U.S. patent application number 11/538319 was filed with the patent office on 2009-08-06 for methods of producing antibodies to zalpha11 cytokine receptors.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Richard D. Holly, Andrew J. Nelson, Julia E. Novak, Scott R. Presnell, Cindy A. Sprecher, James W. West.
Application Number | 20090196882 11/538319 |
Document ID | / |
Family ID | 26890341 |
Filed Date | 2009-08-06 |
United States Patent
Application |
20090196882 |
Kind Code |
A1 |
Sprecher; Cindy A. ; et
al. |
August 6, 2009 |
METHODS OF PRODUCING ANTIBODIES TO ZALPHA11 CYTOKINE RECEPTORS
Abstract
Novel polypeptide combinations, polynucleotides encoding the
polypeptides, and related compositions and methods are disclosed
for soluble zalpha11 receptors that may be used as novel cytokine
antagonists, and within methods for detecting ligands that
stimulate the proliferation and/or development of hematopoietic,
lymphoid and myeloid cells in vitro and in vivo. Ligand-binding
receptor polypeptides can also be used to block zalpha11 Ligand
activity in vitro and in vivo, and may be used in conjunction with
zalpha11 Ligand and other cytokines to selectively stimulate the
immune system. The present invention also includes methods for
producing the protein, uses therefor and antibodies thereto.
Inventors: |
Sprecher; Cindy A.;
(Seattle, WA) ; Novak; Julia E.; (Bainbridge
Island, WA) ; West; James W.; (Seattle, WA) ;
Presnell; Scott R.; (Tacoma, WA) ; Holly; Richard
D.; (Seattle, WA) ; Nelson; Andrew J.;
(Shoreline, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
26890341 |
Appl. No.: |
11/538319 |
Filed: |
October 3, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10872087 |
Jun 18, 2004 |
7189695 |
|
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11538319 |
|
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|
09825561 |
Apr 3, 2001 |
6777539 |
|
|
10872087 |
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60194731 |
Apr 5, 2000 |
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60222121 |
Jul 28, 2000 |
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Current U.S.
Class: |
424/185.1 ;
530/387.9 |
Current CPC
Class: |
A61P 3/10 20180101; C07K
14/7155 20130101; C07K 2319/00 20130101; A61P 25/00 20180101; A61K
38/00 20130101; A61P 37/06 20180101; C07K 16/2866 20130101; C07K
2317/73 20130101; A61P 19/02 20180101; A61P 29/00 20180101; A61P
11/06 20180101 |
Class at
Publication: |
424/185.1 ;
530/387.9 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 16/18 20060101 C07K016/18 |
Claims
1. A method of producing an antibody to a soluble receptor
polypeptide comprising: inoculating an animal with a soluble
receptor polypeptide complex selected from the group consisting of:
(a) a polypeptide comprising a soluble receptor heterodimeric or
multimeric receptor complex comprising SEQ ID NO:6; (b) a
polypeptide comprising a soluble receptor heterodimeric or
multimeric receptor complex comprising SEQ ID NO:6, and further
comprising a soluble Class I cytokine receptor polypeptide; (c) a
polypeptide comprising a soluble receptor heterodimeric or
multimeric receptor complex comprising SEQ ID NO:6, and further
comprising a soluble IL-2R.gamma. receptor polypeptide (SEQ ID
NO:4); (d) a polypeptide comprising a soluble receptor
heterodimeric or multimeric receptor complex comprising SEQ ID
NO:6, and further comprising a soluble IL-13.alpha.'' receptor
polypeptide (SEQ ID NO:82); and wherein the polypeptide elicits an
immune response in the animal to produce the antibody; and
isolating the antibody from the animal; wherein the antibody
specifically binds to a receptor heterodimeric or multimeric
receptor complex comprising SEQ ID NO:6.
2. An antibody produced by the method of claim 1, which
specifically binds to a soluble receptor heterodimeric or
multimeric receptor complex comprising SEQ ID NO:6.
3. The antibody of claim 2, wherein the antibody is a monoclonal
antibody.
4. The antibody of claim 2, wherein the antibody antagonizes the
activity of the zalpha11 Ligand (IL-21) (SEQ ID NO: 10).
5. The antibody of claim 2, wherein the receptor heterodimeric or
multimeric receptor complex to which the antibody specifically
binds further comprises a biotin/avidin label, radionuclide,
enzyme, substrate, cofactor, inhibitor, fluorescent marker,
chemiluminescent marker, toxin, cytotoxic molecule or an
immunoglobulin Fc domain.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of co-pending U.S. patent
application Ser. No. 10/872,087, filed Jun. 18, 2004, which claims
benefit of U.S. Provisional Application 60/194,731, filed on Apr.
5, 2000, and U.S. Provisional Application 60/222,121, filed on Jul.
28, 2000, all of which are incorporated by reference. Under 35
U.S.C. .sctn. 119(e)(1), this application claims benefit of said
Provisional Applications. Additionally, this application claims
benefit of application Ser. No. 09/825,561 filed on Apr. 3, 2001,
issued as U.S. Pat. No. 6,777,539, under 35 U.S.C. 35 .sctn.
120.
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 or binding
partners. The present invention addresses these needs by providing
a new cytokine antagonist or binding partner, a soluble
hematopoietic cytokine receptor, as well as related compositions
and methods.
[0005] The present invention provides such polypeptides for these
and other uses that should be apparent to those skilled in the art
from the teachings herein.
DESCRIPTION OF THE INVENTION
[0006] Within one aspect, the present invention provides 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 as shown in SEQ ID NO:6, and
wherein the soluble receptor polypeptide encoded by the
polynucleotide sequence binds a ligand comprising a polypeptide of
SEQ ID NO:10 or SEQ ID NO:47, or antagonizes the ligand activity.
In one embodiment, the isolated polynucleotide is as disclosed
above, wherein the soluble receptor polypeptide encoded by the
polynucleotide forms a homodimeric receptor complex.
[0007] Within another aspect, the present invention provides 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 as shown in SEQ ID NO:6,
wherein the soluble receptor polypeptide encoded by the
polynucleotide forms a heterodimeric or multimeric receptor
complex. In one embodiment, the isolated polynucleotide is as
disclosed above, wherein the soluble receptor polypeptide encoded
by the polynucleotide forms a heterodimeric or multimeric receptor
complex further comprising a soluble Class I cytokine receptor.
[0008] In one embodiment, the isolated polynucleotide is as
disclosed above, wherein the soluble receptor polypeptide encoded
by the polynucleotide forms a heterodimeric or multimeric receptor
complex further comprising a soluble IL-2R.gamma. receptor
polypeptide (SEQ ID NO:4) or a soluble IL-13.alpha.'
receptorpolypeptide (SEQ ID NO:82). In another embodiment, the
isolated polynucleotide is as disclosed above, wherein the
polypeptide further comprises a WSXWS motif as shown in SEQ ID NO:
13.
[0009] Within another aspect, the present invention provides an
isolated polynucleotide that encodes a soluble receptor polypeptide
comprising a sequence of amino acid residues as shown in SEQ ID
NO:6, wherein the soluble receptor polypeptide encoded by the
polynucleotide forms a heterodimeric or multimeric receptor
complex. In one embodiment, the isolated polynucleotide is as
disclosed above, wherein the soluble receptor polypeptide encoded
by the polynucleotide further comprises a soluble Class I cytokine
receptor. In another embodiment, the isolated polynucleotide is as
disclosed above, wherein the soluble receptor polypeptide encoded
by the polynucleotide forms a heterodimeric or multimeric receptor
complex further comprising a soluble IL-2R.gamma. receptor
polypeptide (SEQ ID NO:4) or a soluble IL-13.alpha.' receptor
polypeptide (SEQ ID NO:82). In another embodiment, the isolated
polynucleotide is as disclosed above, wherein the soluble receptor
polypeptide is encoded by the polynucleotide as shown in SEQ ID
NO:7. In another embodiment, the isolated polynucleotide is as
disclosed above, wherein the soluble receptor polypeptide further
comprises an affinity tag.
[0010] Within a second aspect, the present invention provides an
expression vector comprising the following operably linked
elements: (a) a transcription promoter; a first DNA segment
encoding a soluble receptor polypeptide having an amino acid
sequence as shown in SEQ ID NO:6; and a transcription terminator;
and (b) a second transcription promoter; a second DNA segment
encoding a soluble Class I cytokine receptor polypeptide; and a
transcription terminator; and wherein the first and second DNA
segments are contained within a single expression vector or are
contained within independent expression vectors. In one embodiment,
the expression vector disclosed above further comprises a secretory
signal sequence operably linked to the first and second DNA
segments. In another embodiment, the expression vector is as
disclosed above, wherein the second DNA segment encodes a soluble
IL-2R.gamma. receptor polypeptide (SEQ ID NO:4) or a soluble
IL-13.alpha.' receptor polypeptide (SEQ ID NO:82).
[0011] Within a third aspect, the present invention provides a
cultured cell comprising an expression vector as disclosed above,
wherein the cell expresses the polypeptides encoded by the DNA
segments. In one embodiment, the cultured cell comprising an
expression vector is as disclosed above, wherein the first and
second DNA segments are located on independent expression vectors
and are co-transfected into the cell, and cell expresses the
polypeptides encoded by the DNA segments. In another embodiment,
the cultured cell comprising an expression vector is as disclosed
above, wherein the cell expresses a heterodimeric or multimeric
soluble receptor polypeptide encoded by the DNA segments. In
another embodiment, the cultured cell comprising an expression
vector is as disclosed above, wherein the cell secretes a soluble
receptor polypeptide heterodimer or multimeric complex. In another
embodiment, the cultured cell comprising an expression vector is as
disclosed above, wherein the cell secretes a soluble receptor
polypeptide heterodimer or multimeric complex that binds a ligand
comprising a polypeptide of SEQ ID NO:10 or SEQ ID NO:47, or
antagonizes the ligand activity.
[0012] Within another aspect, the present invention provides a DNA
construct encoding a fusion protein comprising: a first DNA segment
encoding a polypeptide having a sequence of amino acid residues as
shown in SEQ ID NO:6; and at least one other DNA segment encoding a
soluble Class I cytokine receptor polypeptide, wherein the first
and other DNA segments are connected in-frame; and wherein the
first and other DNA segments encode the fusion protein. In one
embodiment, the DNA construct encodes a fusion protein as disclosed
above, wherein at least one other DNA segment encodes a soluble
IL-2R.gamma. receptor polypeptide (SEQ ID NO:4) or a soluble
IL-13.alpha.' receptor polypeptide (SEQ ID NO:82).
[0013] Within another aspect, the present invention provides an
expression vector comprising the following operably linked
elements: a transcription promoter; a DNA construct encoding a
fusion protein as disclosed above; and a transcription terminator,
wherein the promoter is operably linked to the DNA construct, and
the DNA construct is operably linked to the transcription
terminator.
[0014] Within another aspect, the present invention provides a
cultured cell comprising an expression vector as disclosed above,
wherein the cell expresses a polypeptide encoded by the DNA
construct.
[0015] Within another aspect, the present invention provides a
method of producing a fusion protein comprising:culturing a cell as
disclosed above; and isolating the polypeptide produced by the
cell.
[0016] Within another aspect, the present invention provides an
isolated soluble receptor polypeptide comprising a sequence of
amino acid residues that is at least 90% identical to an amino acid
sequence as shown in SEQ ID NO:6, and wherein the soluble receptor
polypeptide binds a ligand comprising a polypeptide of SEQ ID NO:10
or SEQ ID NO:47, or antagonizes the ligand activity. In one
embodiment, the isolated polypeptide is as disclosed above, wherein
the soluble receptor polypeptide forms a homodimeric receptor
complex.
[0017] Within another aspect, the present invention provides an
isolated polypeptide comprising a sequence of amino acid residues
that is at least 90% identical to an amino acid sequence as shown
in SEQ ID NO:6, wherein the soluble receptor polypeptide forms a
heterodimeric or multimeric receptor complex. In one embodiment,
the isolated polypeptide is as disclosed above, wherein the soluble
receptor polypeptide forms a heterodimeric or multimeric receptor
complex further comprising a soluble Class I cytokine receptor. In
another embodiment, the isolated polypeptide is as disclosed above,
wherein the soluble receptor polypeptide forms a heterodimeric or
multimeric receptor complex further comprising a soluble
IL-2R.gamma. receptor polypeptide (SEQ ID NO:4) or a soluble
IL-13.alpha.' receptor polypeptide (SEQ ID NO:82). In another
embodiment, the isolated polypeptide is as disclosed above, wherein
the polypeptide further comprises a WSXWS motif as shown in SEQ ID
NO: 13.
[0018] Within another aspect, the present invention provides an
isolated soluble receptor polypeptide comprising a sequence of
amino acid residues as shown in SEQ ID NO:6, wherein the soluble
receptor polypeptide forms a heterodimeric or multimeric receptor
complex. In one embodiment, the isolated polypeptide is as
disclosed above, wherein the soluble receptor polypeptide forms a
heterodimeric or multimeric receptor complex further comprising a
soluble Class I cytokine receptor. In another embodiment, the
isolated polypeptide is as disclosed above, wherein the soluble
receptor polypeptide forms a heterodimeric or multimeric receptor
complex comprising a soluble IL-2R.gamma. receptor polypeptide (SEQ
ID NO:4) or a soluble IL-13.alpha.' receptor polypeptide (SEQ ID
NO:82). In another embodiment, the isolated polypeptide is as
disclosed above, wherein the soluble receptor polypeptide further
comprises an affinity tag, chemical moiety, toxin, or label.
[0019] Within another aspect, the present invention provides an
isolated heterodimeric or multimetric soluble receptor complex
comprising soluble receptor subunits, wherein at least one of
soluble receptor subunits comprises a soluble receptor polypeptide
comprising a sequence of amino acid residues as shown in SEQ ID
NO:6. In one embodiment, the isolated heterodimeric or multimetric
soluble receptor complex disclosed above further comprises a
soluble Class I cytokine receptor polypeptide. In another
embodiment, the isolated heterodimeric or multimetric soluble
receptor complex disclosed above further comprises a soluble
IL-2R.gamma. receptor polypeptide (SEQ ID NO:4) or a soluble
IL-13.alpha.' receptor polypeptide (SEQ ID NO:82).
[0020] Within another aspect, the present invention provides a
method of producing a soluble receptor polypeptide that form a
heterodimeric or multimeric complex comprising: culturing a cell as
disclosed above; and isolating the soluble receptor polypeptides
produced by the cell.
[0021] Within another aspect, the present invention provides a
method of producing an antibody to a soluble receptor polypeptide
comprising: inoculating an animal with a soluble receptor
polypeptide complex selected from the group consisting of: (a) a
polypeptide comprising a homodimeric soluble receptor complex
comprising SEQ ID NO:6; (b) a polypeptide comprising a soluble
receptor heterodimeric or multimeric receptor complex comprising
SEQ ID NO:6; (b) a polypeptide comprising a soluble receptor
heterodimeric or multimeric receptor complex comprising SEQ ID
NO:6, and further comprising a soluble Class I cytokine receptor
polypeptide; (c) a polypeptide comprising a soluble receptor
heterodimeric or multimeric receptor complex comprising SEQ ID
NO:6, and further comprising a soluble IL-2R.gamma. receptor
polypeptide (SEQ ID NO:4); (d) a polypeptide comprising a soluble
receptor heterodimeric or multimeric receptor complex comprising
SEQ ID NO:6, and further comprising a soluble IL-13.alpha.'
receptor polypeptide (SEQ ID NO:82); and wherein the polypeptide
complex elicits an immune response in the animal to produce the
antibody; and isolating the antibody from the animal.
[0022] Within another aspect, the present invention provides an
antibody produced by the method as disclosed above, which
specifically binds to a homodimeric, heterodimeric or multimeric
receptor complex comprising a soluble receptor polypeptide
comprising SEQ ID NO:6. In one embodiment the antibody disclosed
above is a monoclonal antibody. Within another aspect, the present
invention provides an antibody which specifically binds to a
homodimeric, heterodimeric or multimeric receptor complex as
disclosed above.
[0023] Within another aspect, the present invention provides a
method for inhibiting a ligand comprising a polypeptide of SEQ ID
NO:10 or SEQ ID NO:47, or antagonizing the ligand activity-induced
proliferation of hematopoietic cells and hematopoietic cell
progenitors comprising culturing bone marrow or peripheral blood
cells with a composition comprising an amount of soluble receptor
comprising SEQ ID NO:6 sufficient to reduce proliferation of the
hematopoietic cells in the bone marrow or peripheral blood cells as
compared to bone marrow or peripheral blood cells cultured in the
absence of soluble receptor. In one embodiment the method is as
disclosed above, wherein the hematopoietic cells and hematopoietic
progenitor cells are lymphoid cells. In another embodiment the
method is as disclosed above, wherein the lymphoid cells are NK
cells or cytotoxic T cells.
[0024] Within another aspect, the present invention provides a
method of reducing proliferation of neoplastic B or T cells
comprising administering to a mammal with a B or T cell neoplasm an
amount of a composition of soluble receptor comprising SEQ ID NO:6
sufficient to reduce proliferation of the neoplastic B or T
cells.
[0025] Within another aspect, the present invention provides a
method of suppressing an immune response in a mammal exposed to an
antigen or pathogen comprising: (1) determining a level of an
antigen- or pathogen-specific antibody; (2) administering a
composition of soluble receptor polypeptide comprising SEQ ID NO:6
in an acceptable pharmaceutical vehicle; (3) determining a post
administration level of antigen- or pathogen-specific antibody; (4)
comparing the level of antibody in step (1) to the level of
antibody in step (3), wherein a lack of increase or a decrease in
antibody level is indicative of suppressing an immune response.
[0026] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention.
[0027] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0028] 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.).
[0029] 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.
[0030] 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.
