest software (Becton Dickinson). Results
indicate that both biotinylated zcyto20-cee and zcyto21-cee stain
cells in the myeloid gate of peripheral blood leukocytes. Cells in
the lymphoid gate do not bind zcyto20-cee and zcyto21-cee.
Example 21
[0327] Expression of Zcytor19 by Northern Analysis
[0328] Northern blots were probed to determine the tissue
distribution of zcytor19. A human zcytor19 cDNA fragment was
obtained using PCR with gene specific primers, 5' ZC40285 as shown
in SEQ ID NO: 21; and 3' ZC 40286, as shown in SEQ ID NO: 22. The
template was cloned human zcytor19 cDNA. (SEQ ID NO: 23) The PCR
fragment was gel purified, and .about.25 ng was labeled with
P.sup.32 .alpha.-dCTP using the Prime-It.RTM. RmT random prime
labeling kit (Stratagene, LaJolla, Calif.).
[0329] The following Northern blots (Clontech, Palo Alto, Calif.)
were probed for mRNA expression of zcytor19: (1) a human cancer
cell line blot C, which contains RNA samples from each of the
following cancer cell lines: promyelocytic leukemia HL-60, HELA S3,
chronic myelogenous leukemia k-562, lymphoblastic leukemia MOLT-4,
Burkitt's lymphoma RAJI, colorectal adenocarcinoma SW480, lung
carcinoma A549, and melanoma G-361; (2) a human MTN H blot, which
contains mRNA from the following tissues: heart, whole brain,
placenta, lung, liver, skeletal muscle, kidney, and pancreas; (3) a
human MTN H3 which contains mRNA from the following tissues:
stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland,
and bone marrow; and (4) a human MTN H4, which contains mRNA from
the following tissues: spleen, thymus, prostate testis, uterus,
small intestine, colon, and peripheral blood leukocytes.
Hybridizations were all performed in ULTRAhyb.TM. Ultrasensitive
Hybridization Buffer (Ambion, Austin, Tex.) according the
manufacturer's recommendations, which the exception that an
additional 0.2 mg/ml salmon sperm DNA was added to the
hybridization and prehybridization buffers to lower non-specific
hybridization. Following hybridization, non-specific radioactive
signal was removed by treating the blots with 0.1.times.SSC/0.5%
SDS at 50.degree. C. The blots were exposed using BioMax MR Film
and intensifying screens (Eastman Kodak, Rochester, N.Y.), per the
manufacturer's recommendations for 3 days.
[0330] Expression of a 4.5 kb transcript was in greatest in heart,
skeletal muscle, pancreas and prostate tissue, in addition to in
the Burkitt's lymphoma (RAJI) cell line. Lower levels were seen in
multiple other tissues. In addition, there was an .about.2 kb
transcript which was generally less abundant than the larger
transcript, but also present in many of the tissues and cell lines.
Testis tissue, in addition to having the 2 and 4.5 kb transcripts,
may also have .about.4 kb and 1.4 kb transcripts. Adrenal gland
demonstrated equal levels of expression of the 4.5 kb and 2 kb
transcripts.
Example 22
[0331] Human Zcytor19 Expression Based on RT-PCR Analysis of
Stimulated Versus Non-Stimulated Cells
[0332] Gene expression of zcytor19 was examined using RT-PCR
analysis of the following cell types: Hela, 293, Daudi, CD14+,
U937, and HL-60.
[0333] First-strand cDNA synthesis from total RNA was carried out
using a commercially available first-strand synthesis system for
RT-PCR (Invitrogen life technologies, Carlsbad, Calif.). The
subsequent PCR reactions were set up using zcytor19.times.1 (SEQ ID
NO:1) and zcytor19.times.2 (SEQ ID NO:18) specific oligo primers
ZC40288 (SEQ ID NO:65) and ZC40291 (SEQ ID NO:66) which yield a 806
bp and 892 bp product, respectively, Qiagen HotStarTaq DNA
Polymerase and Buffer, (Qiagen, Inc., Valencia, Calif.), GeneAmp
dNTPs (Applied Biosystems, Foster City, Calif.), RediLoad.TM. dye
(Research Genetics, Inc., Huntville, Ala.) and 2 .mu.l first-strand
cDNA (10% of the first-strand reaction) from the respective cell
types. 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 94.degree. C., 1 minute annealing at
63.degree. C. and 1 minute and 15 second 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.
[0334] Bands of the correct size were seen in Hela.+-.IFN-beta
(only the 892 bp band), 293+Parental Adv, Daudi.+-.IFN-beta,
Daudi.+-.IFN-alpha, CD14+ activated, HL-60 activated. No band was
observed in CD14+ resting, U937 resting and activated, and HL-60
resting. These results show induction of zcytoR19 expression upon
activation or differentiation of monocytes or monocyte cell
lines.
Example 23
[0335] Construct for Generating Hzcytor19/hCRF2-4 Heterodimer
[0336] A cell line expressing a secreted hzcytor19/hCRF2-4
heterodimer was constructed. In this construct, the extracellular
domain of hzcytor19 was fused to the heavy chain of IgG gamma1
(Fc4) (SEQ ID NO:14 and SEQ ID NO:15) with a Glu-Glu tag (SEQ ID
NO:11) at the c-terminus, while the extracellular domain of CRF2-4
(SEQ ID NO:64) was fused to Fc4 with a His tag at the C-terminus.
For both of the hzcytor19 and hCRF2-4 arms of the heterodimer, a
Gly-Ser spacer of 12 amino acids was engineered between the
extracellular portion of the receptor and the n-terminus of Fc4. In
addition, a thrombin cleavage site was engineered between the Fc4
domain and the c-terminal tag to enable possible proteolytic
removal of the tag.
[0337] For construction of the hzcytor19/Fc4-CEE portion of the
heterodimer, the extracellular portion of hzcytor19 was amplified
by PCR from a brain cDNA library with oligos ZC37967 (SEQ ID NO:24)
and ZC37972 (SEQ ID NO:25) with BamHI and Bgl2 restriction sites
engineered at the 5' and 3' ends, respectively, under conditions as
follows: 25 cycles of 94.degree. C. for 60 sec., 57.degree. C. for
60 sec., and 72.degree. C. for 120 sec.; and 72.degree. C. for 7
min. PCR products were purified using QIAquick PCR Purification Kit
(Qiagen), digested with BamHI and Bgl2 (Boerhinger-Mannheim),
separated by gel electrophoresis and purified using a QIAquick gel
extraction kit (Qiagen). The hzcytor19 BamHI/Bgl2 fragment was
ligated into Fc4/pzmp20 vector that had been digested with Bgl2.
The zcytor19 fragment is cloned between a tPA leader peptide and
human Fc4 fragment. Once the sequence is confirmed, the DNA
fragment of zcytor19 with tPA leader peptide was cut out by EcoRI
and Bgl2 digestion, and then cloned into pzp9/zcytor7/Fc4-CEE
vector. This vector has the extracellular portion of hzcytor7 fused
to Fc4 with a CEE tag, and digesting with EcoRI and BamHI removes
the extracellular portion of hzcytor7 and allows substitution of
hzcytor19. Minipreps of the resulting ligation were screened for an
EcoRI/BamHI insert of the correct size and positive minipreps were
sequenced to confirm accuracy of the PCR reaction.
[0338] For construction of the hCRF2-4/Fc4-cHIS portion of the
heterodimer, the extracellular portion of hCRF2-4 was amplified by
PCR from pZP-9 CRF with oligos ZC39319 (SEQ ID NO:68) and ZC39325
(SEQ ID NO:70) under conditions as follows: 30 cycles of 94.degree.
C. for 60 sec., 57.degree. C. for 60 sec., and 72.degree. C. for
120 sec; and 72.degree. C. for 7 min. PCR product were purified as
described above and then digested with EcoRI and BamHI. Because the
PCR product had an internal EcoRI site two bands were obtained upon
digestion: a 0.101 kB EcoRI/EcoRI fragment and a 0.574 kB
EcoRI/BamHI fragment. The 0.574 EcoRI/BamHI fragment was ligated
into vector#249 pHZ-1 DR1/Fc4-TCS-cHIS that had been digested with
EcoRI and BamHI. This vector has the extracellular portion of hDR-1
fused to Fc4 with a C-HIS tag (SEQ ID NO:[#]), and digesting with
EcoRI and BamHI removes the extracellular portion of hDR-1 and
allows substitution of hCRF2-4. Minipreps of the resulting ligation
were screened for an EcoRI/BamHI insert of the correct size, and
positive minipreps, were EcoRI digested and band purified for
further construction. The 0.101 kB EcoRI/EcoRI fragment was ligated
into the EcoRI digested minipreps and clones were screened for
proper orientation of insertion by KpnI/Ndel restriction digestion.
Clones with the correct size insertion were submitted for DNA
sequencing to confirm the accuracy of the PCR reaction.
[0339] About 16 .mu.g each of the hzcytor19/Fc4-cEE and
hCRF2-4/Fc-4-cHIS were co-transfected into BHK-570 (ATCC No.
CRL-10314) cells using Lipofectamine (Gibco/BRL), as per
manufacturer's instructions. The transfected cells were selected
for 10 days in DMEM+5% FBS (Gibco/BRL) containing 1 .mu.M
methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5 mg/ml G418
(Gibco/BRL) for 10 days. The resulting pool of transfectants was
selected again in 10 .mu.M MTX and 0.5 mg./ml G418 for 10 days.
Example 24
[0340] Purification of Zcytor19/CRF2-4 Heterodimer Receptor
[0341] Conditioned culture media zcytor19/CRF2-4 heterodimer was
filtered through 0.2 .mu.m filter and 0.02% (w/v) Sodium Azide was
added. The conditioned media was directly loaded a Poros Protein A
50 Column at 10-20 ml/min. Following load the column was washed
with PBS and the bound protein eluted with 0.1M Glycine pH 3.0. The
eluted fractions containing protein were adjusted to pH 7.2 and
Concentrated to <80 ml using YM30 Stirred Cell Membrane
(Millipore).
[0342] The 80 ml eluate from the Protein A column was loaded onto a
318 ml Superdex 200 HiLoad 26/60 Column (Pharmacia). The column was
eluted with PBS pH 7.2 at 3 ml/min. Protein containing fractions
were pooled to eliminate aggregates. The Superdex 200 pool was
adjusted to 0.5M NaCl, 10 mM Imidazole using solid NaCl and
Imidazole and the pH was adjusted to 7.5 with NaOH. The adjusted
protein solution was loaded onto a 200 ml NiNTA column (Qiagen) at
2 CV/hr. The bound protein was eluted, following PBS wash of the
column, with five concentration steps of Imidazole: 40 mM, 100 mM,
150 mM, 250 mM, 500 mM. The fractions eluted at each step of
imidizole were pooled and analyzed by N-terminal sequencing. Pools
containing heterodimer, determined by sequencing were pooled and
concentrated to 50 ml using a YM30 Stirred Cell Membrane
(Millipore). The 50 ml eluate from the NiNTA column was loaded onto
a 318 ml Superdex 200 HiLoad 26/60 Column (Pharmacia). The column
was eluted with PBS pH 7.2 at 3 ml/min. Protein containing
fractions were pooled to eliminate aggregates, as determined by SEC
MALS analysis.
[0343] Purified proteins were analyzed by N-terminal sequencing,
amino acid analysis, and SEC-MALS. Binding affinities to its ligand
(zcyto20, 21, 22, 24, and 25) and biological activities including
its neutralizing activity were determined.
Example 25
[0344] IL28RA mRNA Expression in Liver and Lymphocyte Subsets.
[0345] In order to further examine the mRNA distribution for
IL28RA, semi-quantitative RT-PCR was performed using the SDS 7900HT
system (Applied Biosystems, Calif.). One-step RT-PCR was performed
using 100 ng total RNA for each sample and gene-specific primers. A
standard curve was generated for each primer set using Bjab RNA and
all sample values were normalized to HPRT. The normalized results
are summarized in Tables 17-19. The normalized values for IFNAR2
and CRF2-4 are also shown. TABLE-US-00017 TABLE 17 B and T cells
express significant levels of IL28RA mRNA. Low levels are seen in
dendritic cells and most monocytes. Cell/Tissue Il28RA IFNAR2
CRF2-4 Dendritic Cells unstim .04 5.9 9.8 Dendritic Cells + IFNg
.07 3.6 4.3 Dendritic Cells .16 7.85 3.9 CD14+ stim'd with LPS/IFNg
.13 12 27 CD14+ monocytes resting .12 11 15.4 Hu CD14+ Unact. 4.2
TBD TBD Hu CD14+ 1 ug/ml LPS act. 2.3 TBD TBD H. Inflamed tonsil 3
12.4 9.5 H. B-cells + PMA/Iono 4 & 24 hrs 3.6 1.3 1.4 Hu CD19+
resting 6.2 TBD TBD Hu CD19+ 4 hr. PMA/Iono 10.6 TBD TBD Hu CD19+
24 hr Act. PMA/Iono 3.7 TBD TBD IgD+ B-cells 6.47 13.15 6.42 IgM+
B-cells 9.06 15.4 2.18 IgD- B-cells 5.66 2.86 6.76 NKCells +
PMA/Iono 0 6.7 2.9 Hu CD3+ Unactivated 2.1 TBD TBD CD4+ resting .9
8.5 29.1 CD4+ Unstim 18 hrs 1.6 8.4 13.2 CD4+ + Poly I/C 2.2 4.5
5.1 CD4+ + PMA/Iono .3 1.8 .9 CD3 neg resting 1.6 7.3 46 CD3 neg
unstim 18 hrs 2.4 13.2 16.8 CD3 neg + Poly I/C 18 hrs 5.7 7 30.2
CD3 neg + LPS 18 hrs 3.1 11.9 28.2 CD8+ unstim 18 hrs 1.8 4.9 13.1
CD8+ stim'd with PMA/Ion 18 hrs .3 .6 1.1
[0346] As shown in Table 18, normal liver tissue and liver derived
cell lines display substantial levels of IL28RA and CRF2-4 mRNA.