[0031] 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'.
[0032] 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).
[0033] 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.
[0034] 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).
[0035] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0036] 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.
[0037] 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.
[0038] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, 0-globin, and myoglobin
are paralogs of each other.
[0039] 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.
[0040] 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".
[0041] "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.
[0042] 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.
[0043] 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.
[0044] The term "receptor" is used herein to denote a
cell-associated protein, or a polypeptide subunit of such a
protein, that binds to a bioactive molecule (the "ligand") and
mediates the effect of the ligand on the cell. Binding of ligand to
receptor results in a conformational change in the receptor (and,
in some cases, receptor multimerization, i.e., association of
identical or different receptor subunits) that causes interactions
between the effector domain(s) and other molecule(s) in the cell.
These interactions in turn lead to alterations in the metabolism of
the cell. Metabolic events that are linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, cell proliferation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. Cell-surface cytokine receptors are
characterized by a multi-domain structure as discussed in more
detail below. These receptors are anchored in the cell membrane by
a transmembrane domain characterized by a sequence of hydrophobic
amino acid residues (typically about 21-25 residues), which is
commonly flanked by positively charged residues (Lys or Arg). In
general, receptors can be membrane bound, cytosolic or nuclear;
monomeric (e.g., thyroid stimulating hormone receptor,
beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,
growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF
receptor, erythropoietin receptor and IL-6 receptor). The term
"receptor polypeptide" is used to denote complete receptor
polypeptide chains and portions thereof, including isolated
functional domains (e.g., ligand-binding domains).
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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%.
[0049] All references cited herein are incorporated by reference in
their entirety.
[0050] The present invention is based in part upon the discovery of
a novel heterodimeric soluble receptor protein having the structure
of a class I cytokine receptor. The heterodimeric soluble receptor
includes at least one zalpha11 soluble receptor subunit, disclosed
in the commonly owned U.S. patent application Ser. No. 09/404,641.
A second soluble receptor polypeptide included in the heterodimeric
soluble receptor belongs to the receptor subfamily that includes
the IL-2 .gamma.-common receptor (IL-2R.gamma., or .gamma..sub.C),
IL-2 receptor .alpha.-subunit, and the .alpha.-common receptor
(i.e., IL3, IL-5, IL-13, IL-15 and GM-CSF receptor
.alpha.-subunits), IL-13a, IL-13.alpha.', IL-15 receptor subunits,
and the like. The soluble human and mouse zalpha11 receptor
(IL-21R) monomer and homodimer was shown to antagonize of the
activity of the natural ligand for the zalpha11 receptor, zalpha11
Ligand (IL-21) (Parrish-Novak, J. et al., Nature 408:57-63, 2000).
The zalpha11 Ligand is disclosed in the commonly owned U.S. patent
application Ser. No. 09/522,217. According to the present
invention, a heterodimeric soluble zalpha11 receptor, as
exemplified by a preferred embodiment of a soluble zalpha11
receptor+soluble IL-2R.gamma. receptor heterodimer
(zalpha11/IL-2R.gamma.), was shown to act as a potent antagonist of
the zalpha11 Ligand. As disclosed in the examples herein, the
preferred zalpha11/IL-2R.gamma. heterodimer was a more effective
antagonist zalpha11 Ligand activity, and hence more superior
antagonist, than a zalpha11 homodimer or monomer.
[0051] Moreover, also contemplated by the present invention are
homodimeric and monomeric zalpha11-comprising soluble receptors; as
well as homodimeric, heterodimeric, and multimeric
zalpha11-comprising receptors that are capable of intracellular
signaling. Such receptors can comprise at least one an
extracellular domain of a zalpha11 receptor, and an intracellular
domain from zalpha11 or another class I cytokine receptor. The
additional heterodimeric or multimeric subunit can comprise the
extracellular domain from IL-2R.gamma. receptor (e.g., SEQ ID
NO:4), IL-13a (also known as IL-13RA2; SEQ ID NO:84), IL-13.alpha.'
(also known as IL-13RA1; SEQ ID NO:82, IL-15 (SEQ ID NO:86)
receptor, or other class I receptor, and an intracellular domain
from zalpha11 or another class I cytokine receptor. The nucleotide
sequence of a representative zalpha11-encoding DNA is described in
SEQ ID NO:1 (from nucleotide 1 to 1614), and its deduced 538 amino
acid sequence is described in SEQ ID NO:2. In its entirety, the
zalpha11 polypeptide (SEQ ID NO:2) represents a full-length
polypeptide segment (residue 1 (Met) to residue 538 (Ser) of SEQ ID
NO:2). The domains and structural features of the zalpha11
polypeptide are further described below.
[0052] Analysis of the zalpha11 polypeptide encoded by the DNA
sequence of SEQ ID NO: 1 revealed an open reading frame encoding
538 amino acids (SEQ ID NO:2) comprising a predicted secretory
signal peptide of 19 amino acid residues (residue 1 (Met) to
residue 19 (Gly) of SEQ ID NO:2), and a mature polypeptide of 519
amino acids (residue 20 (Cys) to residue 538 (Ser) of SEQ ID NO:2).
In addition to the WSXWS motif (SEQ ID NO:13) corresponding to
residues 214 to 218 of SEQ ID NO:2, the receptor comprises a
cytokine-binding domain of approximately 200 amino acid residues
(residues 20 (Cys) to 237 (His) of SEQ ID NO:2); a domain linker
(residues 120 (Pro) to 123 (Pro) of SEQ ID NO:2); a penultimate
strand region (residues 192 (Lys) to 202 (Ala) of SEQ ID NO:2); a
transmembrane domain (residues 238 (Leu) to 255 (Leu) of SEQ ID
NO:2); complete intracellular signaling domain (residues 256 (Lys)
to 538 (Ser) of SEQ ID NO:2) which contains a "Box I" signaling
site (residues 267 (IIe) to 273 (Pro) of SEQ ID NO:2), and a "Box
II" signaling site (residues 301 (Leu) to 304 (Gly) of SEQ ID
NO:2). Moreover, there is a STAT3 binding site (YXXQ) located near
the C-terminus from residues 519 (Tyr) to 522 (Gln) of SEQ ID NO:2.
Those skilled in the art will recognize that these domain
boundaries are approximate, and are based on alignments with known
proteins and predictions of protein folding. In addition to these
domains, conserved receptor features in the encoded receptor
include (as shown in SEQ ID NO:2) a conserved Trp residue at
position 138, and a conserved Arg residue at position 201. Moreover
the zalpha11 contains conserved Cys residues typical of class I
cytokine receptors, shown in residues 25, 35, 65, and 81 of SEQ ID
NO:2, and corresponding regions of SEQ ID NO:6 and SEQ ID NO:69
described below. The corresponding polynucleotides encoding the
zalpha11 polypeptide regions, domains, motifs, residues and
sequences described above are as shown in SEQ ID NO: 1. The human
zalpha11 soluble receptor polypeptide, comprising residues 20 (Cys)
to 237 (His) of SEQ ID NO:2, is shown in SEQ ID NO:6, and the
corresponding polynucleotide sequence for the human zalpha11
soluble receptor polypeptide is shown in SEQ ID NO:5.
[0053] SEQ ID NO:3 is a polynucleotide sequence comprising a
fragment of the human IL-2R.gamma. receptor that encodes a soluble
232 amino acid soluble IL-2R.gamma. receptor polypeptide (SEQ ID
NO:4). Those skilled in the art will recognize that these domain
boundaries for the IL-2R.gamma. receptor extracellular domain are
approximate, and other soluble IL-2R.gamma. receptor polypeptides,
such as those including an IL-2R.gamma. receptor polypeptide
secretory signal sequence or additional IL-2R.gamma. receptor
polypeptide amino acids in the extracellular domain, are
encompassed within the scope of the present invention.
[0054] A variant form of the human zalpha11 polypeptide was
identified (WIPO publication No. WO 00/27822 shown as SEQ ID NO:3
and SEQ ID NO:4 therein) and is shown in the DNA sequence of SEQ ID
NO:64; and corresponding polypeptide sequence shown in (SEQ ID
NO:65). This particular alternative zalpha11 receptor polypeptide
contains 568 amino acids, and comprises a predicted secretory
signal peptide of 20 amino acid residues (residue 1 (Met) to
residue 20 (Gly) of SEQ ID NO:65), and a mature polypeptide of 548
amino acids (residue 21 (Met) to residue 568 (Ser) of SEQ ID
NO:65). In addition to the WSXWS motif (SEQ ID NO:13) corresponding
to residues 244 to 248 of SEQ ID NO:65, the receptor comprises a
cytokine-binding domain of approximately 200 amino acid residues
(residues 21 (Met) to 267 (His) of SEQ ID NO:65); no domain linker;
a penultimate strand region (residues 222 (Lys) to 232 (Ala) of SEQ
ID NO:65); a transmembrane domain (residues 268 (Leu) to 285 (Leu)
of SEQ ID NO:65); complete intracellular signaling domain (residues
286 (Lys) to 568 (Ser) of SEQ ID NO:65) which contains a "Box I"
signaling site (residues 297 (IIe) to 303 (Pro) of SEQ ID NO:65),
and a "Box II" signaling site (residues 331 (Leu) to 334 (Gly) of
SEQ ID NO:65). Moreover, there is a STAT3 binding site (YXXQ)
located near the C-terminus from residues 549 (Tyr) to 552 (Gln) of
SEQ ID NO:65. Those skilled in the art will recognize that these
domain boundaries are approximate, and are based on alignments with
known proteins and predictions of protein folding. In addition to
these domains, conserved receptor features in the encoded receptor
include (as shown in SEQ ID NO:65) a conserved Trp residue at
position 168, and a conserved Arg residue at position 231. The
corresponding polynucleotides encoding the zalpha11 polypeptide
regions, domains, motifs, residues and sequences described above
are as shown in SEQ ID NO:64. This particular human zalpha11
soluble receptor variant polypeptide, comprising residues 21 (Met)
to 267 (His) of SEQ ID NO:65 (SEQ ID NO:69) and the corresponding
polynucleotide sequence for this particular human zalpha11 soluble
receptor polypeptide is shown in SEQ ID NO:68. This variant form of
the human zalpha11 receptor is included in the heterodimeric and
multimeric zalpha11 receptor complexes of the present invention,
disclosed herein.
[0055] In addition, other variant forms of zalpha11 receptor are
contemplated by the present invention, wherein the extracellular
domain of the variant form disclosed above (e.g., 21 (Met) to 267
(His) of SEQ ID NO:65, or SEQ ID NO:69) comprises a domain linker
comprising the amino acids PAPP (SEQ ID NO:70) inserted between
amino acid 161 (Ser) and 162 (Arg) of SEQ ID NO:65, or the
corresponding region of SEQ ID NO:69. A preferred domain linker
comprises a sequence of amino acids from preferably 4 to 14 amino
acids long, most preferably 14 amino acids long, wherein aside from
the PAPP (SEQ ID NO:70) motif sequence any amino acid may be
present. For example, a representative linker-containing variant
zalpha11 soluble receptor is shown in SEQ ID NO:71. Moreover, other
variant zalpha11 sequences can include, in reference to SEQ ID
NO:65 a Gly at position 162 rather than an Arg, or the same Arg to
Gly substitution in the corresponding region of SEQ ID NO:69 or SEQ
ID NO:71, or other variant of SEQ ID NO:65 or SEQ ID NO:69
containing a domain linker as described above. Corresponding DNA
sequences that encode such variants can be readily determined by
one of skill in the art upon using the information present in Table
1 and Table 2.
[0056] The zalpha11 Ligand is a "short-helix" form secreted
four-helical bundle cytokine. The zalpha11 Ligand polynucleotide
sequence is shown in SEQ ID NO:9 and corresponding amino acid
sequence shown in SEQ ID NO:10. The secretory signal sequence
comprises amino acid residues 1 (Met) to 31 (Gly), and the mature
polypeptide comprises amino acid residues 32 (Gln) to 162 (Ser) (as
shown in SEQ ID NO: 10). In general, cytokines are predicted to
have a four-alpha helix structure, with helices A, C and D being
most important in ligand-receptor interactions, and are more highly
conserved among members of the family. Referring to the human
zalpha11 Ligand amino acid sequence shown in SEQ ID NO:10,
alignment of human zalpha11 Ligand, human IL-115, human IL-4, and
human GM-CSF amino acid sequences it is predicted that zalpha11
Ligand helix A is defined by amino acid residues 41-56; helix B by
amino acid residues 69-84; helix C by amino acid residues 92-105;
and helix D by amino acid residues 135-148; as shown in SEQ ID
NO:10. Structural analysis suggests that the A/B loop is long, the
B/C loop is short and the C/D loop is parallel long. Conserved
cysteine residues within zalpha11 Ligand correspond to amino acid
residues 71, 78, 122 and 125 of SEQ ID NO: 10. Consistent cysteine
placement is further confirmation of the four-helical-bundle
structure. Also highly conserved in the family comprising IL-15,
IL-2, IL-4, GM-CSF and zalpha11 Ligand is the Glu-Phe-Leu sequence
as shown in SEQ ID NO: 10 at residues 136-138.
[0057] Further analysis of zalpha11 Ligand based on multiple
alignments of known cytokines predicts that amino acid residues 44,
47 and 135 (as shown in SEQ ID NO: 10) play an important role in
zalpha11 Ligand binding to its cognate receptor. Based on
comparison between sequences of human and murine zalpha11 Ligand
well-conserved residues were found in the regions predicted to
encode alpha helices A and D. The corresponding polynucleotides
encoding the zalpha11 Ligand polypeptide regions, domains, motifs,
residues and sequences described herein are as shown in SEQ ID
NO:9. The murine zalpha11 Ligand is shown in SEQ ID NO:46, and
corresponding polypeptide sequence shown in SEQ ID NO:47.
[0058] The activity of molecules of the present invention can be
measured using a variety of assays that measure proliferation of
and/or binding to cells expressing the zalpha11 receptor. Of
particular interest are changes in zalpha11 Ligand-dependent cells.
A suitable cell line was engineered to be zalpha11 Ligand-dependent
that comprises an IL-3-dependent BaF3 cell line (Palacios and
Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell.
Biol. 6: 4133-4135, 1986). Moreover, other suitable cell lines to
be engineered to be zalpha11 Ligand-dependent include FDC-P1 (Hapel
et al., Blood 64: 786-790, 1984); and MO7e (Kiss et al., Leukemia
7: 235-240, 1993). Growth factor-dependent cell lines can be
established according to published methods (e.g. Greenberger et
al., Leukemia Res. 8: 363-375, 1984; Dexter et al., in Baum et al.
Eds., Experimental Hematology Today, 8th Ann. Mtg. Int. Soc. Exp.
Hematol. 1979, 145-156, 1980).
[0059] Zalpha11 Ligand stimulates proliferation, activation,
differentiation and/or induction or inhibition of specialized cell
function of cells involved homeostasis of hematopoiesis and immune
function. In particular, zalpha11 Ligand polypeptides stimulate
proliferation, activation, differentiation, induction or inhibition
of specialized cell functions of cells of the hematopoietic
lineages, including, but not limited to, T cells, B cells, NK
cells, dendritic cells, monocytes, and macrophages, as well as
epithelial cells. Proliferation and/or differentiation of
hematopoietic cells can be measured in vitro using cultured cells
or in vivo by administering zalpha11 Ligand to the appropriate
animal model. Assays measuring cell proliferation or
differentiation are well known in the art and described herein. For
example, assays measuring proliferation include such assays as
chemosensitivity to neutral red dye (Cavanaugh et al.,
Investigational New Drugs 8:347-354, 1990, incorporated herein by
reference), incorporation of radiolabeled nucleotides (Cook et al.,
Analytical Biochem. 179:1-7, 1989, incorporated herein by
reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the
DNA of proliferating cells (Porstmann et al., J. Immunol. Methods
82:169-179, 1985, incorporated herein by reference), and use of
tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983;
Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth
Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833,
1988; all incorporated herein by reference). Assays measuring
differentiation include, for example, measuring cell-surface
markers associated with stage-specific expression of a tissue,
enzymatic activity, functional activity or morphological changes
(Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75,
1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171,
1989; all incorporated herein by reference). Conversely, these
assays can be used in a competition to assess the antagonist or
zalpha11 Ligand binding activity of the soluble zalpha11 receptor
or soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. receptors of the present invention. Moreover,
the soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. receptors of the
present invention can be used as an antagonist or Ligand binding
agent to modulate the immune system and hematopoietic activities of
the zalpha11 Ligand.
[0060] Zalpha11 Ligand was isolated from tissue known to have
important immunological function and which contain cells that play
a role in the immune system. Zalpha11 Ligand is expressed in CD3+
selected, activated peripheral blood cells, and it has been shown
that zalpha11 Ligand expression increases after T cell activation.
Moreover, results of experiments described in commonly owned U.S.
patent application Ser. No. 09/522,217, and the Examples section
herein, demonstrate that zalpha11 Ligand has an effect on the
growth/expansion and/or differentiated state of NK cells or NK
progenitors. Additional evidence demonstrates that zalpha11 Ligand
affects proliferation and/or differentiation of T cells and B cells
in vivo. Factors that both stimulate proliferation of hematopoietic
progenitors and activate mature cells are generally known. NK cells
are responsive to IL-2 alone, but proliferation and activation
generally require additional growth factors. For example, it has
been shown that IL-7 and Steel Factor (c-kit ligand) were required
for colony formation of NK progenitors. IL-15+IL-2 in combination
with IL-7 and Steel Factor was more effective (Mr6 zek et al.,
Blood 87:2632-2640, 1996). However, unidentified cytokines may be
necessary for proliferation of specific subsets of NK cells and/or
NK progenitors (Robertson et. al., Blood 76:2451-2438, 1990). A
composition comprising zalpha11 Ligand and IL-15 stimulates NK
progenitors and NK cells, with evidence that this composition is
more potent than previously described factors and combinations of
factors. Such compositions can further comprise kit ligand or stem
cell factor. Thus, the soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. receptors of the present invention can be
used as an antagonist or Ligand binding agent to decrease the
activity of the zalpha11 Ligand on NK cells.