TABLE-US-00018 TABLE 18 Cell/Tissue IL28RA IFNAR2 CRF2-4 HepG2 1.6
3.56 2.1 HepG2 UGAR 5/10/02 1.1 1.2 2.7 HepG2, CGAT HKES081501C 4.3
2.1 6 HuH7 5/10/02 1.63 16 2 HuH7 hepatoma - CGAT 4.2 7.2 3.1
Liver, normal - CGAT #HXYZ020801K 11.7 3.2 8.4 Liver, NAT--Normal
adjacent tissue 4.5 4.9 7.7 Liver, NAT--Normal adjacent tissue 2.2
6.3 10.4 Hep SMVC hep vein 0 1.4 6.5 Hep SMCA hep. Artery 0 2.1 7.5
Hep. Fibro 0 2.9 6.2 Hep. Ca. 3.8 2.9 5.8 Adenoca liver 8.3 4.2
10.5 SK-Hep-1 adenoca. Liver .1 1.3 2.5 AsPC-1 Hu. Pancreatic
adenocarc. .7 .8 1.3 Hu. Hep. Stellate cells .025 4.4 9.7
[0347] As shown in Table 19, primary airway epithelial cells
contain abundant levels of IL28RA and CRF2-4. TABLE-US-00019 TABLE
19 Cell/Tissue IL28RA IFNAR2 CRF2-4 U87MG - glioma 0 .66 .99 NHBE
unstim 1.9 1.7 8.8 NHBE + TNF-alpha 2.2 5.7 4.6 NHBE + poly I/C 1.8
nd nd Small Airway Epithelial Cells 3.9 3.3 27.8 NHLF--Normal human
lung fibroblasts 0 nd nd
[0348] As shown in Table 20, ZcytoR19 is present in normal and
diseased liver specimens, with increased expression in tissue from
Hepatitis C and Hepatitis B infected specimens. TABLE-US-00020
TABLE 20 Cell/Tissue IL28RA CRF2-4 IFNAR2 Liver with Coagulation
Necrosis 8.87 15.12 1.72 Liver with Autoimmune Hepatitis 6.46 8.90
3.07 Neonatal Hepatitis 6.29 12.46 6.16 Endstage Liver disease 4.79
17.05 10.58 Fulminant Liver Failure 1.90 14.20 7.69 Fulminant Liver
failure 2.52 11.25 8.84 Cirrhosis, primary biliary 4.64 12.03 3.62
Cirrhosis Alcoholic (Laennec's) 4.17 8.30 4.14 Cirrhosis,
Cryptogenic 4.84 7.13 5.06 Hepatitis C+, with cirrhosis 3.64 7.99
6.62 Hepatitis C+ 6.32 11.29 7.43 Fulminant hepatitis secondary to
Hep A 8.94 21.63 8.48 Hepatitis C+ 7.69 15.88 8.05 Hepatitis B+
1.61 12.79 6.93 Normal Liver 8.76 5.42 3.78 Normal Liver 1.46 4.13
4.83 Liver NAT 3.61 5.43 6.42 Liver NAT 1.97 10.37 6.31 Hu Fetal
Liver 1.07 4.87 3.98 Hepatocellular Carcinoma 3.58 3.80 3.22
Adenocarcinoma Liver 8.30 10.48 4.17 hep. SMVC, hep. Vein 0.00 6.46
1.45 Hep SMCA hep. Artery 0.00 7.55 2.10 Hep. Fibroblast 0.00 6.20
2.94 HuH7 hepatoma 4.20 3.05 7.24 HepG2 Hepatocellular carcinoma
3.40 5.98 2.11 SK-Hep-1 adenocar. Liver 0.03 2.53 1.30 HepG2 Unstim
2.06 2.98 2.28 HepG2 + zcyto21 2.28 3.01 2.53 HepG2 + IFNa 2.61
3.05 3.00 Normal Female Liver - degraded 1.38 6.45 4.57 Normal
Liver - degraded 1.93 4.99 6.25 Normal Liver - degraded 2.41 2.32
2.75 Disease Liver - degraded 2.33 3.00 6.04 Primary Hepatocytes
from Clonetics 9.13 7.97 13.30
[0349] As shown in Tables 21-25, ZcytoR19 is detectable in normal B
cells, B lymphoma cell lines, T cells, T lymphoma cell lines
(Jurkat), normal and transformed lymphocytes (B cells and T cells)
and normal human monocytes. TABLE-US-00021 TABLE 21 HPRT IL28RA
IL28RA IFNR2 CRF2-4 Mean Mean norm IFNAR2 norm CRF2-4 Norm CD14+ 24
hr unstim #A38 13.1 68.9 5.2 92.3 7.0 199.8 15.2 CD14+ 24 hr stim
#A38 6.9 7.6 1.1 219.5 31.8 276.6 40.1 CD14+ 24 hr unstim #A112
17.5 40.6 2.3 163.8 9.4 239.7 13.7 CD14+ 24 hr stim #A112 11.8 6.4
0.5 264.6 22.4 266.9 22.6 CD14+ rest #X 32.0 164.2 5.1 1279.7 39.9
699.9 21.8 CD14+ + LPS #X 21.4 40.8 1.9 338.2 15.8 518.0 24.2 CD14+
24 hr unstim #A39 26.3 86.8 3.3 297.4 11.3 480.6 18.3 CD14+ 24 hr
stim #A39 16.6 12.5 0.8 210.0 12.7 406.4 24.5 HL60 Resting 161.2
0.2 0.0 214.2 1.3 264.0 1.6 HL60 + PMA 23.6 2.8 0.1 372.5 15.8
397.5 16.8 U937 Resting 246.7 0.0 0.0 449.4 1.8 362.5 1.5 U937 +
PMA 222.7 0.0 0.0 379.2 1.7 475.9 2.1 Jurkat Resting 241.7 103.0
0.4 327.7 1.4 36.1 0.1 Jurkat Activated 130.7 143.2 1.1 Colo205
88.8 43.5 0.5 HT-29 26.5 30.5 1.2
[0350] TABLE-US-00022 TABLE 22 HPRT SD IL28RA SD Mono 24 hr unstim
#A38 0.6 2.4 Mono 24 hr stim #A38 0.7 0.2 Mono 24 hr unstim #A112
2.0 0.7 Mono 24 hr stim #A112 0.3 0.1 Mono rest #X 5.7 2.2 Mono +
LPS #X 0.5 1.0 Mono 24 hr unstim #A39 0.7 0.8 Mono 24 hr stim #A39
0.1 0.7 HL60 Resting 19.7 0.1 HL60 + PMA 0.7 0.4 U937 Resting 7.4
0.0 U937 + PMA 7.1 0.0 Jurkat Resting 3.7 1.1 Jurkat Activated 2.4
1.8 Colo205 1.9 0.7 HT-29 2.3 1.7
[0351] TABLE-US-00023 TABLE 23 Mean Mean Mean Mean Hprt IFNAR2
IL28RA CRF CD3+/CD4+ 0 10.1 85.9 9.0 294.6 CD4/CD3+ Unstim 18 hrs
12.9 108.7 20.3 170.4 CD4+/CD3+ + Poly I/C 18 hrs 24.1 108.5 52.1
121.8 CD4+/CD3+ + PMA/Iono 18 hrs 47.8 83.7 16.5 40.8 CD3 neg 0
15.4 111.7 24.8 706.1 CD3 neg unstim 18 hrs 15.7 206.6 37.5 263.0