[0061] Moreover, the tissue distribution of a receptor for a given
cytokine offers a strong indication of the potential sites of
action of that cytokine. Northern analysis of zalpha11 receptor
revealed transcripts in human spleen, thymus, lymph node, bone
marrow, and peripheral blood leukocytes. Specific cell types were
identified as expressing zalpha11 receptors, and strong signals
were seen in a mixed lymphocyte reaction (MLR) and in the Burkitt's
lymphoma Raji. The two monocytic cell lines, THP-1 (Tsuchiya et
al., Int. J. Cancer 26:171-176, 1980) and U937 (Sundstrom et al.,
Int. J. Cancer 17:565-577, 1976), were negative. Zalpha11 receptor
is expressed at relatively high levels in the MLR, in which
peripheral blood mononuclear cells (PBMNC) from two individuals are
mixed, resulting in mutual activation. Detection of high levels of
transcript in the MLR but not in resting T or B cell populations
suggests that zalpha11 receptor expression may be induced in one or
more cell types during activation. Activation of isolated
populations of T and B cells can be artificially achieved by
stimulating cells with PMA and Ionomycin. When sorted cells were
subjected to these activation conditions, levels of zalpha11
receptor transcript increased in both cell types, supporting a role
for this receptor and zalpha11 Ligand in immune responses,
especially in autocrine and paracrine T and B cell expansions
during activation. Zalpha11 Ligand may also play a role in the
expansion of more primitive progenitors involved in lymphopoiesis.
Thus, the soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
receptors of the present invention can be used as an antagonist or
Ligand binding agent to modulate the lymphopoietic activities of
the zalpha11 Ligand.
[0062] Zalpha11 receptor was found to be present at low levels in
resting T and B cells, and was upregulated during activation in
both cell types. Interestingly, the B cells also down-regulate the
message more quickly than do T cells, suggesting that amplitude of
signal and timing of quenching of signal are important for the
appropriate regulation of B cell responses.
[0063] In addition, a large proportion of intestinal lamina propria
cells show positive hybridization signals with zalpha11 receptor.
This tissue consists of a mixed population of lymphoid cells,
including activated CD4+ T cells and activated B cells. Immune
dysfunction, in particular chronic activation of the mucosal immune
response, plays an important role in the etiology of Crohn's
disease and inflammatory bowel disease (IBD); abnormal response to
and/or production of proinflammatory cytokines is also a suspected
factor (Braegger et al., Annals Allergy 72:135-141, 1994; Sartor R
B Am. J. Gastroenterol. 92:5 S-11S, 1997). The zalpha11 Ligand in
concert with IL-15 expands NK cells from bone marrow progenitors
and augments NK cell effector function. Zalpha11 Ligand also
co-stimulates mature B cells stimulated with anti-CD40 antibodies,
but inhibits B cell proliferation to signals through IgM. Zalpha11
Ligand enhances T cell proliferation in concert with a signal
through the T cell receptor, and over expression in transgenic mice
leads to lymphopenia and an expansion of monocytes and
granulocytes. These pleiotropic effects of zalpha11 Ligand suggest
that molecules that antagonize or bind zalpha11 Ligand, such as the
molecules of the present invention, can provide therapeutic utility
for a wide range of diseases arising from defects in the immune
system, including (but not limited to) systemic lupus erythematosus
(SLE), rheumatoid arthritis (RA), multiple sclerosis (MS),
myasthenia gravis, Crohn's Disease, IBD, and diabetes. It is
important to note that these diseases are the result of a complex
network of immune dysfunction (SLE, for example, is the
manifestation of defects in both T and B cells), and that immune
cells are dependent upon interaction with one another to elicit a
potent immune response. Therefore, zalpha11 Ligand (or an
antagonist of the Ligand, such a molecule of the present invention)
that can be used to manipulate more than one type of immune cell is
an attractive therapeutic candidate for intervention at multiple
stages of disease.
[0064] Similarly, the tissue distribution of the mRNA corresponding
to IL-2R.gamma. receptor cDNA shows expression in hematopoietic and
lymphoid cells including CD4+ T-cells, CD8+ T-cells, CD20+ B-cells,
CD56+ NK cells, CD14+ monocytes, as well as granulocytes.
IL-2R.gamma. receptor cDNA is generally not found in other cell
types, including epithelial cells and fibroblast cells. The
expression pattern of this receptor correlates with the activities
of the zalpha11 Ligand and the localization of the zalpha11
receptor. Moreover, antibodies to the IL-2R.gamma. receptor
decrease or ablate the effect of zalpha11 Ligand in B-cells and
BaF3/zalpha11 receptor cells, demonstrating that the zalpha11
receptor and IL-2R.gamma. receptor can heterodimerize in vivo and
in vitro.
[0065] The zalpha11 Ligand both promotes expansion of NK cell
populations from bone marrow and regulates the proliferation of
mature T and B cells in response to activating stimuli. The
zalpha11 Ligand acts through a receptor complex that includes at
least one zalpha11 receptor subunit and the .gamma..sub.C subunit
of IL2R.gamma., even though the cytoplasmic domain of zalpha11
receptor is capable of transducing signal in a homodimeric
configuration (commonly owned U.S. patent application Ser. No.
09/522,217). IL4R.alpha. is also capable of signaling as a
homodimer (Kammer, W. et al., J. Biol. Chem. 271:23634-23637,
1996), although the true functional IL4 receptor complex is a
IL4R.alpha./.gamma..sub.C heterodimer. Signaling in BaF3/zalpha11
receptor could have resulted from interactions of the human
zalpha11 receptor with endogenous murine .gamma..sub.C, and
Examples herein show that antibodies to the .gamma..sub.C subunit
decrease zalpha11 Ligand signaling in these cells.
[0066] Moreover, the IL2 receptor has been studied in detail and is
composed of an .alpha.-.beta.-.gamma..sub.C heterotrimer. The
.beta. and .gamma..sub.C subunits are both essential for signal
transduction and are members of the hematopoietin receptor
superfamily (Cosman, D., Cytokine 5:95-106, 1993), whereas the
.alpha. subunit appears to primarily be involved in high-affinity
binding conversion and is structurally distinct from the
hematopoietin receptor family. The .gamma..sub.C subunit has been
shown to participate in forming the receptors for IL4, IL7, IL9,
and IL15, in addition to IL2 (for review, see Sugamura, K., et al.,
Annu. Rev. Immunol. 14:179-205, 1996), and null mutations in the
.gamma..sub.C gene have been shown to cause X-linked severe
combined immunodeficiency (X--SCID) (Noguchi, M. et al., Cell
73:147-157, 1993).
[0067] Zalpha11 Ligand antagonism of anti-IgM and IL4-induced B
cell proliferation (commonly owned U.S. patent application Ser. No.
09/522,217, and examples herein) could be due to competition for
.gamma..sub.C; however, it is clear that IL4 can signal through a
.gamma..sub.C-independent receptor (IL4R.alpha.+IL13R.alpha.')
(Murata, T. et al., Blood 91:3884-3891, 1998). B cells from human
SCID patients proliferate normally in response to anti-IgM and IL4
(Matthews, D. J. et al., Blood 85:38-42, 1995), and IL4
responsiveness in normal human B cells is associated with both IL13
responsiveness and levels of IL13R (Ford, D. et al., J. Immunol.
163:3185-3193, 1999). Similarly, Zalpha11 Ligand may signal through
a heterodimeric, heterotrimeric or multimeric complex that includes
zalpha11 receptor and a non-.gamma..sub.C-subunit. As such, the
present invention contemplates soluble zalpha11 receptor
heterodimeric antagonists and binding agents to the zalpha11 Ligand
that do not include the .gamma..sub.C subunit, but include an
additional Class I cytokine subunit, for example, IL13R.alpha.' and
the like.
[0068] The soluble receptors of the present invention are useful as
antagonists of the zalpha11 Ligand cytokine. Such antagonistic
effects can be achieved by direct neutralization or binding of the
zalpha11 Ligand. In addition to antagonistic uses, the soluble
receptors of the present invention can bind zalpha11 Ligand and act
as carrier proteins for the zalpha11 Ligand cytokine, in order to
transport the Ligand to different tissues, organs, and cells within
the body.
[0069] As such, the soluble receptors of the present invention can
be fused or coupled to molecules, polypeptides or chemical moieties
that direct the soluble-receptor-Ligand complex to a specific site,
such as a tissue, specific immune cell, or tumor. Thus, the soluble
receptors of the present invention can be used to specifically
direct the action of the zalpha11 Ligand. See, Cosman, D. Cytokine
5: 95-106, 1993; and Fernandez-Botran, R. Exp. Opin. Invest. Drugs
9:497-513, 2000.
[0070] Moreover, the soluble receptors of the present invention can
be used to stabilize the zalpha11 Ligand, to increase the
bio-availability, therapeutic longevity, and/or efficacy of the
Ligand by stabilizing the Ligand from degradation or clearance, or
by targeting the ligand to a site of action within the body. For
example the naturally occurring IL-6/soluble IL-6R complex
stabilizes IL-6 and can signal through the gp130 receptor. See,
Cosman, D. supra., and Fernandez-Botran, R. supra.
[0071] For example, the Zalpha11 Ligand will be useful in treating
tumorgenesis, and therefore would be useful in the treatment of
cancer. Zalpha11 Ligand inhibits IL-4 stimulated proliferation of
anti-IgM stimulated normal B-cells and a similar effect is observed
in B-cell tumor lines suggesting that there may be therapeutic
benefit in treating patients with the zalpha11 Ligand in order to
induce the B cell tumor cells into a less proliferative state. The
ligand could be administered in combination with other agents
already in use including both conventional chemotherapeutic agents
as well as immune modulators such as interferon alpha.
[0072] Alpha/beta interferons have been shown to be effective in
treating some leukemias and animal disease models, and the growth
inhibitory effects of interferon-alpha and zalpha11 Ligand are
additive for at least one B-cell tumor-derived cell line. Moreover,
stabilization of the zalpha11 Ligand or ability to target the
Ligand to specific sites of action with the soluble receptors of
the present invention would be desirable in this therapeutic
endeavor.
[0073] The present invention provides a method of reducing
proliferation of neoplastic B or T cells comprising administering
to a mammal with a B or T cell neoplasm an amount of a composition
of zalpha11 Ligand antagonist, such as the soluble receptors of the
present invention, sufficient to reduce proliferation of the
neoplastic B or T cells. In other embodiments, the composition can
comprise at least one other cytokine selected from the group
consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3 ligand or stem cell
factor. Furthermore, the zalpha11 Ligand antagonist can be a toxic
fusion. Similarly, soluble receptor-toxic fusions and soluble
receptors of the present invention can be used to reduce
proliferation of lymphoid and hematopoietic neoplasms that
over-express or grow in response to zalpha11 Ligand. Moreover,
indirect effects of the soluble receptors of the present invention
can modulate NK cell function induced by zalpha11 Ligand, and hence
indirectly enhance tumor cell killing.
[0074] The present invention provides polynucleotide molecules,
including DNA and RNA molecules that encode the heterodimeric
zalpha11 receptor 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:7 is a degenerate DNA
sequence that encompasses all DNAs that encode the soluble zalpha11
receptor polypeptide of SEQ ID NO:6. SEQ ID NO:66 is a degenerate
DNA sequence that encompasses all DNAs that encode the soluble
zalpha11 receptor polypeptide of SEQ ID NO:69. SEQ ID NO:8 is a
degenerate DNA sequence that encompasses all DNAs that encode the
soluble human IL-2R.gamma. polypeptide of SEQ ID NO:4. Those
skilled in the art will recognize that the degenerate sequence of
SEQ ID NO:7, SEQ ID NO:66 and SEQ ID NO:8 also provide all RNA
sequences encoding SEQ ID NO:6, SEQ ID NO:69 and SEQ ID NO:4
respectively by substituting U for T. Thus, zalpha11
polypeptide-encoding polynucleotides comprising nucleotide 1 to
nucleotide 654 of SEQ ID NO:7 or comprising nucleotide 1 to
nucleotide 741 of SEQ ID NO:66, soluble human IL-2R.gamma.
polypeptide-encoding polynucleotides comprising nucleotide 1 to
nucleotide 696 of SEQ ID NO:8, and their RNA equivalents are
contemplated by the present invention. Table 1 sets forth the
one-letter codes used within SEQ ID NO:7, SEQ ID NO:66 and SEQ ID
NO:8 to denote degenerate nucleotide positions. "Resolutions" are
the nucleotides denoted by a code letter. "Complement" indicates
the code for the complementary nucleotide(s). For example, the code
Y denotes either C or T, and its complement R denotes A or G, A
being complementary to T, and G being complementary to C.
TABLE-US-00001 TABLE 1 Nucleotide Resolution Complement Resolution
A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T
K G|T M A|C S C|G S C|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G
V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T
[0075] The degenerate codons used in SEQ ID NO:7, SEQ ID NO:66 and
SEQ ID NO:8 encompass all possible codons for a given amino acid,
are set forth in Table 2.
TABLE-US-00002 TABLE 2 Amino One 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
[0076] 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:6,
SEQ ID NO:69 or SEQ ID NO:4. Variant sequences can be readily
tested for functionality as described herein.
[0077] 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:7, SEQ ID NO:66 and SEQ ID NO:8 serves as a
template for optimizing expression of polynucleotides and
polypeptides 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.
[0078] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NO:5, SEQ ID NO:68 or SEQ ID NO:3, 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 at., (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 T.sub.m,
hybridization and wash solutions used, and are routinely determined
empirically by one of skill in the art. 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 zalpha11 receptor RNA or the RNA for the
heterodimeric component of the receptor, such as IL-2R.gamma., or
other class I cytokine receptor. Such tissues and cells are
identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA
77:5201, 1980), and include PBLs, spleen, thymus, and lymph
tissues, Raji cells, human erythroleukemia cell lines (e.g., TF-1),
acute monocytic leukemia cell lines, other lymphoid and
hematopoietic cell lines, and the like, for the zalpha11 receptor.
RNA for a heterodimeric component of the receptor, such as
IL-2R.gamma., or other class I cytokine receptor can be isolated
from lymphoid cells, such as those described above, and other cells
and tissues as is known in the art for these receptors. 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)+ 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
zalpha11 polypeptides are then identified and isolated by, for
example, hybridization or polymerase chain reaction (PCR) (Mullis,
U.S. Pat. No. 4,683,202).
[0079] The polynucleotides of the present invention can also be
synthesized using DNA synthesis machines. Currently the method of
choice is the phosphoramidite method. If chemically synthesized
double stranded DNA is required for an application such as the
synthesis of a gene or a gene fragment, then each complementary
strand is made separately. The production of short polynucleotides
(60 to 80 bp) is technically straightforward and can be
accomplished by synthesizing the complementary strands and then
annealing them. However, for producing longer polynucleotides
(>300 bp), special strategies are usually employed, because the
coupling efficiency of each cycle during chemical DNA synthesis is
seldom 100%. To overcome this problem, synthetic genes
(double-stranded) are assembled in modular form from
single-stranded fragments that are from 20 to 100 nucleotides in
length.
[0080] An alternative way to prepare a full-length gene is to
synthesize a specified set of overlapping oligonucleotides (40 to
100 nucleotides). After the 3' and 5' short overlapping
complementary regions (6 to 10 nucleotides) are annealed, large
gaps still remain, but the short base-paired regions are both long
enough and stable enough to hold the structure together. The gaps
are filled and the DNA duplex is completed via enzymatic DNA
synthesis by E. coli DNA polymerase I. After the enzymatic
synthesis is completed, the nicks are sealed with T4 DNA ligase.
Double-stranded constructs are sequentially linked to one another
to form the entire gene sequence which is verified by DNA sequence
analysis. See Glick and Pasternak, Molecular Biotechnology,
Principles & Applications of Recombinant DNA, (ASM Press,
Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53:
323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA
87:633-7, 1990. Moreover, other sequences are generally added that
contain signals for proper initiation and termination of
transcription and translation.
[0081] 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 heterodimeric
soluble receptor complexes combining soluble zalpha11 receptor and
soluble human IL-2R.gamma. or other soluble Class I cytokine
receptor polypeptides from other mammalian species, including
murine, porcine, ovine, bovine, canine, feline, equine, and other
primate polypeptides. Known and unknown orthologs of human soluble
zalpha11 receptor and soluble human IL-2R.gamma. or other soluble
Class I cytokine receptors can be cloned using information and
compositions provided by the present invention in combination with
conventional cloning techniques. For example, a cDNA can be cloned
using mRNA obtained from a tissue or cell type, such as lymphoid
cells, that expresses zalpha11receptor, human IL-2R.gamma. or other
Class I cytokine receptors. Moreover, suitable sources of mRNA can
be identified by probing Northern blots with probes designed from
the sequences disclosed herein. A library is then prepared from
mRNA of a positive tissue or cell line. A zalpha11-encoding cDNA
can then be isolated by a variety of methods, such as by probing
with a complete or partial human cDNA or with one or more sets of
degenerate probes based on the disclosed sequences. A cDNA can also
be cloned using PCR (Mullis, supra.), using primers designed from
the representative human zalpha11 sequence, or soluble human
IL-2R.gamma. sequence, disclosed herein. Within an additional
method, the cDNA library can be used to transform or transfect host
cells, and expression of the cDNA of interest can be detected with
an antibody to zalpha11 polypeptide. Similar techniques can also be
applied to the isolation of genomic clones.
[0082] 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 the
zalpha11 receptor is a ligand-binding domain, that binds zalpha11
Ligand, and the intracellular domain is an effector domain involved
in signal transduction, although ligand-binding and effector
functions can 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,
IL-13, IL-15 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: 13). 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
.alpha.-subunit, GM-CSF receptor .alpha.-subunit, GM-CSF receptor
.alpha.-subunit; and G-CSF, EPO, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-9, IL-13 and IL-15 receptors); TNF receptor family (including
TNF (p80) TNF (p60) receptors, CD27, CD30, CD40, Fas, and NGF
receptor).
[0083] Analysis of the zalpha11 receptor sequence suggests that it
is a member of the same receptor subfamily as the IL-2 receptor
.alpha.-subunit, IL-4, and IL-9, receptors. Certain receptors in
this subfamily (e.g., EPO-R or MPL) associate to form homodimers
that transduce a signal. Other members of the subfamily (e.g.,
IL-6, IL-11, and LIF receptors) combine with a second subunit
(termed a .alpha.-subunit) to bind ligand and transduce a signal.
Specific .alpha.-subunits associate with a plurality of specific
cytokine receptor subunits. For example, the .alpha.-subunit gp130
(Hibi et al., Cell 63:1149-1157, 1990) associates with receptor
subunits specific for IL-6, IL-11, and LIF (Gearing et al., EMBO J.
10:2839-2848, 1991; Gearing et al., U.S. Pat. No. 5,284,755).
Oncostatin M binds to a heterodimer of LIF receptor and gp130. CNTF
binds to trimeric receptors comprising CNTF receptor, LIF receptor,
and gp130 subunits. Moreover, IL-4 and IL-13 elicit responses
through the IL-4 and IL-13 receptors by acting upon various
functional heterodimeric receptor complexes, e.g. with and without
the .gamma..sub.C subunit, and such heterodimeric receptor
complexes may affect whether the cytokines act upon hematopoietic
or non-hematopoietic cells (Andersson, A. et al., Eur. J. Immunol.
27:1762-1768, 1997; Murata, T. et al., Blood 10:3884-3891, 1998).
Moreover, binding affinity of IL-4 on its receptor is increased
when the .gamma..sub.C subunit of the IL4R complex is replaced by
an IL-13R.alpha.' subunit (Murata, T. et al., supra.). Thus, the
soluble receptors of the present invention include zalpha11
receptor homodimers; and heterodimers that have a zalpha11 receptor
component, such as soluble zalpha11/IL-2R.gamma. or soluble
zalpha11 receptor heterodimerized with another soluble Class I
cytokine receptor, such as IL-13R.alpha. (SEQ ID NO:84),
IL-13R.alpha.' (SEQ ID NO:82) or an IL-15 (SEQ ID NO:86) receptor
subunit.
[0084] For example, suitable Class I cytokine soluble receptors
that can heterodimerize with a soluble zalpha11 receptor component
(e.g., SEQ ID NO:6), include a soluble receptor for IL-13R.alpha.
as shown in SEQ ID NO:84, IL-13R.alpha.' as shown in SEQ ID NO:82,
or IL-15 as shown in SEQ ID NO:86. Moreover, functional
sub-fragments, such as minimal cytokine binding fragments, of these
Class I cytokine soluble receptors can be used.
[0085] Such functional fragments include 1 to 322, 7 to 322, and
105 to 322 of SEQ ID NO:82; 1 to 317, 10 to 317, and 105 to 317 of
SEQ ID NO:84; and 1 to 173 of SEQ ID NO:86. The corresponding
polynucleotide sequences are as shown in SEQ ID NO:81, SEQ ID NO:83
and SEQ ID NO:85 respectively. It is well within the level of one
of skill in the art to delineate what sequences of a known class I
cytokine sequence comprise the extracellular cytokine binding
domain free of a transmembrane domain and intracellular domain.
[0086] A polynucleotide sequence for the mouse ortholog of human
zalpha11 receptor has been identified and is shown in SEQ ID NO: 11
and the corresponding amino acid sequence shown in SEQ ID NO: 12.
Analysis of the mouse zalpha11 polypeptide encoded by the DNA
sequence of SEQ ID NO: 11 revealed an open reading frame encoding
529 amino acids (SEQ ID NO:12) comprising a predicted secretory
signal peptide of 19 amino acid residues (residue 1 (Met) to
residue 19 (Ser) of SEQ ID NO:12), and a mature polypeptide of 510
amino acids (residue 20 (Cys) to residue 529 (Ser) of SEQ ID NO:2).
In addition to the WSXWS motif (SEQ ID NO:13) corresponding to
residues 214 to 218 of SEQ ID NO:12, the receptor comprises a
cytokine-binding domain of approximately 200 amino acid residues
(residues 20 (Cys) to 237 (His) of SEQ ID NO:12); a domain linker
(residues 120 (Pro) to 123 (Pro) of SEQ ID NO:12); a penultimate
strand region (residues 192 (Lys) to 202 (Ala) of SEQ ID NO:12); a
transmembrane domain (residues 238 (Met) to 254 (Leu) of SEQ ID NO:
12); complete intracellular signaling domain (residues 255 (Lys) to
529 (Ser) of SEQ ID NO:12) which contains a "Box I" signaling site
(residues 266 (IIe) to 273 (Pro) of SEQ ID NO:12), and a "Box II"
signaling site (residues 301 (IIe) to 304 (Val) of SEQ ID NO:2). A
comparison of the human and mouse amino acid sequences reveals that
both the human and orthologous polypeptides contain corresponding
structural features described above. The mature sequence for the
mouse zalpha11 begins at Cys.sub.20 (as shown in SEQ ID NO: 12),
which corresponds to Cys.sub.20 (as shown in SEQ ID NO:2) in the
human sequence. There is about 69% identity a between the mouse and
human zalpha11 sequences over the extracellular cytokine binding
domain corresponding to residues 20 (Cys) to 237 (His) of SEQ ID
NO:2 (SEQ ID NO:6) and residues 20 (Cys) to 237 (His) of SEQ ID NO:
12. The above percent identities were determined using a FASTA
program with ktup=1, gap opening penalty=12, gap extension
penalty=2, and substitution matrix=BLOSUM62, with other FASTA
program parameters set as default. The corresponding
polynucleotides encoding the mouse zalpha11 polypeptide regions,
domains, motifs, residues and sequences described above are as
shown in SEQ ID NO: 11.
[0087] The present invention also provides for a heterodimeric
soluble receptor, wherein the isolated soluble zalpha11 receptor
polypeptide therein is substantially similar to the polypeptides of
SEQ ID NO:6 and their orthologs. Moreover, in a preferred
embodiment, the present invention also provides for a heterodimeric
soluble receptor, wherein an isolated soluble IL-2R.gamma. receptor
polypeptide therein is substantially similar to the polypeptides of
SEQ ID NO:4 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:6 or their orthologs. Such polypeptides will more
preferably be at least 90% identical, and most preferably 95% or
more identical to SEQ ID NO:6 or SEQ ID NO:4 their orthologs.)
Percent sequence identity is determined by conventional methods.
See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616,
1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915-10919, 1992. Briefly, two amino acid sequences are aligned
to optimize the alignment scores using a gap opening penalty of 10,
a gap extension penalty of 1, and the "blosum 62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as:
Total number of identical matches [ length of the longer sequence
plus the number of gaps introduced into the longer sequence in
order to align the two sequences ] .times. 100 ##EQU00001##
TABLE-US-00003 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4
R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0
2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3
-3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3
1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3
-3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1
-2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1
-1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2
-3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2
-2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0088] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0089] 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 zsig57. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzmol. 183:63
(1990).
[0090] Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:6) 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
program 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 FASTA
program 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).
[0091] 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 program parameters set as default.
[0092] 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).
[0093] Variant zalpha11 polypeptides or substantially homologous
zalpha11 polypeptides are characterized as having one or more amino
acid substitutions, deletions or additions. These changes are
preferably of a minor nature, that is conservative amino acid
substitutions (see Table 4) and other substitutions that do not
significantly affect the folding or activity of the polypeptide;
small deletions, typically of one to about 30 amino acids; and
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide of up to
about 20-25 residues, or an affinity tag. The present invention
thus includes zalpha11 soluble receptor polypeptides of from about
190 to about 245 amino acid residues 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:6.
Polypeptides comprising affinity tags can further comprise a
proteolytic cleavage site between the zalpha11 polypeptide and the
affinity tag. Suitable sites include thrombin cleavage sites and
factor Xa cleavage sites.
TABLE-US-00004 TABLE 4 Conservative amino acid substitutions Basic:
arginine lysine histidine Acidic: glutamic acid aspartic acid
Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine
serine threonine methionine
[0094] The present invention further provides a variety of other
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a soluble zalpha11
receptor or soluble zalpha11 heterodimeric polypeptide, such as
soluble zalpha11/IL-2R.gamma. can be prepared as a fusion to a
dimerizing protein as disclosed in U.S. Pat. Nos. 5,155,027 and
5,567,584. Preferred dimerizing proteins in this regard include
immunoglobulin constant region domains, e.g., IgG.gamma.1, and the
human .kappa. light chain. Immunoglobulin-soluble zalpha11 receptor
or immunoglobulin-soluble zalpha11 heterodimeric polypeptide, such
as immunoglobulin-soluble zalpha11/IL-2R.gamma. fusions can be
expressed in genetically engineered cells to produce a variety of
multimeric zalpha11 receptor analogs. Auxiliary domains can be
fused to soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma. to
target them to specific cells, tissues, or macromolecules (e.g.,
collagen, or cells expressing the zalpha11 Ligand). A zalpha11
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. 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).
[0095] 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 zalpha11 amino acid residues.
[0096] 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.
[0097] 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.
[0098] Amino acid sequence changes are made in soluble zalpha11
receptor or soluble zalpha11 heterodimeric polypeptide, such as
soluble zalpha11/IL-2R.gamma., so as to minimize disruption of
higher order structure essential to biological activity. For
example, where the soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
comprises one or more helices, changes in amino acid residues will
be made so as not to disrupt the helix geometry and other
components of the molecule where changes in conformation abate some
critical function, for example, binding of the molecule to the
zalpha11 Ligand, or antagonizing zalpha11 Ligand activity. The
effects of amino acid sequence changes can be predicted by, for
example, computer modeling as disclosed above or determined by
analysis of crystal structure (see, e.g., Lapthorn et al., Nat.
Struct. Biol. 2:266-268, 1995). Other techniques that are well
known in the art compare folding of a variant protein to a standard
molecule (e.g., the native protein). For example, comparison of the
cysteine pattern in a variant and standard molecules can be made.
Mass spectrometry and chemical modification using reduction and
alkylation provide methods for determining cysteine residues which
are associated with disulfide bonds or are free of such
associations (Bean et al., Anal. Biochem. 201:216-226, 1992; Gray,
Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal. Chem.
66:3727-3732, 1994). It is generally believed that if a modified
molecule does not have the same disulfide bonding pattern as the
standard molecule folding would be affected. Another well known and
accepted method for measuring folding is circular dichrosism (CD).
Measuring and comparing the CD spectra generated by a modified
molecule and standard molecule is routine (Johnson, Proteins
7:205-214, 1990). Crystallography is another well known method for
analyzing folding and structure. Nuclear magnetic resonance MR),
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).
[0099] A Hopp/Woods hydrophilicity profile of the zalpha11 protein
sequence as shown in SEQ ID NO:6 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 the soluble zalpha11 receptor, hydrophilic regions
include amino acid residues 55 through 60 of SEQ ID NO: 2, amino
acid residues 56 through 61 of SEQ ID NO: 2, amino acid residues
139 through 144 of SEQ ID NO: 2, and amino acid residues 227
through 232 of SEQ ID NO: 2. The corresponding hydrophilic regions
in reference to SEQ ID NO:6 can be made with cross-reference to the
above amino acid residues of SEQ ID NO:2.
[0100] 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 soluble zalpha11
receptor or soluble zalpha11 heterodimeric polypeptide, such as
soluble zalpha11/IL-2R.gamma., 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:6,
SEQ ID NO:69 and SEQ ID NO:4. However, Cysteine residues could be
relatively intolerant of substitution.
[0101] The identities of essential amino acids can also be inferred
from analysis of sequence similarity between Class I cytokine
receptor family members with soluble zalpha11 receptor or soluble
IL-2R.gamma. receptor. 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 extracellular domain zalpha11 polynucleotide on the basis
of structure is to determine whether a nucleic acid molecule
encoding a potential variant zalpha11 polynucleotide can hybridize
to a nucleic acid molecule having the nucleotide sequence of SEQ ID
NO:5, or SEQ ID NO:68 as discussed above. Likewise, variants of
soluble class I cytokine receptor contained within a zalpha11
heterodimeric polypeptide, such as the soluble IL-2R.gamma.
receptor component in soluble zalpha11/IL-2R.gamma., can be
identified as described above.
[0102] Other methods of identifying essential amino acids in the
polypeptides of the present invention are procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Natl Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity as
disclosed below to identify amino acid residues that are critical
to the activity of the molecule. See also, Hilton et al., J. Biol.
Chem. 271:4699 (1996).
[0103] The present invention also includes functional fragments of
soluble zalpha11 receptor polypeptides or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
polypeptides and nucleic acid molecules encoding such functional
fragments. A "functional" soluble zalpha11 receptor polypeptide or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. polypeptide, or fragment thereof defined
herein is characterized by its by its ability to bind to an
anti-zalpha11 antibody, or to zalpha11 Ligand (either soluble or
immobilized), or to antagonize zalpha11 Ligand activity in, for
example, a biological or binding assay. As previously described
herein, zalpha11 receptor is characterized by a class I cytokine
receptor structure. Thus, the present invention further provides
fusion proteins encompassing: (a) homodimeric or multimeric
polypeptide molecules comprising an extracellular 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 I cytokine receptor, for
example, IL-2R.gamma., IL-2 receptor .alpha.-subunit and the
.beta.-common receptor (i.e., IL3, IL-5, and GM-CSF receptor
.alpha.-subunits), IL-13.alpha., IL-13.alpha.', or IL-15 receptor
subunits, or by a non-native and/or an unrelated secretory signal
peptide that facilitates secretion of the soluble fusion
protein.
[0104] Routine deletion analyses of nucleic acid molecules can be
performed to obtain functional fragments of a nucleic acid molecule
that encode a soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma..
As an illustration, DNA molecules having the nucleotide sequence of
SEQ ID NO:1 or fragments thereof, can be digested with Bal31
nuclease to obtain a series of nested deletions. These DNA
fragments are then inserted into expression vectors in proper
reading frame, and the expressed polypeptides are isolated and
tested for antagonizing zalpha11 Ligand biological or zalpha11
Ligand binding activity; or for the ability to bind anti-soluble
zalpha11 receptor or anti-soluble zalpha11 heterodimeric
polypeptide antibodies; or for the ability to bind zalpha11 Ligand.
One alternative to exonuclease digestion is to use
oligonucleotide-directed mutagenesis to introduce deletions or stop
codons to specify production of a desired polypeptide fragment.
Alternatively, particular fragments of a soluble zalpha11 receptor
or soluble zalpha11 heterodimeric polypeptide polynucleotide can be
synthesized using the polymerase chain reaction.
[0105] Standard methods for identifying functional domains, such as
Ligand binding domains, are routine for 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).
[0106] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner
et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO
92/062045) and region-directed mutagenesis (Derbyshire et al., Gene
46:145, 1986; Ner et al., DNA 7:127, 1988).
[0107] Variants of the disclosed soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide DNA and polypeptide
sequences can be generated through DNA shuffling as disclosed by
Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci.
USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly,
variant DNAs are generated by in vitro homologous recombination by
random fragmentation of a parent DNA followed by reassembly using
PCR, resulting in randomly introduced point mutations. This
technique can be modified by using a family of parent DNAs, such as
allelic variants or DNAs from different species, to introduce
additional variability into the process. Selection or screening for
the desired activity, followed by additional iterations of
mutagenesis and assay provides for rapid "evolution" of sequences
by selecting for desirable mutations while simultaneously selecting
against detrimental changes.
[0108] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect zalpha11
Ligand antagonist or binding activity in host cells of cloned,
mutagenized soluble zalpha11 receptor or soluble zalpha11
heterodimeric receptor polypeptides. Preferred assays in this
regard include cell proliferation assays and biosensor-based
ligand-binding assays, which are described below and in the
Examples. Mutagenized DNA molecules that encode active receptors or
portions thereof (e.g., ligand-binding fragments, and the like) can
be recovered from the host cells and rapidly sequenced using modern
equipment. These methods allow the rapid determination of the
importance of individual amino acid residues in a polypeptide of
interest, and can be applied to polypeptides of unknown
structure.
[0109] The present invention thus provides a series of novel,
hybrid molecules in which a segment comprising one or more of the
domains of soluble zalpha11 receptor is fused to another soluble
receptor 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 pharmicodynamics. Such hybrid molecules may further
comprise additional amino acid residues (e.g. a polypeptide linker)
between the component proteins or polypeptides.
[0110] 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:6 that retain zlpha11 Ligand
binding or antagonist activity. For example, one can make a
zalpha11 soluble receptor by preparing a variety of polypeptides
that are substantially homologous to the cytokine-binding domain
(residues 20 (Cys) to 237 (His) of SEQ ID NO:2 (SEQ ID NO:6), or a
subsequence therein that binds zalpha11 Ligand, or allelic variants
or species orthologs thereof) and retain ligand-binding activity of
the wild-type zalpha11 protein. Such polypeptides may include
additional amino acids from, for example, part or all of the signal
peptide sequence, transmembrane and intracellular domains. Such
polypeptides may also include additional polypeptide segments as
generally disclosed herein such as labels, affinity tags, and the
like. Similarly, one of ordinary skill in the art can identify
and/or prepare a variety of polypeptide fragments or variants of
SEQ ID NO:4, or other soluble class I cytokine receptors that form
heterodimers with zalpha11 receptor.