CD3 neg + Poly I/C 18 hrs 9.6 67.0 54.7 289.5 CD3 neg + LPS 18 hrs
14.5 173.2 44.6 409.3 CD8+ Unstim. 18 hrs 6.1 29.7 11.1 79.9 CD8+ +
PMA/Iono 18 hrs 78.4 47.6 26.1 85.5 12.8.1 - NHBE Unstim 47.4 81.1
76.5 415.6 12.8.2 - NHBE + TNF-alpha 42.3 238.8 127.7 193.9 SAEC
15.3 49.9 63.6 426.0
[0352] TABLE-US-00024 TABLE 24 IL28RA CRF IFNAR2 IL28RA CRF IFNAR2
Norm Norm Norm SD SD SD CD3+/CD4+ 0 0.9 29.1 8.5 0.1 1.6 0.4
CD4/CD3+ Unstim 18 hrs 1.6 13.2 8.4 0.2 1.6 1.4 CD4+/CD3+ + Poly
I/C 18 hrs 2.2 5.1 4.5 0.1 0.3 0.5 CD4+/CD3+ + PMA/Iono 18 hrs 0.3
0.9 1.8 0.0 0.1 0.3 CD3 neg 0 1.6 46.0 7.3 0.2 4.7 1.3 CD3 neg
unstim 18 hrs 2.4 16.8 13.2 0.4 2.7 2.3 CD3 neg + Poly I/C 18 hrs
5.7 30.2 7.0 0.3 1.7 0.8 CD3 neg + LPS 18 hrs 3.1 28.2 11.9 0.4 5.4
2.9 CD8+ Unstim. 18 hrs 1.8 13.1 4.9 0.1 1.1 0.3 CD8+ + PMA/Iono 18
hrs 0.3 1.1 0.6 0.0 0.1 0.0 12.8.1 - NHBE Unstim 1.6 8.8 1.7 0.1
0.4 0.1 12.8.2 - NHBE + TNF-alpha 3.0 4.6 5.7 0.1 0.1 0.1 SAEC 4.1
27.8 3.3 0.2 1.1 0.3
[0353] TABLE-US-00025 TABLE 25 SD SD SD SD Hprt IFNAR2 IL28RA CRF
CD3+/CD4+ 0 0.3 3.5 0.6 12.8 CD4/CD3+ Unstim 18 hrs 1.4 13.7 1.1
8.5 CD4+/CD3+ + Poly I/C 18 hrs 1.3 9.8 1.6 3.4 CD4+/CD3+ +
PMA/Iono 18 hrs 4.0 10.3 0.7 3.7 CD3 neg 0 1.4 16.6 1.6 28.6 CD3
neg unstim 18 hrs 2.4 16.2 2.7 12.6 CD3 neg + Poly I/C 18 hrs 0.5
7.0 1.0 8.3 CD3 neg + LPS 18 hrs 1.0 39.8 5.6 73.6 CD8+ Unstim. 18
hrs 0.2 1.6 0.5 6.1 CD8+ + PMA/Iono 18 hrs 1.3 1.7 0.2 8.1 12.8.1 -
NHBE Unstim 2.4 5.6 2.7 2.8 12.8.2 - NHBE + TNF-alpha 0.5 3.4 3.5
3.4 SAEC 0.5 4.8 1.8 9.9
Example 26
[0354] Inhibition of IIL28A, IL29, and Zcyto24 Signalling with the
Soluble Heterodimer (ZcytoR19/CRF2-4), and with the Soluble
Homodimer.
[0355] Signal Transduction Reporter Assay
[0356] A signal transduction reporter assay can be used to show the
inhibitor properties of zcytor19-Fc4 homodimeric and
zcytor19-Fc/CRF2-4-Fc heterodimeric soluble receptors on zcyto20,
zcyto21 and zcyto24 signaling. Human embryonal kidney (HEK) cells
overexpressing the zcytor19 receptor are transfected with a
reporter plasmid containing an interferon-stimulated response
element (ISRE) driving transcription of a luciferase reporter gene.
Luciferase activity following stimulation of transfected cells with
ligands (including zcyto20 (SEQ ID NO:52), zcyto21 (SEQ ID NO:55),
and zcyto24 (SEQ ID NO:60) reflects the interaction of the ligand
with soluble receptor.
[0357] Cell Transfections
[0358] 293 HEK cells overexpressing zcytor19 were transfected as
follows: 700,000 293 cells/well (6 well plates) were plated
approximately 18 h prior to transfection in 2 milliliters DMEM+10%
fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA
(Stratagene) and 1 microgram pIRES2-EGFP DNA (Clontech,) were added
to 6 microliters Fugene 6 reagent (Roche Biochemicals) in a total
of 100 microliters DMEM. This transfection mix was added 30 minutes
later to the pre-plated 293 cells. Twenty-four hours later the
transfected cells were removed from the plate using trypsin-EDTA
and replated at approximately 25,000 cells/well in 96 well
microtiter plates. Approximately 18 h prior to ligand stimulation,
media was changed to DMEM+0.5% FBS.
[0359] Signal Transduction Reporter Assays
[0360] The signal transduction reporter assays were done as
follows: Following an 18 h incubation at 37.degree. C. in DMEM+0.5%
FBS, transfected cells were stimulated with 10 ng/ml zcyto20,
zcyto21 or zcyto24 and 10 micrograms/ml of the following soluble
receptors; human zcytor19-Fc homodimer, human zcytor19-Fc/human
CRF2-4-Fc heterodimer, human CRF2-4-Fc homodimer, murine
zcytor19-Ig homodimer. Following a 4-hour incubation at 37.degree.
C., the cells were lysed, and the relative light units (RLU) were
measured on a luminometer after addition of a luciferase substrate.
The results obtained are shown as the percent inhibition of
ligand-induced signaling in the presence of soluble receptor
relative to the signaling in the presence of PBS alone. Table 26
shows that the human zcytor19-Fc/human CRF2-4 heterodimeric soluble
receptor is able to inhibit zcyto20, zcyto21 and zcyto24-induced
signaling between 16 and 45% of control. The human zcytor19-Fc
homodimeric soluble receptor is also able to inhibit
zcyto21-induced signaling by 45%. No significant effects were seen
with huCRF2-4-Fc or muzcytor19-Ig homodimeric soluble receptors.
TABLE-US-00026 TABLE 26 Percent Inhibition of Ligand-induced
Interferon Stimulated Response Element (ISRE) Signaling by Soluble
Receptors Huzcytor19-Fc/ Huzcytor19- HuCRF2-4- Muzcytor19- Ligand
huCRF2-4-Fc Fc Fc Ig Zcyto20 16% 92% 80% 91% Zcyto21 16% 45% 79%
103% Zcyto24 47% 90% 82% 89%
[0361] 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
69 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 sequence 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 VARIANT (1)...(5) Xaa = Any Amino Acid VARIANT 3 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 Zcytor19-Fc4 fusion construct 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 Zcytor19-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 sequence 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
nggngayggn 1260 caycargarw snytnccncc nccngartty wsnaargayw
snggnttyyt ngargarytn 1320 ccngargaya ayytnwsnws ntgggcnacn
tggggnacny tnccnccnga rccnaayytn 1380 gtnccnggng gnccnccngt
nwsnytncar acnytnacnt tytgytggga rwsnwsnccn 1440 gargargarg
argargcnmg ngarwsngar athgargayw sngaygcngg nwsntggggn 1500
gcngarwsna cncarmgnac ngargaymgn ggnmgnacny tnggncayta yatggcnmgn
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 51 618
DNA Homo sapiens CDS (1)...(618) 51 atg act ggg gac tgc acg cca gtg
ctg gtg ctg atg gcc gca gtg ctg 48 Met Thr Gly Asp Cys Thr Pro Val
Leu Val Leu Met Ala Ala Val Leu 1 5 10 15 acc gtg act gga gca gtt
cct gtc gcc agg ctc cac ggg gct ctc ccg 96 Thr Val Thr Gly Ala Val
Pro Val Ala Arg Leu His Gly Ala Leu Pro 20 25 30 gat gca agg ggc
tgc cac ata gcc cag ttc aag tcc ctg tct cca cag 144 Asp Ala Arg Gly
Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln 35 40 45 gag ctg
cag gcc ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt 192 Glu Leu
Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu 50 55 60
ctg ctg aag gac tgc agg tgc cac tcc cgc ctc ttc ccc agg acc tgg 240
Leu Leu Lys Asp Cys Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp 65
70 75 80 gac ctg agg cag ctg cag gtg agg gag cgc ccc atg gct ttg
gag gct 288 Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu
Glu Ala 85 90 95 gag ctg gcc ctg acg ctg aag gtt ctg gag gcc acc
gct gac act gac 336 Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr
Ala Asp Thr Asp 100 105 110 cca gcc ctg gtg gac gtc ttg gac cag ccc
ctt cac acc ctg cac cat 384 Pro Ala Leu Val Asp Val Leu Asp Gln Pro
Leu His Thr Leu His His 115 120 125 atc ctc tcc cag ttc cgg gcc tgt
gtg agt cgt cag ggc ctg ggc acc 432 Ile Leu Ser Gln Phe Arg Ala Cys
Val Ser Arg Gln Gly Leu Gly Thr 130 135 140 cag atc cag cct cag ccc
acg gca ggg ccc agg acc cgg ggc cgc ctc 480 Gln Ile Gln Pro Gln Pro
Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu 145 150 155 160 cac cat tgg
ctg tac cgg ctc cag gag gcc cca aaa aag gag tcc cct 528 His His Trp
Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro 165 170 175 ggc
tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc ctc ctc acg 576 Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 180 185
190 cga gac ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc tga 618 Arg
Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val * 195 200 205 52
205 PRT Homo sapiens 52 Met Thr Gly Asp Cys Thr Pro Val Leu Val Leu
Met Ala Ala Val Leu 1 5 10 15 Thr Val Thr Gly Ala Val Pro Val Ala
Arg Leu His Gly Ala Leu Pro 20 25 30 Asp Ala Arg Gly Cys His Ile
Ala Gln Phe Lys Ser Leu Ser Pro Gln 35 40 45 Glu Leu Gln Ala Phe
Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu 50 55 60 Leu Leu Lys
Asp Cys Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp 65 70 75 80 Asp
Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala 85 90
95 Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp
100 105 110 Pro Ala Leu Val Asp Val Leu Asp Gln Pro Leu His Thr Leu
His His 115 120 125 Ile Leu Ser Gln Phe Arg Ala Cys Val Ser Arg Gln
Gly Leu Gly Thr 130 135 140 Gln Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Thr Arg Gly Arg Leu 145 150 155 160 His His Trp Leu Tyr Arg Leu
Gln Glu Ala Pro Lys Lys Glu Ser Pro 165 170 175 Gly Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 180 185 190 Arg Asp Leu
Asn Cys Val Ala Ser Gly Asp Leu Cys Val 195 200 205 53 615 DNA
Artificial Sequence degenerate sequence misc_feature (1)...(615) n
= A,T,C or G 53 atgacnggng aytgyacncc ngtnytngtn ytnatggcng
cngtnytnac ngtnacnggn 60 gcngtnccng tngcnmgnyt ncayggngcn
ytnccngayg cnmgnggntg ycayathgcn 120 carttyaarw snytnwsncc
ncargarytn cargcnttya armgngcnaa rgaygcnytn 180 gargarwsny
tnytnytnaa rgaytgymgn tgycaywsnm gnytnttycc nmgnacntgg 240
gayytnmgnc arytncargt nmgngarmgn ccnatggcny tngargcnga rytngcnytn
300 acnytnaarg tnytngargc nacngcngay acngayccng cnytngtnga
ygtnytngay 360 carccnytnc ayacnytnca ycayathytn wsncarttym
gngcntgygt nwsnmgncar 420 ggnytnggna cncarathca rccncarccn
acngcnggnc cnmgnacnmg nggnmgnytn 480 caycaytggy tntaymgnyt
ncargargcn ccnaaraarg arwsnccngg ntgyytngar 540 gcnwsngtna
cnttyaayyt nttymgnytn ytnacnmgng ayytnaaytg ygtngcnwsn 600
ggngayytnt gygtn 615 54 603 DNA Homo sapiens CDS (1)...(603) 54 atg
gct gca gct tgg acc gtg gtg ctg gtg act ttg gtg cta ggc ttg 48 Met
Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10
15 gcc gtg gca ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag
96 Ala Val Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys
20 25 30 ggc tgc cac att ggc agg ttc aaa tct ctg tca cca cag gag
cta gcg 144 Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu
Leu Ala 35 40 45 agc ttc aag aag gcc agg gac gcc ttg gaa gag tca
ctc aag ctg aaa 192 Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser
Leu Lys Leu Lys 50 55 60 aac tgg agt tgc agc tct cct gtc ttc ccc