[0111] For any soluble zalpha11 receptor polypeptide, including
variants, 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.
[0112] The soluble zalpha11 receptor or soluble zalpha11
heterodimeric receptor polypeptides of the present invention,
including full-length soluble receptor 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.
[0113] In general, a DNA sequence encoding a zalpha11 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.
[0114] To direct a soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
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 zalpha11 receptor disclosed herein,
the IL-2R.gamma. (amino acid 1 (Met) to 22 (Gly) of SEQ ID NO: 18),
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 zalpha11 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).
[0115] 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, Rockville, Md. In general, strong
transcription promoters are preferred, such as promoters from SV-40
or cytomegalovirus (CMV). 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.
[0116] 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, flow cytometry, or magnetic bead separation
technology.
[0117] 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. (an alore)
11:47-58, 1987.
[0118] 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 zalpha11 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, pFastBac.TM. (Life Technologies)
containing a Tn7 transposon to move the DNA encoding the zalpha11
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 zalpha11 polypeptide, for
example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc.
Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known in the
art, a transfer vector containing zalpha11 is transformed into E.
Coli, and screened for bacmids which contain an interrupted lacZ
gene indicative of recombinant baculovirus. The bacmid DNA
containing the recombinant baculovirus genome is isolated, using
common techniques, and used to transfect Spodoptera frugiperda
cells, e.g. Sf9 cells. Recombinant virus that expresses zalpha11 is
subsequently produced. Recombinant viral stocks are made by methods
commonly used in the art.
[0119] 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.
[0120] 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 zalpha11 polypeptide from the supernatant can
be achieved using methods described herein.
[0121] 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 methanotica. 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.
[0122] The use of Pichia methanolica as host for the production of
recombinant proteins is disclosed in WIPO Publications WO 97/17450,
WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in
transforming P. methanolica will commonly be prepared as
double-stranded, circular plasmids, which are preferably linearized
prior to transformation. For polypeptide production in P.
methanolica, it is preferred that the promoter and terminator in
the plasmid be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A preferred selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), which allows ade2 host cells to grow in the absence of
adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, it is preferred to use
host cells in which both methanol utilization genes (AUG1 and AUG2)
are deleted. For production of secreted proteins, host cells
deficient in vacuolar protease genes (PEP4 and PRB1) are preferred.
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. It is preferred to transform P. methanolica
cells by electroporation using an exponentially decaying, pulsed
electric field having a field strength of from 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (t) of from 1 to
40 milliseconds, most preferably about 20 milliseconds. Prokaryotic
host cells, including strains of the bacteria Escherichia coli,
Bacillus and other genera are also useful host cells within the
present invention. Techniques for transforming these hosts and
expressing foreign DNA sequences cloned therein are well known in
the art (see, e.g., Sambrook et al., ibid.). When expressing a
zalpha11 polypeptide in bacteria such as E. coli, the polypeptide
may be retained in the cytoplasm, typically as insoluble granules,
or may be directed to the periplasmic space by a bacterial
secretion sequence. In the former case, the cells are lysed, and
the granules are recovered and denatured using, for example,
guanidine isothiocyanate or urea. The denatured polypeptide can
then be refolded and dimerized by diluting the denaturant, such as
by dialysis against a solution of urea and a combination of reduced
and oxidized glutathione, followed by dialysis against a buffered
saline solution. In the latter case, the polypeptide can be
recovered from the periplasmic space in a soluble and functional
form by disrupting the cells (by, for example, sonication or
osmotic shock) to release the contents of the periplasmic space and
recovering the protein, thereby obviating the need for denaturation
and refolding.
[0123] 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).
[0124] Mammalian cells suitable for use in assaying antagonist
activity of the novel soluble receptors of the present invention
express a zalpha11 receptor or receptor fusion capable of signaling
and transducing a receptor-mediated signal of the zalpha11 Ligand.
Such cells include cells that express a .alpha.-subunit, such as
gp130, IL-2R.gamma. and cells that co-express receptors (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 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 .alpha.-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) has been used to produce a cell line
responsive to the zalpha11 Ligand (see Examples). Other such lines
include a baby hamster kidney (BHK) cell line, or the CTLL-2 cell
line (ATCC TIB-214) can be transfected to express an IL-2R.gamma.
subunit in addition to zalpha11 receptor. 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 zalpha11 ligand. Such cells
can be used as described herein in the presence of zalpha11 Ligand
to assess the antagonist activity of soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. on zalpha11 Ligand signaling and
proliferative activity.
[0125] Cells expressing functional soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. 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 to the
zalpha11 Ligand in the presence or absence of the soluble receptors
of the present invention. One such assay is a cell proliferation
assay. Cells are cultured in the presence or absence of zalpha11
Ligand with or without the addition other cytokines or
proliferative agents, and cell proliferation is detected by, for
example, measuring incorporation of tritiated thymidine or by
colorimetric assay based on the metabolic breakdown of Alamar
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, and the assay detects
activation of transcription of the reporter gene. DNA response
elements can include, but are not limited to, cyclic AMP response
elements (CRE), hormone response elements (HRE) insulin response
element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7,
1990) and serum response elements (SRE) (Shaw et al. Cell 56:
563-72, 1989). Cyclic AMP response elements are reviewed in
Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener,
Molec. Endocrinol. 4 (8):1087-94; 1990. Hormone response elements
are reviewed in Beato, Cell 56:335-44; 1989. A preferred promoter
element in this regard is a serum response element, or SRE (see,
for example, Shaw et al., Cell 56:563-572, 1989). A preferred such
reporter gene is a luciferase gene (de Wet et al., Mol. Cell. Biol.
7:725, 1987). Expression of the luciferase gene is detected by
luminescence using methods known in the art (e.g., Baumgartner et
al., J. Biol. Chem. 269:19094-29101, 1994; Schenbom and Goiffin,
Promega Notes 41:11, 1993). Luciferase assay kits are commercially
available from, for example, Promega Corp., Madison, Wis. Target
cell lines of this type can be used to screen libraries of
chemicals, cell-conditioned culture media, fungal broths, soil
samples, water samples, and the like for antagonist activity. Such
cells can be used as described herein in the presence of zalpha11
Ligand in a competitive inhibition type assay to assess the
antagonist activity of soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. on zalpha11 Ligand signaling and
proliferative activity.
[0126] T- and B-cell proliferation assay methods can also be used
to assess the antagonist activity of soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. on zalpha11 Ligand signaling and
proliferative activity in the presence of other cytokines, for
example, IL-15, Flt3 and the like. Such assays are described in the
examples herein, and are know in the art. Briefly, using flow
cytometry, mature or immature subsets of T-cells or B-cells are
isolated based on the presence or absence of various cell surface
molecules (e.g., CD4, CD8, CD19, CD3, CD40, CD28, etc.). Cells can
be selected prior to or after exposure to zalpha11 Ligand,
depending on the cell type being studied, and the effect of
zalpha11 Ligand thereon. The soluble receptors or antibodies of the
present invention can be added at a range of concentrations to
assess the antagonistic or binding activity on the Ligand in the
T-cell or B-cell proliferation assay. Such assays are well known in
the art, and described herein.
[0127] Moreover, a secretion trap method employing soluble zalpha11
receptor or soluble zalpha11 heterodimeric receptor polypeptides
can be used to isolate transfected cells that express zalpha11
Ligand. For the method, see, Aldrich, et al., Cell 87: 1161-1169,
1996. A cDNA expression library prepared from a known or suspected
ligand source is transfected into COS-7 cells. The cDNA library
vector generally has an SV40 origin for amplification in COS-7
cells, and a CMV promoter for high expression. The transfected
COS-7 cells are grown in a monolayer and then fixed and
permeabilized. Tagged or biotin-labeled soluble zalpha11 receptor
or soluble zalpha11 heterodimeric receptor polypeptides, described
herein, is then placed in contact with the cell layer and allowed
to bind cells in the monolayer that express an anti-complementary
molecule, i.e., a zalpha11 Ligand. A cell expressing a ligand will
thus be bound with receptor molecules. An anti-tag antibody
(anti-Ig for Ig fusions, M2 or anti-FLAG for FLAG-tagged fusions,
streptavidin, and the like) which is conjugated with horseradish
peroxidase (HRP) is used to visualize these cells to which the
tagged or biotin-labeled soluble zalpha11 receptor or soluble
zalpha11 heterodimeric receptor polypeptides has bound. The HRP
catalyzes deposition of a tyramide reagent, for example,
tyramide-FITC. A commercially-available kit can be used for this
detection (for example, Renaissance TSA-Direct.TM. Kit; NEN Life
Science Products, Boston, Mass.). Cells which express zalpha11
receptor Ligand will be identified under fluorescence microscopy as
green cells and picked for subsequent cloning of the ligand using
procedures for plasmid rescue as outlined in Aldrich, et al.,
supra., followed by subsequent rounds of secretion trap assay until
single clones are identified.
[0128] Moreover, histologic and immunohistochemical methods
employing soluble zalpha11 receptor or soluble zalpha11
heterodimeric receptor polypeptides can be used to identify cells
and tissues cells that express zalpha11 Ligand. Such methods are
known in the art and described herein.
[0129] Additional assays to detect the antagonist or binding
activity of soluble zalpha11 receptor or soluble zalpha11
heterodimeric receptor polypeptides 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 zalpha11, comprising
approximately residues 256 (Lys) to 528 (Ser) of SEQ ID NO:2, is
joined to the ligand-binding domain of a second receptor. It is
preferred that the second receptor be a hematopoietic cytokine
receptor, such as mpl receptor (Souyri et al., Cell 63:1137-1147,
1990). The hybrid receptor will further comprise a transmembrane
domain, which may be derived from either receptor. A DNA construct
encoding the hybrid receptor is then inserted into a host cell.
Cells expressing the hybrid receptor are cultured in the presence
of a ligand for the binding domain and assayed for a response. This
system provides a means for analyzing signal transduction mediated
by zalpha11 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 zalpha11. A second class of
hybrid receptor polypeptides comprise the extracellular
(ligand-binding) domain of zalpha11 (approximately residues 20
(Cys) to 237 (His) of SEQ ID NO:2) (SEQ ID NO:6) 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. Such hybrid receptors are expressed in cells known
to be capable of responding to signals transduced by the receptor
comprising the extracellular domain, such as in the presence of the
zalpha11 Ligand. Addition of the soluble zalpha11 receptor or
soluble zalpha11 heterodimeric receptor polypeptides, in the
presence of the zalpha11 Ligand, is used to assess the zalpha11
Ligand antagonist or binding activity of the soluble zalpha11
receptor or soluble zalpha11 heterodimeric receptor polypeptides to
the zalpha11 Ligand.
[0130] The tissue specificity and biological activities of zalpha11
Ligand expression suggest a role in early NK cell and thymocyte
development, mature B-cell expansion, general immune response
stimulation, and immune response regulation. These processes
involve stimulation of cell proliferation and differentiation in
response to the binding of the zalpha11 Ligand to its cognate
receptor, comprising at least one zalpha11 receptor subunit. In
view of the biological activity observed for this Ligand,
antagonists have enormous potential in both in vitro and in vivo
applications. As antagonists of the zalpha11 Ligand, soluble
zalpha11 receptor or soluble zalpha11 heterodimeric receptor
polypeptides can find utility in the suppression of the immune
system, such as in the treatment of autoimmune diseases, including
rheumatoid arthritis, multiple sclerosis, diabetes mellitus,
inflammatory bowel disease, Crohn's disease, and the like. Immune
suppression can also be used to reduce rejection of tissue or organ
transplants and grafts and to treat B-cell malignancies, T-cell
specific leukemias or lymphomas by inhibiting proliferation of the
affected cell type.
[0131] Soluble zalpha11 receptor or soluble zalpha11 heterodimeric
receptor polypeptides may also be used within diagnostic systems
for the detection of circulating levels of zalpha11 Ligand. Within
a related embodiment, antibodies or other agents that specifically
bind to soluble zalpha11 receptor or soluble zalpha11 heterodimeric
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).
[0132] A ligand-binding polypeptide of a zalpha11 receptor, such as
soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. can be prepared
by expressing a truncated DNA encoding the zalpha11 cytokine
binding domain (approximately residue 20 (Cys) through residue 237
(His) of the human receptor (SEQ ID NO:2) (SEQ ID NO:6)) or the
corresponding region of a non-human receptor (e.g., SEQ ID NO: 12).
A soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. can be prepared by co-expressing a truncated
DNA encoding the zalpha11 cytokine binding domain (SEQ ID NO:6) and
the truncated DNA encoding the extracellular domain of another
class I cytokine receptor, such as IL-2R.gamma. (SEQ ID NO:4). It
is preferred that the extracellular domains of the soluble zalpha11
homodimer or heterodimer be prepared in a form substantially free
of transmembrane and intracellular polypeptide segments. Moreover,
ligand-binding polypeptide fragments within the soluble zalpha11
receptor or soluble zalpha11 heterodimeric polypeptide (e.g.,
soluble zalpha11/IL-2R.gamma.), or cytokine-binding domain,
described above, can also serve as zalpha11 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, secretory peptide from another cytokine
receptor, other secreted molecule, or a zalpha11 receptor secretory
peptide. To facilitate purification of the secreted receptor
polypeptide, a C-terminal extension, such as a poly-histidine tag,
substance P, Flag.TM. peptide (Hopp et al., Bio/Technology
6:1204-1210, 1988; available from Eastman Kodak Co., New Haven,
Conn.), Glu-glu tag (SEQ ID NO: 14) or another polypeptide or
protein for which an antibody or other specific binding agent is
available, can be fused to the soluble receptor polypeptide.
[0133] 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 zalpha11-Ig
chimera (e.g., Zalpha11-Fc4 described herein), 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, 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.
[0134] Moreover, soluble zalpha11 receptor or soluble zalpha11
heterodimeric receptor polypeptides, such as soluble
zalpha11/IL-2R.gamma., 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 amounts of
bioactive zalpha11 Ligand, soluble zalpha11 receptor or soluble
zalpha11 heterodimeric receptor polypeptides, such as soluble
zalpha11/IL-2R.gamma. can be used as a direct antagonist of the
ligand in vivo, and may aid in reducing progression and symptoms
associated with the disease, and can be used in conjunction with
other therapies (e.g., chemotherapy) to enhance the effect of the
therapy in reducing progression and symptoms, and preventing
relapse. Moreover, soluble zalpha11 receptor or soluble zalpha11
heterodimeric receptor polypeptides, such as soluble
zalpha11/IL-2R.gamma. can be used to slow the progression of
cancers that over-express zalpha11 receptors, by binding ligand in
vivo that would otherwise enhance proliferation of those
cancers.
[0135] Moreover, soluble zalpha11 receptor or soluble zalpha11
heterodimeric receptor polypeptides, such as soluble
zalpha11/IL-2R.gamma. can be used in vivo or in diagnostic
applications to detect zalpha11 Ligand-expressing cancers in vivo
or in tissue samples. For example, the soluble zalpha11 receptor or
soluble zalpha11 heterodimeric receptor polypeptides, such as
soluble zalpha11/IL-2R.gamma. can be conjugated to a radio-label or
fluorescent label as described herein, and used to detect the
presence of the zalpha11 Ligand in a tissue sample using an in
vitro ligand-receptor type binding assay, or fluorescent imaging
assay.
[0136] Moreover, a radiolabeled soluble zalpha11 receptor or
soluble zalpha11 heterodimeric receptor polypeptides, such as
soluble zalpha11/IL-2R.gamma. could be administered in vivo to
detect Ligand-expressing solid tumors through a radio-imaging
method known in the art.
[0137] 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.
[0138] Expressed recombinant soluble zalpha11 receptor or soluble
zalpha11 heterodimeric receptor polypeptides, such as soluble
zalpha11/IL-2R.gamma. (or zalpha11 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.
[0139] 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. Moreover zalpha11 Ligand
affinity columns can be used to purify soluble zalpha11 receptor or
soluble zalpha11 heterodimeric receptor polypeptides, such as
soluble zalpha11/IL-2R.gamma.. Such affinity chromatography methods
are well known in the art.
[0140] Moreover, using methods described in the art, polypeptide
fusions, or hybrid zalpha11 proteins, are constructed using regions
or domains of the inventive zalpha11 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.
[0141] Soluble receptor 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 zalpha11 of
the present invention with the functionally equivalent domain(s)
from another cytokine family member. Such domains include, but are
not limited to, the extracellular cytokine binding domain, ligand
binding domain and residues, transmembrane 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.
[0142] Standard molecular biological and cloning techniques can be
used to swap the equivalent domains between the zalpha11
polypeptide and those polypeptides to which they are fused.
Generally, a DNA segment that encodes a domain of interest, e.g., a
zalpha11 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
the IL-2R.gamma. 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. Such
fusion proteins can be expressed, isolated, and assayed for
activity as described herein.
[0143] Soluble zalpha11 receptor or soluble zalpha11 heterodimeric
receptor polypeptides, such as soluble zalpha11/IL-2R.gamma.
polypeptides, or fragments thereof may also be prepared through
chemical synthesis. Such polypeptides may be monomers or multimers;
glycosylated or non-glycosylated; pegylated or non-pegylated; and
may or may not include an initial methionine amino acid
residue.
[0144] Polypeptides of the present invention can also be
synthesized by exclusive solid phase synthesis, partial solid phase
methods, fragment condensation or classical solution synthesis.
Methods for synthesizing polypeptides are well known in the art.
See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; and
Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire
synthesis of the desired peptide on a solid support, the
peptide-resin is with a reagent that cleaves the polypeptide from
the resin and removes most of the side-chain protecting groups.
Such methods are well established in the art.
[0145] The activity of molecules of the present invention can be
measured using a variety of assays that measure cell
differentiation and proliferation. Such assays are well known in
the art and described herein.
[0146] Proteins of the present invention are useful for example, in
treating lymphoid, immune, hematopoietic, inflammatory disorders
and the like, and can be measured in vitro using cultured cells or
in vivo by administering molecules of the claimed invention to the
appropriate animal model. For instance, host cells expressing a
soluble zalpha11 receptor or soluble zalpha11 heterodimeric
receptor polypeptides, such as soluble zalpha11/IL-2R.gamma. can be
embedded in an alginate environment and injected (implanted) into
recipient animals. Alginate-poly-L-lysine microencapsulation,
permselective membrane encapsulation and diffusion chambers are a
means to entrap transfected mammalian cells or primary mammalian
cells. These types of non-immunogenic "encapsulations" permit the
diffusion of proteins and other macromolecules secreted or released
by the captured cells to the recipient animal. Most importantly,
the capsules mask and shield the foreign, embedded cells from the
recipient animal's immune response. Such encapsulations can extend
the life of the injected cells from a few hours or days (naked
cells) to several weeks (embedded cells). Alginate threads provide
a simple and quick means for generating embedded cells.
[0147] The materials needed to generate the alginate threads are
known in the art. In an exemplary procedure, 3% alginate is
prepared in sterile H.sub.2O, and sterile filtered. Just prior to
preparation of alginate threads, the alginate solution is again
filtered. An approximately 50% cell suspension (containing about
5.times.10.sup.5 to about 5.times.10.sup.7 cells/ml) is mixed with
the 3% alginate solution. One ml of the alginate/cell suspension is
extruded into a 100 mM sterile filtered CaCl.sub.2 solution over a
time period of .about.15 min, forming a "thread". The extruded
thread is then transferred into a solution of 50 mM CaCl.sub.2, and
then into a solution of 25 mM CaCl.sub.2. The thread is then rinsed
with deionized water before coating the thread by incubating in a
0.01% solution of poly-L-lysine. Finally, the thread is rinsed with
Lactated Ringer's Solution and drawn from solution into a syringe
barrel (without needle). A large bore needle is then attached to
the syringe, and the thread is intraperitoneally injected into a
recipient in a minimal volume of the Lactated Ringer's
Solution.
[0148] Adenoviral and other viral systems, such as vaccinia virus
can be used to express and produce the proteins of the present
invention. For example, using adenovirus vectors where portions of
the adenovirus genome are deleted, inserts are incorporated into
the viral DNA by direct ligation or by homologous recombination
with a co-transfected plasmid. In an exemplary system, the
essential E1 gene has been deleted from the viral vector, and the
virus will not replicate unless the E1 gene is provided by the host
cell (the human 293 cell line is exemplary). If the adenoviral
delivery system has an E1 gene deletion, the virus cannot replicate
in human cells, but will express and process (and, if a secretory
signal sequence is present, secrete) the heterologous protein.
Moreover, by deleting the entire adenovirus genome, very large
inserts of heterologous DNA can be accommodated. Generation of so
called "gutless" adenoviruses where all viral genes are deleted are
particularly advantageous for insertion of large inserts of
heterologous DNA. For review, see Yeh, P. and Perricaudet, M.,
FASEB J. 11:615-623, 1997.
[0149] The adenovirus system can be used for protein production in
vitro. By culturing adenovirus-infected non-293 cells under
conditions where the cells are not rapidly dividing, the cells can
produce proteins for extended periods of time. For instance, BHK
cells are grown to confluence in cell factories, are exposed to the
adenoviral vector encoding the secreted protein of interest. The
cells are then grown under serum-free conditions, which allows
infected cells to survive for several weeks without significant
cell division. Alternatively, adenovirus vector infected 293 cells
can be grown as adherent cells or in suspension culture at
relatively high cell density to produce significant amounts of
protein (See Garnier et al., Cytotechnol. 15:145-55, 1994). With
either protocol, an expressed, secreted heterologous protein can be
repeatedly isolated from the cell culture supernatant, lysate, or
membrane fractions depending on the disposition of the expressed
protein in the cell. Within the infected 293 cell production
protocol, non-secreted proteins may also be effectively
obtained.
[0150] Soluble zalpha11 receptor or soluble zalpha11 heterodimeric
receptor polypeptides, such as soluble zalpha11/IL-2R.gamma.
receptor antagonists can be used in vitro in an assay to measure a
decrease in stimulation of colony formation by zalpha11 Ligand from
isolated primary bone marrow cultures. Such assays are disclosed
herein and are well known in the art.
[0151] Zalpha11 Ligand antagonists and binding agents are also
useful as research reagents for characterizing sites of
ligand-receptor interaction. Inhibitors of zalpha11 Ligand activity
(zalpha11 Ligand antagonists) include anti-soluble zalpha11
receptor or anti-soluble zalpha11 heterodimeric receptor
polypeptide antibodies, such as anti-soluble zalpha11/IL-2R.gamma.
antibodies and soluble zalpha11 receptor or soluble zalpha11
heterodimeric receptor polypeptides, such as soluble
zalpha11/IL-2R.gamma. receptors, as well as other peptidic and
non-peptidic agents (including ribozymes).
[0152] A soluble zalpha11 receptor or soluble zalpha11
heterodimeric receptor polypeptides, such as soluble
zalpha11/IL-2R.gamma. ligand-binding polypeptide of the present
invention, can also be used for purification of zalpha11 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.
[0153] An assay system that uses a ligand-binding receptor (or an
antibody, one member of a complement/anti-complement pair) or a
binding fragment thereof, and a commercially available biosensor
instrument may be advantageously employed (e.g., BIAcore.TM.,
Pharmacia Biosensor, Piscataway, N.J.; or SELDI.TM. technology,
Ciphergen, Inc., Palo Alto, Calif.). Such receptor, antibody,
member of a complement/anti-complement pair or fragment is
immobilized onto the surface of a receptor chip. Use of this
instrument is disclosed by Karlsson, J. Immunol. Methods
145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol.
234:554-63, 1993. A receptor, antibody, member or fragment is
covalently attached, using amine or sulfhydryl chemistry, to
dextran fibers that are attached to gold film within the flow cell.
A test sample is passed through the cell. If a ligand, epitope, or
opposite member of the complement/anti-complement pair is present
in the sample, it will bind to the immobilized receptor, antibody
or member, respectively, causing a change in the refractive index
of the medium, which is detected as a change in surface plasmon
resonance of the gold film. This system allows the determination of
on- and off-rates, from which binding affinity can be calculated,
and assessment of stoichiometry of binding.
[0154] Ligand-binding receptor polypeptides, such as those of the
present invention, 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).
[0155] Soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. polypeptides can
also be used to prepare antibodies that bind to epitopes, peptides,
or polypeptides contained within the antigen. The zalpha11 receptor
or soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. polypeptides 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 zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. polypeptides (e.g., SEQ ID NO:2, SEQ ID NO:6,
SEQ ID NO:4). Polypeptides comprising a larger portion of a
zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide,
such as soluble zalpha11/IL-2R.gamma. polypeptides 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 zalpha11 polypeptide encoded by SEQ
ID NO:2 from amino acid number 20 (Cys) to amino acid number 237
(His) (SEQ ID NO:6), or a contiguous 9 to 218 AA amino acid
fragment thereof. Preferred peptides to use as antigens are the
cytokine binding domain, disclosed herein, and zalpha11 hydrophilic
peptides such as those predicted by one of skill in the art from a
hydrophobicity plot, determined for example, from a Hopp/Woods
hydrophilicity profile based on a sliding six-residue window, with
buried G, S, and T residues and exposed H, Y, and W residues
ignored. For example, zalpha11 hydrophilic peptides include
peptides comprising amino acid sequences selected from the group
consisting of: (1) amino acid number 51 (Trp) to amino acid number
61 (Glu) of SEQ ID NO:2; (2) amino acid number 136 (IIe) to amino
acid number 143 (Glu) of SEQ ID NO:2; (3) amino acid number 187
(Pro) to amino acid number 195 (Ser) of SEQ ID NO:2; and (4) amino
acid number 223 (Phe) to amino acid number 232 (Glu) of SEQ ID
NO:2. The corresponding hydrophilic regions in reference to SEQ ID
NO:6 can be made with cross-reference to the above amino acid
residues of SEQ ID NO:2. Moreover, antigenic epitope-bearing
polypeptides 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 zalpha11 soluble receptor are suitable
antigens. Suitable antigens also include the zalpha11 polypeptides
disclosed above in combination with another class I cytokine
extracellular domain, such as those that form soluble zalpha11
heterodimeric polypeptides, such as soluble zalpha11/IL-2R.gamma..
Moreover, corresponding regions of the mouse soluble zalpha11
receptor polypeptide (residues 20 (Cys) to 237 (His) SEQ ID NO:12)
can be used to generate antibodies against the soluble mouse
zalpha11 receptor. In addition 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.
[0156] 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 soluble zalpha11 receptor
or soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. polypeptide or a fragment thereof. The
immunogenicity of a zalpha11 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
zalpha11 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.
[0157] 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.
[0158] 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-soluble zalpha11
receptor or anti-soluble zalpha11 heterodimeric polypeptide, such
as anti-soluble zalpha11/IL-2R.gamma. antibodies herein bind to a
soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. polypeptide,
peptide or epitope with an affinity at least 10-fold greater than
the binding affinity to control (non-soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma.) polypeptide. It is preferred that the
antibodies exhibit a binding affinity (Ka) of 10.sup.6 M.sup.-1 or
greater, preferably 10.sup.7 M or greater, more preferably 10.sup.8
M.sup.-1 or greater, and most preferably 109 M 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).
[0159] Whether anti-soluble zalpha11 receptor or anti-soluble
zalpha11 heterodimeric polypeptide, such as anti-soluble
zalpha11/IL-2R.gamma. antibodies do not significantly cross-react
with related polypeptide molecules is shown, for example, by the
antibody detecting soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
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.
[0160] Screening can also be done using non-human soluble zalpha11
receptor or soluble zalpha11 heterodimeric polypeptide, such as
soluble zalpha11/IL-2R.gamma., and soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. mutant polypeptides. Moreover, antibodies can
be "screened against" known related polypeptides, to isolate a
population that specifically binds to the soluble zalpha11 receptor
or soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. polypeptides. For example, antibodies raised
to soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. are adsorbed to
related polypeptides adhered to insoluble matrix; antibodies
specific to soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
will flow through the matrix under the proper buffer conditions.
Screening allows isolation of polyclonal and monoclonal antibodies
non-crossreactive to known closely related polypeptides
(Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold
Spring Harbor Laboratory Press, 1988; Current Protocols in
Immunology, Cooligan, et al. (eds.), National Institutes of Health,
John Wiley and Sons, Inc., 1995). Screening and isolation of
specific antibodies is well known in the art. See, Fundamental
Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in
Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and
Practice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjamin
et al., Ann. Rev. Immunol. 2: 67-101, 1984. Specifically binding
anti-soluble zalpha11 receptor or anti-soluble zalpha11
heterodimeric polypeptide, such as anti-soluble
zalpha11/IL-2R.gamma. antibodies can be detected by a number of
methods in the art, and disclosed below.
[0161] A variety of assays known to those skilled in the art can be
utilized to detect antibodies that bind to soluble zalpha11
receptor or soluble zalpha11 heterodimeric polypeptide, such as
soluble zalpha11/IL-2R.gamma. proteins or polypeptides. Exemplary
assays are described in detail in Antibodies: A Laboratory Manual,
Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988.
Representative examples of such assays include: concurrent
immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,
enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot
assay, inhibition or competition assay, and sandwich assay. In
addition, antibodies can be screened for binding to wild-type
versus mutant soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
protein or polypeptide.
[0162] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. protein or
peptide, and selection of antibody display libraries in phage or
similar vectors (for instance, through use of immobilized or
labeled soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. protein or
peptide). Genes encoding polypeptides having potential binding
domains for soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
polypeptide, can be obtained by screening random peptide libraries
displayed on phage (phage display) or on bacteria, such as E. coli.
Nucleotide sequences encoding the polypeptides can be obtained in a
number of ways, such as through random mutagenesis and random
polynucleotide synthesis. These random peptide display libraries
can be used to screen for peptides which interact with a known
target which can be a protein or polypeptide, such as a ligand or
receptor, a biological or synthetic macromolecule, or organic or
inorganic substances. Techniques for creating and screening such
random peptide display libraries are known in the art (Ladner et
al., U.S. Pat. No. 5,223,409; Ladner et al., U.S. Pat. No.
4,946,778; Ladner et al., U.S. Pat. No. 5,403,484 and Ladner et
al., U.S. Pat. No. 5,571,698) and random peptide display libraries
and kits for screening such libraries are available commercially,
for instance from Clontech (Palo Alto, Calif.), Invitrogen Inc.
(San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) and
Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide
display libraries can be screened using the soluble zalpha11
receptor or soluble zalpha11 heterodimeric polypeptide, such as
soluble zalpha11/IL-2R.gamma. sequences disclosed herein to
identify proteins which bind to soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma.. These "binding polypeptides" which interact
with soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. polypeptides can
be used for tagging cells; for isolating homolog polypeptides by
affinity purification; they can be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like. These
binding polypeptides can also be used in analytical methods such as
for screening expression libraries and neutralizing activity, e.g.,
for blocking interaction between ligand and receptor, or viral
binding to a receptor. The binding polypeptides can also be used
for diagnostic assays for determining circulating levels of soluble
zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide,
such as soluble zalpha11/IL-2R.gamma. polypeptides; for detecting
or quantitating soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
polypeptides as marker of underlying pathology or disease. These
binding polypeptides can also act as zalpha11 receptor or zalpha11
heterodimeric polypeptide, such as zalpha11/IL-2R.gamma.
"antagonists" to block zalpha11 receptor or zalpha11 heterodimeric
polypeptide, such as zalpha11/IL-2R.gamma. binding and signal
transduction in vitro and in vivo. Again, these anti-soluble
zalpha11 receptor or anti-soluble zalpha11 heterodimeric
polypeptide, such as anti-soluble zalpha11/IL-2R.gamma. binding
polypeptides would be useful for inhibiting zalpha11 Ligand
activity, as well as receptor activity or protein-binding.
Antibodies raised to the heterodimer or multimeric combinations of
the present invention are preferred embodiments, as they may act
more specifically against the zalpha11 Ligand, or more potently
than antibodies raised to only one subunit. Moreover, the
antagonistic and binding activity of the antibodies of the present
invention can be assayed in the zalpha11 Ligand proliferation and
other biological assays described herein.
[0163] Antibodies to soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
may be used for tagging cells that express zalpha11 receptor or
zalpha11 heterodimeric polypeptides, such as zalpha11/IL-2R.gamma.;
for isolating soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
polypeptide by affinity purification; for diagnostic assays for
determining circulating levels of soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. polypeptides; for detecting or quantitating
soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. as marker of
underlying pathology or disease; in analytical methods employing
FACS; for screening expression libraries; for generating
anti-idiotypic antibodies; and as neutralizing antibodies or as
antagonists to block zalpha11 receptor or zalpha11 heterodimeric
polypeptide, such as zalpha11/IL-2R.gamma., or zalpha11 Ligand
activity in vitro and in vivo. Suitable direct tags or labels
include radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent markers, chemiluminescent markers, magnetic particles
and the like; indirect tags or labels may feature use of
biotin-avidin or other complement/anti-complement pairs as
intermediates. Antibodies herein may also be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like, and these
conjugates used for in vivo diagnostic or therapeutic applications.
Moreover, antibodies to soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. or fragments thereof may be used in vitro to
detect denatured or non-denatured soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. or fragments thereof in assays, for example,
Western Blots or other assays known in the art.
[0164] Antibodies to soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
are useful for tagging cells that express the corresponding
receptors and assaying their expression levels, for affinity
purification, within diagnostic assays for determining circulating
levels of soluble receptor polypeptides, analytical methods
employing fluorescence-activated cell sorting. Moreover, divalent
antibodies, and anti-idiotypic antibodies may be used as agonists
to mimic the effect of the zalpha11 Ligand.
[0165] Antibodies herein can also be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like, and these
conjugates used for in vivo diagnostic or therapeutic applications.
For instance, antibodies or binding polypeptides which recognize
soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. polypeptides of
the present invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary molecule
(i.e., a zalpha11 receptor, or zalpha11 heterodimeric receptor,
such as zalpha11/IL-2R.gamma.). More specifically, anti-soluble
zalpha11 receptor or anti-soluble zalpha11 heterodimeric
polypeptide, such as anti-soluble zalpha11/IL-2R.gamma. antibodies,
or bioactive fragments or portions thereof, can be coupled to
detectable or cytotoxic molecules and delivered to a mammal having
cells, tissues or organs that express the zalpha11 receptor or a
zalpha11 heterodimeric receptor, such as zalpha11/IL-2R.gamma.
receptor molecules.
[0166] Suitable detectable molecules may be directly or indirectly
attached to polypeptides that bind soluble zalpha11 receptor or
soluble zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. ("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.
[0167] In another embodiment, binding polypeptide-toxin fusion
proteins or antibody-toxin fusion proteins can be used for targeted
cell or tissue inhibition or ablation (for instance, to treat
cancer cells or tissues). Alternatively, if the binding polypeptide
has multiple functional domains (i.e., an activation domain or a
ligand binding domain, plus a targeting domain), a fusion protein
including only the targeting domain may be suitable for directing a
detectable molecule, a cytotoxic molecule or a complementary
molecule to a cell or tissue type of interest. In instances where
the fusion protein including only a single domain includes a
complementary molecule, the anti-complementary molecule can be
conjugated to a detectable or cytotoxic molecule. Such
domain-complementary molecule fusion proteins thus represent a
generic targeting vehicle for cell/tissue-specific delivery of
generic anti-complementary-detectable/cytotoxic molecule
conjugates.