ggg aat tgg gac ctg agg 240 Asn Trp Ser Cys Ser Ser Pro Val Phe Pro
Gly Asn Trp Asp Leu Arg 65 70 75 80 ctt ctc cag gtg agg gag cgc cct
gtg gcc ttg gag gct gag ctg gcc 288 Leu Leu Gln Val Arg Glu Arg Pro
Val Ala Leu Glu Ala Glu Leu Ala 85 90 95 ctg acg ctg aag gtc ctg
gag gcc gct gct ggc cca gcc ctg gag gac 336 Leu Thr Leu Lys Val Leu
Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp 100 105
110 gtc cta gac cag ccc ctt cac acc ctg cac cac atc ctc tcc cag ctc
384 Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu
115 120 125 cag gcc tgt atc cag cct cag ccc aca gca ggg ccc agg ccc
cgg ggc 432 Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro
Arg Gly 130 135 140 cgc ctc cac cac tgg ctg cac cgg ctc cag gag gcc
ccc aaa aag gag 480 Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala
Pro Lys Lys Glu 145 150 155 160 tcc gct ggc tgc ctg gag gca tct gtc
acc ttc aac ctc ttc cgc ctc 528 Ser Ala Gly Cys Leu Glu Ala Ser Val
Thr Phe Asn Leu Phe Arg Leu 165 170 175 ctc acg cga gac ctc aaa tat
gtg gcc gat ggg gac ctg tgt ctg aga 576 Leu Thr Arg Asp Leu Lys Tyr
Val Ala Asp Gly Asp Leu Cys Leu Arg 180 185 190 acg tca acc cac cct
gag tcc acc tga 603 Thr Ser Thr His Pro Glu Ser Thr * 195 200 55
200 PRT Homo sapiens 55 Met Ala Ala Ala Trp Thr Val Val Leu Val Thr
Leu Val Leu Gly Leu 1 5 10 15 Ala Val Ala Gly Pro Val Pro Thr Ser
Lys Pro Thr Thr Thr Gly Lys 20 25 30 Gly Cys His Ile Gly Arg Phe
Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40 45 Ser Phe Lys Lys Ala
Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50 55 60 Asn Trp Ser
Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg 65 70 75 80 Leu
Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala 85 90
95 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
100 105 110 Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln Leu 115 120 125 Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Pro Arg Gly 130 135 140 Arg Leu His His Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys Glu 145 150 155 160 Ser Ala Gly Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170 175 Leu Thr Arg Asp Leu
Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg 180 185 190 Thr Ser Thr
His Pro Glu Ser Thr 195 200 56 615 DNA Homo sapiens CDS (1)...(615)
56 atg acc ggg gac tgc atg cca gtg ctg gtg ctg atg gcc gca gtg ctg
48 Met Thr Gly Asp Cys Met Pro Val Leu Val Leu Met Ala Ala Val Leu
1 5 10 15 acc gtg act gga gca gtt cct gtc gcc agg ctc cgc ggg gct
ctc ccg 96 Thr Val Thr Gly Ala Val Pro Val Ala Arg Leu Arg Gly Ala
Leu Pro 20 25 30 gat gca agg ggc tgc cac ata gcc cag ttc aag tcc
ctg tct cca cag 144 Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser
Leu Ser Pro Gln 35 40 45 gag ctg cag gcc ttt aag agg gcc aaa gat
gcc tta gaa gag tcg ctt 192 Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp
Ala Leu Glu Glu Ser Leu 50 55 60 ctg ctg aag gac tgc aag tgc cgc
tcc cgc ctc ttc ccc agg acc tgg 240 Leu Leu Lys Asp Cys Lys Cys Arg
Ser Arg Leu Phe Pro Arg Thr Trp 65 70 75 80 gac ctg agg cag ctg cag
gtg agg gag cgc ccc gtg gct ttg gag gct 288 Asp Leu Arg Gln Leu Gln
Val Arg Glu Arg Pro Val Ala Leu Glu Ala 85 90 95 gag ctg gcc ctg
acg ctg aag gtt ctg gag gcc acc gct gac act gac 336 Glu Leu Ala Leu
Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp 100 105 110 cca gcc
ctg ggg gat gtc ttg gac cag ccc ctt cac acc ctg cac cat 384 Pro Ala
Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu His His 115 120 125
atc ctc tcc cag ctc cgg gcc tgt gtg agt cgt cag ggc ccg ggc acc 432
Ile Leu Ser Gln Leu Arg Ala Cys Val Ser Arg Gln Gly Pro Gly Thr 130
135 140 cag atc cag cct cag ccc acg gca ggg ccc agg acc cgg ggc cgc
ctc 480 Gln Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg
Leu 145 150 155 160 cac cat tgg ctg cac cgg ctc cag gag gcc cca aaa
aag gag tcc cct 528 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys
Lys Glu Ser Pro 165 170 175 ggc tgc ctc gag gcc tct gtc acc ttc aac
ctc ttc cgc ctc ctc acg 576 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn
Leu Phe Arg Leu Leu Thr 180 185 190 cga gac ctg aat tgt gtt gcc agc
ggg gac ctg tgt gtc 615 Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu
Cys Val 195 200 205 57 205 PRT Homo sapiens 57 Met Thr Gly Asp Cys
Met Pro Val Leu Val Leu Met Ala Ala Val Leu 1 5 10 15 Thr Val Thr
Gly Ala Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro 20 25 30 Asp
Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln 35 40
45 Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu
50 55 60 Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg
Thr Trp 65 70 75 80 Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val
Ala Leu Glu Ala 85 90 95 Glu Leu Ala Leu Thr Leu Lys Val Leu Glu
Ala Thr Ala Asp Thr Asp 100 105 110 Pro Ala Leu Gly Asp Val Leu Asp
Gln Pro Leu His Thr Leu His His 115 120 125 Ile Leu Ser Gln Leu Arg
Ala Cys Val Ser Arg Gln Gly Pro Gly Thr 130 135 140 Gln Ile Gln Pro
Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu 145 150 155 160 His
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro 165 170
175 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr
180 185 190 Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 195
200 205 58 615 DNA Artificial Sequence degenerate sequence
misc_feature (1)...(615) n = A,T,C or G 58 atgacnggng aytgyatgcc
ngtnytngtn ytnatggcng cngtnytnac ngtnacnggn 60 gcngtnccng
tngcnmgnyt nmgnggngcn ytnccngayg cnmgnggntg ycayathgcn 120
carttyaarw snytnwsncc ncargarytn cargcnttya armgngcnaa rgaygcnytn
180 gargarwsny tnytnytnaa rgaytgyaar tgymgnwsnm gnytnttycc
nmgnacntgg 240 gayytnmgnc arytncargt nmgngarmgn ccngtngcny
tngargcnga rytngcnytn 300 acnytnaarg tnytngargc nacngcngay