[0168] In another embodiment, soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. binding polypeptide-cytokine or
antibody-cytokine fusion proteins can be used for enhancing in vivo
killing of target tissues (for example, blood, lymphoid, colon, and
bone marrow cancers), if the binding polypeptide-cytokine or
anti-soluble zalpha11 receptor or anti-soluble zalpha11
heterodimeric polypeptide, such as anti-soluble
zalpha11/IL-2R.gamma. antibody targets the hyperproliferative cell
(See, generally, Homick et al., Blood 89:4437-47, 1997). The
described fusion proteins enable targeting of a cytokine to a
desired site of action, thereby providing an elevated local
concentration of cytokine. Suitable anti-zalpha11 homodimer and
heterodimer 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.
[0169] Alternatively, soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
binding polypeptide or antibody fusion proteins described herein
can be used for enhancing in vivo killing of target tissues by
directly stimulating a zalpha11 receptor-modulated apoptotic
pathway, resulting in cell death of hyperproliferative cells
expressing zalpha11 receptor or a zalpha11 heterodimeric receptor,
such as soluble zalpha11/IL-2R.gamma. receptor.
[0170] Four-helix bundle cytokines that bind to cytokine receptors
as well as other proteins produced by activated lymphocytes play an
important biological role in cell differentiation, activation,
recruitment and homeostasis of cells throughout the body.
Therapeutic utility includes treatment of diseases that require
immune regulation including autoimmune diseases, such as,
rheumatoid arthritis, multiple sclerosis, myasthenia gravis,
systemic lupus erythomatosis and diabetes. Zalpha11 Ligand
antagonists, including soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma., may be important in the regulation of
inflammation, and therefore would be useful in treating rheumatoid
arthritis, asthma, ulcerative colitis, inflammatory bowel disease,
Crohn's disease, and sepsis. There may be a role of zalpha11 Ligand
antagonists, including soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma., in mediating tumorgenesis, and therefore
would be useful in the treatment of cancer. Zalpha11 Ligand
antagonists, including soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma., may be a potential therapeutic in
suppressing the immune system that would be important for reducing
graft rejection. Soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
may have usefulness in prevention of graft vs. host disease.
[0171] Alternatively, zalpha11 Ligand antagonists, including
soluble zalpha11 receptor or soluble zalpha11 heterodimeric
polypeptide, such as soluble zalpha11/IL-2R.gamma. receptors in
conjunction with other cytokines may enable selective activation,
enhancement, or selective suppression, of the immune system in
conjunction with zalpha11 Ligand on other cytokines which would be
important in boosting immunity to infectious diseases, treating
immunocompromised patients, such as HIV+ patient, or in improving
vaccines. In particular, zalpha11 antagonists, including soluble
zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide,
such as soluble zalpha11/IL-2R.gamma., could prevent the expansion
of a subset of the immune system involving zalpha11 Ligand (e.g.,
NK cells and mature B-cells), while enabling expansion of
progenitors induced by other cytokines (e.g., T-cells), and would
provide therapeutic value in treatment of viral infection and other
infection. For example, with Dengue virus infection, which causes
dengue hemorrhagic fever/Dengue Shock syndrome (DHF/DSS) it is
believed that severe DHF/DSS occurs as a result of "immune
enhancement" i.e., enhanced replication of the virus in the
presence of preexisting antibodies against another serotype. In the
second infection by a different Dengue virus serotype, the immune
system raises antibodies against the first virus that cross-react
but do not neutralize the virus, and that potentially aid its entry
into macrophages. Thus, suppression of the antibody immune
response, or B cell response, during a second or third Dengue
infection may help the immune system react appropriately in the
second infection to neutralize the virus by suppressing the
"enhancing" antibodies from the first serotype infection, and
consequently avoiding severe DHF/DSS. For review, see White, D. O.
and Fenner F. J. (Eds.) Medical Virology, 3.sup.rd ed., Academic
Press, Orlando Fla., 1986, pages 479-508). Similarly, suppression
of maternal antibody responses against fetal antigens by soluble
receptors of the present invention can aid in preventing birth
defects and spontaneous abortion. Moreover, in such applications
the soluble receptors of the present invention can be used in
conjunction with other cytokines to suppress some immune system
activities (e.g., B-cell proliferation, using the soluble
receptors) but allowing others to increase, e.g., in the presence
of other cytokines described herein and known in the art.
[0172] 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. For pharmaceutical use, the soluble
zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide,
such as soluble zalpha11/IL-2R.gamma. 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 zalpha11 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 zalpha11 soluble receptor
polypeptide is an amount sufficient to produce a clinically
significant effect.
[0173] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Construction of Expression Vector Expressing Full-Length
zalpha11
[0174] The entire zalpha11 receptor was isolated from a plasmid
containing zalpha11 receptor cDNA (SEQ ID NO:1) using PCR with
primers ZC19,905 (SEQ ID NO:19) and ZC19,906 (SEQ ID NO:20). The
reaction conditions were as follows: 95.degree. C. for 1 min; 35
cycles at 95.degree. C. for 1 min, 55.degree. C. for 1 min,
72.degree. C. for 2 min; followed by 72.degree. C. at 10 min; then
a 10.degree. C. soak. The PCR product was run on a 1% low melting
point agarose (Boerhinger Mannheim) gel and the approximately 1.5
kb zalpha11 cDNA isolated using Qiaquick.TM. gel extraction kit
(Qiagen) as per manufacturer's instructions.
[0175] The purified zalpha11 cDNA was digested with BamHI
(Boerhinger Mannheim) and EcoRI (BRL) as per manufacturer's
instructions. The entire digest was run on a 1% low melting point
agarose (Boerhinger Mannheim) gel and the cleaved zalpha11 fragment
was purified the using Qiaquick.TM. gel extraction kit as per
manufacturer's instructions. The resultant cleaved zalpha11
fragment was inserted into an expression vector as described
below.
[0176] Recipient expression vector pZP-5N was digested with BamHI
(Boerhinger Mannheim) and EcoRI (BRL) as per manufacturer's
instructions, and gel purified as described above. This vector
fragment was combined with the BamHI and EcoRI cleaved zalpha11
fragment isolated above in a ligation reaction using T4 Ligase
(BRL). The ligation was incubated at 15.degree. C. overnight. A
sample of the ligation was electroporated in to DH10B
electroMAX.TM. electrocompetent E. coli cells (25 .mu.F,
200.OMEGA., 2.3V). Transformants were plated on LB+Ampicillin
plates and single colonies screened by PCR to check for the
zalpha11 sequence using ZC19,905 (SEQ ID NO:19) and ZC19,906 (SEQ
ID NO:20) using the PCR conditions as described above. Confirmation
of the zalpha11 sequence was made by sequence analysis. The insert
was approximately 1.6 kb, and was full-length.
Example 2
Zalpha11 Based Proliferation in BAF3 Assay Using Alamar Blue
[0177] BaF3 cells expressing the full-length zalpha11 receptor were
constructed, using the zalpha11 expression vector, described in
Example 1. The BaF3 cells expressing the zalpha11 receptor mRNA
were designated BaF3/zalpha11. These cells provide an assay system
for detecting zalpha11 Ligand activity as described in numerous
Examples below. Conversely, these cells provide also an assay
system for detecting zalpha11 Ligand antagonist or inhibitory
activity by the soluble receptors and antibodies of the present
invention.
A. Construction of BaF3 Cells Expressing Human Zalpha11
Receptor
[0178] 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 (m/L-3) (R
& D, Minneapolis, Minn.), 2 mM L-glutaMax-1.TM. (Gibco BRL), 1
mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics (GIBCO BRL)).
Prior to electroporation, pZP-5N/zalpha11 plasmid DNA (Example 1)
was prepared and purified using a Qiagen Maxi Prep kit (Qiagen) as
per manufacturer's instructions. BaF3 cells for electroporation
were washed once in RPMI media and then resuspended in RPMI media
at a cell density of 10.sup.7 cells/ml. One ml of resuspended BaF3
cells was mixed with 30 .mu.g of the pZP-5N/zalpha11 plasmid DNA
and transferred to separate disposable electroporation chambers
(GIBCO BRL).
[0179] Following a 15 minute incubation at room temperature the
cells were given two serial shocks (800 lFad/300 V.; 1180 1Fad/300
V.) delivered by an electroporation apparatus (CELL-PORATOR.TM.;
GIBCO BRL). After a 5 minute recovery time, the electroporated
cells were transferred to 50 ml of complete media and placed in an
incubator for 15-24 hours (37.degree. C., 5% CO.sub.2). The cells
were then spun down and resuspended in 50 ml of complete media
containing Geneticin.TM. (Gibco) selection (500 .mu.g/ml G418) in a
T-162 flask to isolate the G418-resistant pool. Pools of the
transfected BaF3 cells, hereinafter called BaF3/zalpha11 cells,
were assayed for signaling capability as described below.
B. Testing the Signaling Capability of the BaF3/zalpha11 Cells
Using an Alamar Blue Proliferation Assay
[0180] BaF3/zalpha11 cells were spun down and washed in the
complete media, described above, but without mIL-3 (hereinafter
referred to as "mIL-3 free media"). The cells were spun and washed
3 times to ensure the removal of the mIL-3. Cells were then counted
in a hemacytometer. Cells were plated in a 96-well format at 5000
cells per well in a volume of 100 .mu.l per well using the mIL-3
free media.
[0181] Proliferation of the BaF3/zalpha11 cells was assessed using
conditioned media from zalpha11 Ligand-expressing cells diluted
with mIL-3 free media to 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%,
0.75% and 0.375% concentrations; or purified zalpha11 Ligand
(commonly owned U.S. patent application Ser. No. 09/522,217)
diluted with mIL-3 free media to 500 ng/ml, 250 ng/ml, 125 ng/ml,
62 ng/ml, 30 ng/ml, 15 ng/ml, 7.5 ng/ml, 3.75 ng/ml, 1.8 ng/ml, 0.9
ng/ml, 0.5 ng/ml and 0.25 ng/ml concentrations. 100 .mu.l of the
diluted mTPO was added to the BaF3/zalpha11 cells. The total assay
volume is 200 .mu.l. Negative controls were run in parallel using
mIL-3 free media only. The assay plates were incubated at
37.degree. C., 5% CO.sub.2 for 3 days at which time Alamar Blue
(Accumed, Chicago, Ill.) was added at 20 .mu.l/well. Alamar Blue
gives a fluourometric readout based on the metabolic activity of
cells, and is thus a direct measurement of cell proliferation in
comparison to a negative control. Plates were again incubated at
37.degree. C., 5% CO.sub.2 for 24 hours. Plates were read on the
Fmax.TM. plate reader (Molecular Devices Sunnyvale, Calif.) using
the SoftMax.TM. Pro program, at wavelengths 544 (Excitation) and
590 (Emmission). Results confirmed the signaling capability of the
zalpha11 receptor, as the zalpha11 Ligand significantly induced
proliferation over background levels.
Example 3
Screening for zalpha11 Ligand Using BaF3/Zalpha11 Cells Using an
Alamar Blue Proliferation Assay
[0182] A. Activation of Primary Monkey Splenocytes to Test for
Presence of zalpha11 Ligand
[0183] Monkey splenocytes were stimulated in vitro to produce
conditioned media to test for the presence of zalpha11 Ligand
activity as described below. Monkey spleens were obtained from 8
year old female M. nesestrian monkeys. The spleens were teased part
to produce a single cell suspension. The mononuclear cells were
isolated by Ficoll-Paque.RTM. PLUS (Pharmacia Biotech, Uppsala,
Sweden) density gradient. The mononuclear cells were seeded at
2.times.10.sup.6 cells/ml in RPMI-1640 media supplemented with 10%
FBS and activated with 5 ng/ml Phorbol-12-myristate-13-acetate
(PMA) (Calbiochem, San Diego, Calif.), and 0.5 mg/ml Ionomycin.TM.
(Calbiochem) for 48 h. The supernatant from the stimulated monkey
spleen cells was used to assay proliferation of the BaF3/zalpha11
cells as described below.
B. Screening for zalpha11 Ligand Using BaF3/Zalpha11 cells using an
Alamar Blue Proliferation Assay
[0184] BaF3/Zalpha11 cells were spun down and washed in mIL-3 free
media. The cells were spun and washed 3 times to ensure the removal
of the mIL-3. Cells were then counted in a hemacytometer. Cells
were plated in a 96-well format at 5000 cells per well in a volume
of 100 .mu.l per well using the mIL-3 free media.
[0185] Proliferation of the BaF3/Zalpha11 cells was assessed using
conditioned media from activated monkey spleen (see Example 3A).
Conditioned media was diluted with mIL-3 free media to 50%, 25%,
12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations. 100
.mu.l of the diluted conditioned media was added to the
BaF3/Zalpha11 cells. The total assay volume is 200 .mu.l. The assay
plates were incubated at 37.degree. C., 5% CO.sub.2 for 3 days at
which time Alamar Blue (Accumed, Chicago, Ill.) was added at 20
.mu.l/well. Plates were again incubated at 37.degree. C., 5%
CO.sub.2 for 24 hours. Plates were read on the Fmax.TM. plate
reader (Molecular devices) as described above (Example 2).
[0186] Results confirmed the proliferative response of the
BaF3/Zalpha11 cells to a factor present in the activated monkey
spleen conditioned media. The response, as measured, was
approximately 4-fold over background at the 50% concentration. The
untransfected BaF3 cells did not proliferate in response to this
factor, showing that this factor is specific for the Zalpha11
receptor.
C. Human Primary Source used to Isolate zalpha11 Ligand
[0187] 100 ml blood draws were taken from each of six donors. The
blood was drawn using 10.times.10 ml vacutainer tubes containing
heparin. Blood was pooled from six donors (600 ml), diluted 1:1 in
PBS, and separated using a Ficoll-Paque.TM. PLUS (Pharmacia
Biotech). The isolated primary human cell yield after separation on
the ficoll gradient was 1.2.times.10.sup.9 cells.
[0188] Cells were suspended in 9.6 ml MACS buffer (PBS, 0.5% EDTA,
2 mM EDTA). 1.6 ml of cell suspension was removed and 0.4 ml CD3
microbeads (Miltenyi Biotec, Auburn, Calif.) added. The mixture was
incubated for 15 min. at 4.degree. C. These cells labeled with CD3
beads were washed with 30 ml MACS buffer, and then resuspended in 2
ml MACS buffer.
[0189] A VS+ column (Miltenyi) was prepared according to the
manufacturer's instructions. The VS+ column was then placed in a
VarioMACS.TM. magnetic field (Miltenyi). The column was
equilibrated with 5 ml MACS buffer. The isolated primary human
cells were then applied to the column. The CD3 negative cells were
allowed to pass through. The column was rinsed with 9 ml (3.times.3
ml) MACS buffer. The column was then removed from the magnet and
placed over a 15 ml falcon tube. CD3+ cells were eluted by adding 5
ml MACS buffer to the column and bound cells flushed out using the
plunger provided by the manufacturer. The incubation of the cells
with the CD3 magnetic beads, washes, and VS+ column steps
(incubation through elution) above were repeated five more times.
The resulting CD3+ fractions from the six column separations were
pooled. The yield of CD3+ selected human cells were
3.times.10.sup.8 total cells.
[0190] A sample of the pooled CD3+ selected human cells was removed
for staining and sorting on a fluorescent antibody cell sorter
(FACS) to assess their purity. The human CD3+ selected cells were
91% CD3+ cells.
[0191] The human CD3+ selected cells were activated by incubating
in RPMI+5% FBS+PMA 10 ng/ml and Ionomycin 0.5 .mu.g/ml (Calbiochem)
for 13 hours 37.degree. C. The supernatant from these activated
CD3+ selected human cells was tested for zalpha11 Ligand activity
as described below. Moreover, the activated CD3+ selected human
cells were used to prepare a cDNA library, as described in commonly
owned U.S. patent application Ser. No. 09/522,217.
D. Testing Supernatant from Activated CD3+ Selected Human Cells for
zalpha11 Ligand using BaF3/Zalpha11 cells and an Alamar Blue
Proliferation Assay
[0192] BaF3/Zalpha11 cells were spun down and washed in mIL-3 free
media. The cells were spun and washed 3 times to ensure the removal
of the mIL-3. Cells were then counted in a hemacytometer. Cells
were plated in a 96-well format at 5000 cells per well in a volume
of 100 .mu.l per well using the mIL-3 free media.
[0193] Proliferation of the BaF3/Zalpha11 cells was assessed using
conditioned media from activated CD3+ selected human cells (see
Example 5C) diluted with mIL-3 free media to 50%, 25%, 12.5%,
6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations. 100 .mu.l of
the diluted conditioned media was added to the BaF3/Zalpha11 cells.
The total assay volume is 200 .mu.l. The assay plates were
incubated and assayed as described in Example 5B.
[0194] Results confirmed the proliferative response of the
BaF3/Zalpha11 cells to a factor present in the activated CD3+
selected human cell conditioned media. The response, as measured,
was approximately 10-fold over background at the 50% concentration.
The untransfected BaF3 cells did not proliferate in response to
this factor, showing that this factor is specific for the Zalpha11
receptor. Moreover soluble zalpha11 receptor blocked this
proliferative activity in the BaF3/Zalpha11 cells (see, Example
16).
Example 4
Construction of Mammalian Expression Vectors That Express zalpha11
Soluble Receptors: zalpha11CEE zalpha11CFLG zalpha11CHIS and
zalph11-Fc4
[0195] A. Construction of zalpha11 Mammalian Expression Vector
containing zalph11CEE, zalph11CFLG and zalph11CHIS
[0196] An expression vector was prepared for the expression of the
soluble, extracellular domain of the zalpha11 polypeptide,
pC4zalph11CEE, wherein the construct is designed to express a
zalpha11 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: 14).