acngayccng cnytnggnga ygtnytngay 360 carccnytnc ayacnytnca
ycayathytn wsncarytnm gngcntgygt nwsnmgncar 420 ggnccnggna
cncarathca rccncarccn acngcnggnc cnmgnacnmg nggnmgnytn 480
caycaytggy tncaymgnyt ncargargcn ccnaaraarg arwsnccngg ntgyytngar
540 gcnwsngtna cnttyaayyt nttymgnytn ytnacnmgng ayytnaaytg
ygtngcnwsn 600 ggngayytnt gygtn 615 59 633 DNA Homo sapiens CDS
(22)...(630) 59 tcacagaccc cggagagcaa c atg aag cca gaa aca gct ggg
ggc cac atg 51 Met Lys Pro Glu Thr Ala Gly Gly His Met 1 5 10 ctc
ctc ctg ctg ttg cct ctg ctg ctg gcc gca gtg ctg aca aga acc 99 Leu
Leu Leu Leu Leu Pro Leu Leu Leu Ala Ala Val Leu Thr Arg Thr 15 20
25 caa gct gac cct gtc ccc agg gcc acc agg ctc cca gtg gaa gca aag
147 Gln Ala Asp Pro Val Pro Arg Ala Thr Arg Leu Pro Val Glu Ala Lys
30 35 40 gat tgc cac att gct cag ttc aag tct ctg tcc cca aaa gag
ctg cag 195 Asp Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Lys Glu
Leu Gln 45 50 55 gcc ttc aaa aag gcc aag gat gcc atc gag aag agg
ctg ctt gag aag 243 Ala Phe Lys Lys Ala Lys Asp Ala Ile Glu Lys Arg
Leu Leu Glu Lys 60 65 70 gac ctg agg tgc agt tcc cac ctc ttc ccc
agg gcc tgg gac ctg aag 291 Asp Leu Arg Cys Ser Ser His Leu Phe Pro
Arg Ala Trp Asp Leu Lys 75 80 85 90 cag ctg cag gtc caa gag cgc ccc
aag gcc ttg cag gct gag gtg gcc 339 Gln Leu Gln Val Gln Glu Arg Pro
Lys Ala Leu Gln Ala Glu Val Ala 95 100 105 ctg acc ctg aag gtc tgg
gag aac atg act gac tca gcc ctg gcc acc 387 Leu Thr Leu Lys Val Trp
Glu Asn Met Thr Asp Ser Ala Leu Ala Thr 110 115 120 atc ctg ggc cag
cct ctt cat aca ctg agc cac att cac tcc cag ctg 435 Ile Leu Gly Gln
Pro Leu His Thr Leu Ser His Ile His Ser Gln Leu 125 130 135 cag acc
tgt aca cag ctt cag gcc aca gca gag ccc agg tcc ccg agc 483 Gln Thr
Cys Thr Gln Leu Gln Ala Thr Ala Glu Pro Arg Ser Pro Ser 140 145 150
cgc cgc ctc tcc cgc tgg ctg cac agg ctc cag gag gcc cag agc aag 531
Arg Arg Leu Ser Arg Trp Leu His Arg Leu Gln Glu Ala Gln Ser Lys 155
160 165 170 gag acc cct ggc tgc ctg gag gcc tct gtc acc tcc aac ctg
ttt cgc 579 Glu Thr Pro Gly Cys Leu Glu Ala Ser Val Thr Ser Asn Leu
Phe Arg 175 180 185 ctg ctc acc cgg gac ctc aag tgt gtg gcc aat gga
gac cag tgt gtc 627 Leu Leu Thr Arg Asp Leu Lys Cys Val Ala Asn Gly
Asp Gln Cys Val 190 195 200 tga cct 633 * 60 202 PRT Homo sapiens
60 Met Lys Pro Glu Thr Ala Gly Gly His Met Leu Leu Leu Leu Leu Pro
1 5 10 15 Leu Leu Leu Ala Ala Val Leu Thr Arg Thr Gln Ala Asp Pro
Val Pro 20 25 30 Arg Ala Thr Arg Leu Pro Val Glu Ala Lys Asp Cys
His Ile Ala Gln 35 40 45 Phe Lys Ser Leu Ser Pro Lys Glu Leu Gln
Ala Phe Lys Lys Ala Lys 50 55 60 Asp Ala Ile Glu Lys Arg Leu Leu
Glu Lys Asp Leu Arg Cys Ser Ser 65 70 75 80 His Leu Phe Pro Arg Ala
Trp Asp Leu Lys Gln Leu Gln Val Gln Glu 85 90 95 Arg Pro Lys Ala
Leu Gln Ala Glu Val Ala Leu Thr Leu Lys Val Trp 100 105 110 Glu Asn
Met Thr Asp Ser Ala Leu Ala Thr Ile Leu Gly Gln Pro Leu 115 120 125
His Thr Leu Ser His Ile His Ser Gln Leu Gln Thr Cys Thr Gln Leu 130
135 140 Gln Ala Thr Ala Glu Pro Arg Ser Pro Ser Arg Arg Leu Ser Arg
Trp 145 150 155 160 Leu His Arg Leu Gln Glu Ala Gln Ser Lys Glu Thr
Pro Gly Cys Leu 165 170 175 Glu Ala Ser Val Thr Ser Asn Leu Phe Arg
Leu Leu Thr Arg Asp Leu 180 185 190 Lys Cys Val Ala Asn Gly Asp Gln
Cys Val 195 200 61 632 DNA Homo sapiens CDS (22)...(630) 61
tcacagaccc cggagagcaa c atg aag cca gaa aca gct ggg ggc cac atg 51
Met Lys Pro Glu Thr Ala Gly Gly His Met 1 5 10 ctc ctc ctg ctg ttg
cct ctg ctg ctg gcc gca gtg ctg aca aga acc 99 Leu Leu Leu Leu Leu
Pro Leu Leu Leu Ala Ala Val Leu Thr Arg Thr 15 20 25 caa gct gac
cct gtc ccc agg gcc acc agg ctc cca gtg gaa gca aag 147 Gln Ala Asp
Pro Val Pro Arg Ala Thr Arg Leu Pro Val Glu Ala Lys 30 35 40 gat
tgc cac att gct cag ttc aag tct ctg tcc cca aaa gag ctg cag 195 Asp
Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Lys Glu Leu Gln 45 50
55 gcc ttc aaa aag gcc aag ggt gcc atc gag aag agg ctg ctt gag aag
243 Ala Phe Lys Lys Ala Lys Gly Ala Ile Glu Lys Arg Leu Leu Glu Lys
60 65 70 gac atg agg tgc agt tcc cac ctc atc tcc agg gcc tgg gac
ctg aag 291 Asp Met Arg Cys Ser Ser His Leu Ile Ser Arg Ala Trp Asp
Leu Lys 75 80 85 90 cag ctg cag gtc caa gag cgc ccc aag gcc ttg cag
gct gag gtg gcc 339 Gln Leu Gln Val Gln Glu Arg Pro Lys Ala Leu Gln
Ala Glu Val Ala 95 100 105 ctg acc ctg aag gtc tgg gag aac ata aat
gac tca gcc ctg acc acc 387 Leu Thr Leu Lys Val Trp Glu Asn Ile Asn
Asp Ser Ala Leu Thr Thr 110 115 120 atc ctg ggc cag cct ctt cat aca
ctg agc cac att cac tcc cag ctg 435 Ile Leu Gly Gln Pro Leu His Thr
Leu Ser His Ile His Ser Gln Leu 125 130 135 cag acc tgt aca cag ctt
cag gcc aca gca gag ccc aag ccc ccg agt 483 Gln Thr Cys Thr Gln Leu
Gln Ala Thr Ala Glu Pro Lys Pro Pro Ser 140 145 150 cgc cgc ctc tcc
cgc tgg ctg cac agg ctc cag gag gcc cag agc aag 531 Arg Arg Leu Ser
Arg Trp Leu His Arg Leu Gln Glu Ala Gln Ser Lys 155 160 165 170 gag
act cct ggc tgc ctg gag gac tct gtc acc tcc aac ctg ttt caa 579 Glu
Thr Pro Gly Cys Leu Glu Asp Ser Val Thr Ser Asn Leu Phe Gln 175 180
185 ctg ctc ctc cgg gac ctc aag tgt gtg gcc agt gga gac cag tgt gtc
627 Leu Leu Leu Arg Asp Leu Lys Cys Val Ala Ser Gly Asp Gln Cys Val
190 195 200 tga cc 632 * 62 202 PRT Homo sapiens 62 Met Lys Pro Glu
Thr Ala Gly Gly His Met Leu Leu Leu Leu Leu Pro 1 5 10 15 Leu Leu
Leu Ala Ala Val Leu Thr Arg Thr Gln Ala Asp Pro Val Pro 20 25 30
Arg Ala Thr Arg Leu Pro Val Glu Ala Lys Asp Cys His Ile Ala Gln 35
40 45 Phe Lys Ser Leu Ser Pro Lys Glu Leu Gln Ala Phe Lys Lys Ala
Lys 50 55 60 Gly Ala Ile Glu Lys Arg Leu Leu Glu Lys Asp Met Arg
Cys Ser Ser 65 70 75 80 His Leu Ile Ser Arg Ala Trp Asp Leu Lys Gln
Leu Gln Val Gln Glu 85 90 95 Arg Pro Lys Ala Leu Gln Ala Glu Val
Ala Leu Thr Leu Lys Val Trp 100 105 110 Glu Asn Ile Asn Asp Ser Ala
Leu Thr Thr Ile Leu Gly Gln Pro Leu 115 120 125 His Thr Leu Ser His
Ile His Ser Gln Leu Gln Thr Cys Thr Gln Leu 130 135 140 Gln Ala Thr
Ala Glu Pro Lys Pro Pro Ser Arg Arg Leu Ser Arg Trp 145 150 155 160
Leu His Arg Leu Gln Glu Ala Gln Ser Lys Glu Thr Pro Gly Cys Leu 165
170 175 Glu Asp Ser Val Thr Ser Asn Leu Phe Gln Leu Leu Leu Arg Asp
Leu 180 185 190 Lys Cys Val Ala Ser Gly Asp Gln Cys Val 195 200 63
1013 DNA Homo sapiens CDS (14)...(991) 63 ccagcgtccg tcc atg gcg
tgg agc ctt ggg agc tgg ctg ggt ggc tgc 49 Met Ala Trp Ser Leu Gly
Ser Trp Leu Gly Gly Cys 1 5 10 ctg ctg gtg tca gca ttg gga atg gta
cca cct ccc gaa aat gtc aga 97 Leu Leu Val Ser Ala Leu Gly Met Val
Pro Pro Pro Glu Asn Val Arg 15 20 25 atg aat tct gtt aat ttc aag
aac att cta cag tgg gag tca cct gct 145 Met Asn Ser Val Asn Phe Lys
Asn Ile Leu Gln Trp Glu Ser Pro Ala 30 35 40 ttt gcc aaa ggg aac
ctg act ttc aca gct cag tac cta agt tat agg 193 Phe Ala Lys Gly Asn
Leu Thr Phe Thr Ala Gln Tyr Leu Ser Tyr Arg 45 50 55 60 ata ttc caa
gat aaa tgc atg aat act acc ttg acg gaa tgt gat ttc 241 Ile Phe Gln
Asp Lys Cys Met Asn Thr Thr Leu Thr Glu Cys Asp Phe 65 70 75 tca
agt ctt tcc aag tat ggt gac cac acc ttg aga gtc agg gct gaa 289 Ser
Ser Leu Ser Lys Tyr Gly Asp His Thr Leu Arg Val Arg Ala Glu 80 85
90 ttt gca gat gag cat tca gac tgg gta aac atc acc ttc tgt cct gtg
337 Phe Ala Asp Glu His Ser Asp Trp Val Asn Ile Thr Phe Cys Pro Val
95 100 105 gat gac acc att att gga ccc cct gga atg caa gta gaa gta
ctt gct 385 Asp Asp Thr Ile Ile Gly Pro Pro Gly Met Gln Val Glu Val
Leu Ala 110 115 120 gat tct tta cat atg cgt ttc tta gcc cct aaa att
gag aat gaa tac 433 Asp Ser Leu His Met Arg Phe Leu Ala Pro Lys Ile
Glu Asn Glu Tyr 125 130 135 140 gaa act tgg act atg aag aat gtg tat
aac tca tgg act tat aat gtg 481 Glu Thr Trp Thr Met Lys Asn Val Tyr
Asn Ser Trp Thr Tyr Asn Val 145 150 155 caa tac tgg aaa aac ggt act
gat gaa aag ttt caa att act ccc cag 529 Gln Tyr Trp Lys Asn Gly Thr
Asp Glu Lys Phe Gln Ile Thr Pro Gln 160 165 170 tat gac ttt gag gtc
ctc aga aac ctg gag cca tgg aca act tat tgt 577 Tyr Asp Phe Glu Val
Leu Arg Asn Leu Glu Pro Trp Thr Thr Tyr Cys 175 180
185 gtt caa gtt cga ggg ttt ctt cct gat cgg aac aaa gct ggg gaa tgg
625 Val Gln Val Arg Gly Phe Leu Pro Asp Arg Asn Lys Ala Gly Glu Trp
190 195 200 agt gag cct gtc tgt gag caa aca acc cat gac gaa acg gtc
ccc tcc 673 Ser Glu Pro Val Cys Glu Gln Thr Thr His Asp Glu Thr Val
Pro Ser 205 210 215 220 tgg atg gtg gcc gtc atc ctc atg gcc tcg gtc
ttc atg gtc tgc ctg 721 Trp Met Val Ala Val Ile Leu Met Ala Ser Val
Phe Met Val Cys Leu 225 230 235 gca ctc ctc ggc tgc ttc tcc ttg ctg
tgg tgc gtt tac aag aag aca 769 Ala Leu Leu Gly Cys Phe Ser Leu Leu
Trp Cys Val Tyr Lys Lys Thr 240 245 250 aag tac gcc ttc tcc cct agg
aat tct ctt cca cag cac ctg aaa gag 817 Lys Tyr Ala Phe Ser Pro Arg
Asn Ser Leu Pro Gln His Leu Lys Glu 255 260 265 ttt ttg ggc cat cct
cat cat aac aca ctt ctg ttt ttc tcc ttt cca 865 Phe Leu Gly His Pro
His His Asn Thr Leu Leu Phe Phe Ser Phe Pro 270 275 280 ttg tcg gat
gag aat gat gtt ttt gac aag cta agt gtc att gca gaa 913 Leu Ser Asp
Glu Asn Asp Val Phe Asp Lys Leu Ser Val Ile Ala Glu 285 290 295 300
gac tct gag agc ggc aag cag aat cct ggt gac agc tgc agc ctc ggg 961
Asp Ser Glu Ser Gly Lys Gln Asn Pro Gly Asp Ser Cys Ser Leu Gly 305
310 315 acc ccg cct ggg cag ggg ccc caa agc tag gctctgagaa
ggaaacacac 1011 Thr Pro Pro Gly Gln Gly Pro Gln Ser * 320 325 tc
1013 64 325 PRT Homo sapiens 64 Met Ala Trp Ser Leu Gly Ser Trp Leu
Gly Gly Cys Leu Leu Val Ser 1 5 10 15 Ala Leu Gly Met Val Pro Pro
Pro Glu Asn Val Arg Met Asn Ser Val 20 25 30 Asn Phe Lys Asn Ile
Leu Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly 35 40 45 Asn Leu Thr
Phe Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp 50 55 60 Lys
Cys Met Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu Ser 65 70
75 80 Lys Tyr Gly Asp His Thr Leu Arg Val Arg Ala Glu Phe Ala Asp
Glu 85 90 95 His Ser Asp Trp Val Asn Ile Thr Phe Cys Pro Val Asp
Asp Thr Ile 100 105 110 Ile Gly Pro Pro Gly Met Gln Val Glu Val Leu
Ala Asp Ser Leu His 115 120 125 Met Arg Phe Leu Ala Pro Lys Ile Glu
Asn Glu Tyr Glu Thr Trp Thr 130 135 140 Met Lys Asn Val Tyr Asn Ser
Trp Thr Tyr Asn Val Gln Tyr Trp Lys 145 150 155 160 Asn Gly Thr Asp
Glu Lys Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu 165 170 175 Val Leu
Arg Asn Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg 180 185 190
Gly Phe Leu Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro Val 195
200 205 Cys Glu Gln Thr Thr His Asp Glu Thr Val Pro Ser Trp Met Val
Ala 210 215 220 Val Ile Leu Met Ala Ser Val Phe Met Val Cys Leu Ala
Leu Leu Gly 225 230 235 240 Cys Phe Ser Leu Leu Trp Cys Val Tyr Lys
Lys Thr Lys Tyr Ala Phe 245 250 255 Ser Pro Arg Asn Ser Leu Pro Gln
His Leu Lys Glu Phe Leu Gly His 260 265 270 Pro His His Asn Thr Leu
Leu Phe Phe Ser Phe Pro Leu Ser Asp Glu 275 280 285 Asn Asp Val Phe
Asp Lys Leu Ser Val Ile Ala Glu Asp Ser Glu Ser 290 295 300 Gly Lys
Gln Asn Pro Gly Asp Ser Cys Ser Leu Gly Thr Pro Pro Gly 305 310 315
320 Gln Gly Pro Gln Ser 325 65 40 DNA Artificial Sequence
oligonucleotide ZC19372 65 tgtcgatgaa gccctgaaag acgcgcagac
taattcgagc 40 66 60 DNA Artificial Sequence oligonucleotide ZC19351
66 acgcgcagac taattcgagc tcccaccatc accatcacca cgcgaattcg
gtaccgctgg 60 67 60 DNA Artificial Sequence oligonucleotide ZC19352
67 actcactata gggcgaattg cccgggggat ccacgcggaa ccagcggtac
cgaattcgcg 60 68 37 DNA Artificial Sequence oligonucleotide ZC39319
68 atcggaattc gcagaagcca tggcgtggag ccttggg 37 69 28 DNA Artificial
Sequence oligonucleotide ZC39325 69 cagtggatcc ggaggggacc gtttcgtc
28
* * * * *
References