[0197] A 700 bp PCR generated zalpha11 DNA fragment was created
using ZC 19,931 (SEQ ID NO:21) and ZC19,932 (SEQ ID NO:22) as PCR
primers to add Asp718 and BamHI restriction sites. A plasmid
containing the zalpha11 receptor cDNA (SEQ ID NO:1) was used as a
template. PCR amplification of the zalpha11 fragment was performed
as follows: Twenty five cycles at 94C for 0.5 minutes; five cycles
at 94.degree. C. for 10 seconds, 50.degree. C. for 30 seconds,
68.degree. C. for 45 seconds, followed by a 4.degree. C. hold. The
reaction was purified by chloroform/phenol extraction and
isopropanol precipitation, and digested with Asp718 and BamHI
(Gibco BRL) following manufacturer's protocol. A band of the
predicted size, 700 bp, was visualized by 1% agarose gel
electrophoresis, excised and the DNA was purified using a
QiaexII.TM. purification system (Qiagen) according the
manufacturer's instructions.
[0198] The excised DNA was subcloned into plasmid pC4EE which had
been cut with BamHI and Asp718. The pC4zalph11CEE expression vector
uses the native zalpha11 signal peptide and attaches the Glu-Glu
tag (SEQ ID NO: 14) to the C-terminus of the zalpha11
polypeptide-encoding polynucleotide sequence. Plasmid pC4EE, is a
mammalian expression vector containing an expression cassette
having the mouse metallothionein-1 promoter, multiple restriction
sites for insertion of coding sequences, a stop codon and a human
growth hormone terminator. The plasmid also has an E. coli origin
of replication, a mammalian selectable marker expression unit
having an SV40 promoter, enhancer and origin of replication, a DHFR
gene and the SV40 terminator.
[0199] About 30 ng of the restriction digested zalpha11 insert and
about 12 ng of the digested vector were ligated overnight at
16.degree. C. One microliter of each ligation reaction was
independently electroporated into 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 were screened by restriction analysis of DNA
prepared from 2 ml liquid cultures of individual colonies. The
insert sequence of positive clones was verified by sequence
analysis. A large scale plasmid preparation was done using a
QIAGEN.RTM. Maxi prep kit (Qiagen) according to manufacturer's
instructions.
[0200] The same process was used to prepare the zalpha11 soluble
receptors with a C-terminal his tag, composed of 6 His residues in
a row; and a C-terminal flag (SEQ ID NO:23) tag, zalpha11CFLAG. To
construct these constructs, the aforementioned vector has either
the HIS or the FLAG.RTM. tag in place of the glu-glu tag (SEQ ID
NO: 14).
B. Mammalian Expression Construction of Soluble zalpha11 receptor
zalpha11-Fc4
[0201] An expression plasmid containing all or part of a
polynucleotide encoding zalpha11 was constructed via homologous
recombination. A fragment of zalpha11 cDNA was isolated using PCR
that includes the polynucleotide sequence from extracellular domain
of the zalpha11 receptor. The two primers used in the production of
the zalpha11 fragment were: (1) The primers for PCR each include
from 5' to 3' end: 40 bp of the vector flanking sequence (5' of the
insert) and 17 bp corresponding to the 5' end of the zalpha11
extracellular domain (SEQ ID NO:24); and (2) 40 bp of the 5' end of
the Fc4 polynucleotide sequence (SEQ ID NO:25) and 17 bp
corresponding to the 3' end of the zalpha11 extracellular domain
(SEQ ID NO:26). The fragment of Fc4 for fusion with the zalpha11
was generated by PCR in a similar fashion. The two primers used in
the production of the Fc4 fragment were: (1) a 5' primer consisting
of 40 bp of sequence from the 3' end of zalpha11 extracellular
domain and 17 bp of the 5' end of Fc4 (SEQ ID NO:27); and (2) a 3'
primer consisting of 40 bp of vector sequence (3' of the insert)
and 17 bp of the 3' end of Fc4 (SEQ ID NO:28).
[0202] PCR amplification of the each of the reactions described
above was performed as follows: one cycle at 94.degree. C. for 2
minutes; twenty-five cycles at 94.degree. C. for 30 seconds,
60.degree. C. for 30 seconds, 72.degree. C. for 1 minute; one cycle
at 72.degree. C. for 5 minutes; followed by a 4.degree. C. hold.
Ten .mu.l of the 100 .mu.l PCR reaction was run on a 0.8% LMP
agarose gel (Seaplaque GTG) with 1.times.TBE buffer for analysis.
The remaining 90 .mu.l of PCR reaction is precipitated with the
addition of 5 .mu.l 1 M NaCl and 250 .mu.l of absolute ethanol. The
expression vector used was derived from the plasmid pCZR199
(deposited at the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209, designated No.
98668), and was cut with SmaI (BRL). The expression vector was
derived from the plasmid pCZR199, and is a mammalian expression
vector containing an expression cassette having the CMV immediate
early promoter, a consensus intron from the variable region of
mouse immunoglobulin heavy chain locus, multiple restriction sites
for insertion of coding sequences, a stop codon and a human growth
hormone terminator. The expression vector also has an E. coli
origin of replication, a mammalian selectable marker expression
unit having an SV40 promoter, enhancer and origin of replication, a
DHFR gene and the SV40 terminator. The expression vector used was
constructed from pCZR199 by the replacement of the metallothionein
promoter with the CMV immediate early promoter.
[0203] One hundred microliters of competent yeast cells (S.
cerevisiae) were combined with 10 .mu.l containing approximately 1
.mu.g each of the zalpha11 and Fc4 inserts, and 100 ng of SmaI
(BRL) digested expression vector and transferred to a 0.2 cm
electroporation cuvette. The yeast/DNA mixtures were electropulsed
at 0.75 kV (5 kV/cm), "infinite" ohms, 25 .mu.F. To each cuvette is
added 600 .mu.l of 1.2 M sorbitol and the yeast was plated in two
300 .mu.l aliquots onto two URA-D plates and incubated at
30.degree. C.
[0204] 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 200 .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.
[0205] Transformation of electrocompetent E. coli cells (DH10B,
GibcoBRL) is done with 0.5-2 ml yeast DNA prep and 40 ul of DH10B
cells. The cells were electropulsed at 2.0 kV, 25 mF and 400 ohms.
Following electroporation, 1 ml SOC (2% Bacto.RTM. 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 plated in 250
.mu.l aliquots on four LB AMP plates (LB broth (Lennox), 1.8% Bacto
Agar (Difco), 100 mg/L Ampicillin).
[0206] Individual clones harboring the correct expression construct
for zalpha11-Fc4 were identified by restriction digest to verify
the presence of the zalpha11-Fc4 insert and to confirm that the
various DNA sequences have been joined correctly to one another.
The insert of positive clones were subjected to sequence analysis.
Larger scale plasmid DNA is isolated using the Qiagen Maxi kit
(Qiagen) according to manufacturer's instructions.
Example 5
Transfection And Expression Of Zalpha11 Soluble Receptor
Polypeptides
[0207] BHK 570 cells (ATCC No. CRL-10314), passage 27, were plated
at 1.2.times.10.sup.6 cells/well (6-well plate) in 800 .mu.l of
serum free (SF) DMEM media (DMEM, Gibco/BRL High Glucose) (Gibco
BRL, Gaithersburg, Md.). The cells were transfected with expression
plasmids containing zalpha11CEE, zalpha11CFLG or zalpha11CHIS
described above (see, Example 4), using Lipofectin.TM. (Gibco BRL),
in serum free (SF) DMEM. Three micrograms of zalpha11CEE,
zalpha11CFLG or zalpha11CHIS each were separately diluted into 1.5
ml tubes to a total final volume of 100 .mu.l SF DMEM. In separate
tubes, 15 .mu.l of Lipofectin.TM. (Gibco BRL) was mixed with 100
.mu.l of SF DMEM. The Lipofectin.TM. mix was incubated at room
temperature for 30-45 minutes then the DNA mix was added and
allowed to incubate approximately 10-15 minutes at room
temperature.
[0208] The entire DNA: Lipofectin.TM. mixture was added to the
plated cells and distributed evenly over them. The cells were
incubated at 37.degree. C. for approximately five hours, then
transferred to separate 150 mm MAXI plates in a final volume of 30
ml DMEM/5% fetal bovine serum (FBS) (Hyclone, Logan, Utah). The
plates were incubated at 37.degree. C., 5% CO.sub.2, overnight and
the DNA: Lipofectin.TM. mixture was replaced with selection media
(5% FBS/DMEM with 1 .mu.M methotrexate (MTX) the next day.
[0209] Approximately 10-12 days post-transfection, the plates were
washed with 10 ml SF DMEM. The wash media was aspirated and
replaced with 7.25 ml serum-free DMEM. Sterile Teflon meshes
(Spectrum Medical Industries, Los Angeles, Calif.) pre-soaked in SF
DMEM were then placed over the clonal cell colonies. A sterile
nitrocellulose filter pre-soaked in SF DMEM was then placed over
the mesh. Orientation marks on the nitrocellulose were transferred
to the culture dish. The plates were then incubated for 5-6 hours
in a 37.degree. C., 5% CO.sub.2 incubator.
[0210] Following incubation, the filters/meshes were removed, and
the media aspirated and replaced with 5% FBS/DMEM with 1 .mu.M MTX.
The filters were then blocked in 10% nonfat dry milk/Western A
buffer (Western A: 50 mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150
mM NaCl and 0.25% gelatin) for 15 minutes at room temperature on a
rotating shaker. The filters were then incubated with an
anti-Glu-Glu, anti-FLAG.RTM., or anti-HIS antibody-HRP conjugates,
respectively, in 2.5% nonfat dry milk/Western A buffer for one hour
at room temperature on a rotating shaker. The filters were then
washed three times at room temperature with Western A for 5-10
minutes per wash. The filters were developed with 30 ultra ECL
reagent (Amersham Corp., Arlington Heights, Ill.) according the
manufacturer's directions and visualized on the Lumi-Imager (Roche
Corp.)
[0211] Positive expressing clonal colonies were mechanically picked
to 12-well plates in one ml of 5% FCS/DMEM with 5 .mu.M MTX, then
grown to confluence. Conditioned media samples were then tested for
expression levels via SDS-PAGE and Western analysis. The three
highest expressing clones for each construct were picked; two out
of three were frozen down as back up and one was expanded for
mycoplasma testing and large-scale factory seeding.
B. Mammalian Expression of Soluble zalpha11 Receptor
zalpha11-Fc4
[0212] BHK 570 cells (ATCC NO: CRL-10314) were plated in 10 cm
tissue culture dishes and allowed to grow to approximately 50 to
70% confluency overnight at 37FC, 5% CO.sub.2, in DMEM/FBS media
(DMEM, Gibco/BRL High Glucose, (Gibco BRL, Gaithersburg, Md.), 5%
fetal bovine serum (Hyclone, Logan, Utah), 1 mM L-glutamine (JRH
Biosciences, Lenexa, Kans.), 1 mM sodium pyruvate (Gibco BRL)). The
cells were then transfected with the plasmid containing
zalpha11-Fc4 (see, Example 9), 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). The plasmid containing zalpha11-Fc4 was diluted into 15
ml tubes to a total final volume of 640 ml with SF media. 35 ml of
Lipofectamine.TM. (Gibco BRL) was mixed with 605 ml of SF medium.
The Lipofectamine.TM. mix was added to the DNA mix and allowed to
incubate approximately 30 minutes at room temperature. Five
milliliters of SF media was added to the DNA:Lipofectamine.TM.
mixture. The cells were rinsed once with 5 ml of SF media,
aspirated, and the DNA:Lipofectamine.TM. mixture is added. The
cells were incubated at 37.degree. C. for five hours, then 6.4 ml
of DMEM/10% FBS, 1% PSN media was added to each plate. The plates
were incubated at 370.degree. C. overnight and the
DNA:Lipofectamine.TM. mixture was replaced with fresh 5% FBS/DMEM
media the next day. On day 2 post-transfection, the cells were
split into the selection media (DMEM/FBS media from above with the
addition of 1 mM methotrexate (Sigma Chemical Co., St. Louis, Mo.))
in 150 mm plates at 1:10, 1:20 and 1:50. The media on the cells was
replaced with fresh selection media at day 5 post-transfection.
Approximately 10 days post-transfection, two 150 mm culture dishes
of methotrexate resistant colonies from each transfection were
trypsinized and the cells are pooled and plated into a T-162 flask
and transferred to large scale culture.
Example 6
Purification of zalpha11 Soluble Receptors From BHK 570 Cells
[0213] A. Purification of zalpha11CEE Polypeptide from BHK 570
[0214] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
zalpha11 polypeptide containing C-terminal GluGlu (EE) tags. Thirty
liters of cell factory conditioned media was concentrated to 1.6
liters with an Amicon S10Y3 spiral cartridge on a ProFlux A30. A
Protease inhibitor solution was added to the concentrated 1.6
liters of cell factory conditioned media from transfected BHK 570
cells (Example 5) to final concentrations of 2.5 mM
ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.
Louis, Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis,
Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc
(Boehringer-Mannheim). Samples were removed for analysis and the
bulk volume was frozen at -80.degree. C. until the purification was
started. Total target protein concentrations of the concentrated
cell factory conditioned media was determined via SDS-PAGE and
Western blot analysis with the anti-EE HRP conjugated antibody.
[0215] A 100 ml column of anti-EE G-Sepharose (prepared as
described below) was poured in a Waters AP-5, 5 cm.times.10 cm
glass column. The column was flow packed and equilibrated on a
BioCad Sprint (PerSeptive BioSystems, Framingham, Mass.) with
phosphate buffered saline (PBS) pH 7.4. The concentrated cell
factory conditioned media was thawed, 0.2 micron sterile filtered,
pH adjusted to 7.4, then loaded on the column overnight with 1
ml/minute flow rate. The column washed with 10 column volumes (CVs)
of phosphate buffered saline (PBS, pH 7.4), then plug eluted with
200 ml of PBS (pH 6.0) containing 0.5 mg/ml EE peptide (Anaspec,
San Jose, Calif.) at 5 ml minute. The EE peptide used has the
sequence EYMPME (SEQ ID NO:14). The column washed for 10 CVs with
PBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The pH of the
glycine-eluted column was adjusted to 7.0 with 2 CVs of
5.times.PBS, then equilibrated in PBS (pH 7.4). Five ml fractions
were collected over the entire elution chromatography and
absorbance at 280 and 215 nM were monitored; the pass through and
wash pools were also saved and analyzed. The EE-polypeptide elution
peak fractions were analyzed for the target protein via SDS-PAGE
Silver staining and Western Blotting with the anti-EE HRP
conjugated antibody. The polypeptide elution fractions of interest
were pooled and concentrated from 60 ml to 5.0 ml using a 10,000
Dalton molecular weight cutoff membrane spin concentrator
(Millipore, Bedford, Mass.) according to the manufacturer's
instructions.
[0216] To separate zalpha11CEE from other co-purifying proteins,
the concentrated polypeptide elution pooled fractions were
subjected to a POROS HQ-50 (strong anion exchange resin from
PerSeptive BioSystems, Framingham, Mass.) at pH 8.0. A
1.0.times.6.0 cm column was poured and flow packed on a BioCad
Sprint. The column was counter ion charged then equibrated in 20 mM
TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). The sample was
diluted 1:13 (to reduce the ionic strength of PBS) then loaded on
the Poros HQ column at 5 ml/minute. The column washed for 10 CVs
with 20 mM Tris pH 8.0 then eluted with a 40 CV gradient of 20 mM
Tris/1 M sodium chloride (NaCl) at 10 ml/minute. 1.5 ml fractions
were collected over the entire chromatography and absorbance at 280
and 215 nM were monitored. The elution peak fractions were analyzed
via SDS-PAGE Silver staining. Fractions of interest were pooled and
concentrated to 1.5-2 ml using a 10,000 Dalton molecular weight
cutoff membrane spin concentrator (Millipore, Bedford, Mass.)
according to the manufacturer's instructions.
[0217] To separate zalpha11CEE polypeptide from free EE peptide and
any contaminating co-purifying proteins, the pooled concentrated
fractions were subjected to chromatography on a 1.5.times.90 cm
Sephadex S200 (Pharmacia, Piscataway, N.J.) column equilibrated and
loaded in PBS at a flow rate of 1.0 ml/min using a BioCad Sprint.
1.5 ml fractions were collected across the entire chromatography
and the absorbance at 280 and 215 nM were monitored. The peak
fractions were characterized via SDS-PAGE Silver staining, and only
the most pure fractions were pooled. This material represented
purified zalpha11CEE polypeptide.
[0218] This purified material was finally subjected to a 4 ml
ActiClean Etox (Sterogene) column to remove any remaining
endotoxins. The sample was passed over the PBS equilibrated gravity
column four times then the column washed with a single 3 ml volume
of PBS, which was pooled with the "cleaned" sample. The material
was then 0.2 micron sterile filtered and stored at -80.degree. C.
until it was aliquoted.
[0219] On Western blotted, Coomassie Blue and Silver stained
SDS-PAGE gels, the zalpha11CEE polypeptide was one major band of an
apparent molecular weight of 50,000 Daltons. The mobility of this
band was the same on reducing and non-reducing gels.
[0220] The protein concentration of the purified material was
performed by BCA analysis (Pierce, Rockford, Ill.) and the protein
was aliquoted, and stored at -80.degree. C. according to our
standard procedures. On IEF (isoelectric focusing) gels the protein
runs with a PI of less than 4.5. The concentration of zalpha11CEE
polypeptide was 1.0 mg/ml.
[0221] Purified zalpha11CEE polypeptide was prepared for injection
into rabbits and sent to R & R Research and Development
(Stanwood, Wash.) for antibody production. Rabbits were injected to
produce anti-huzalpha11-CEE-BHK serum (Example 10, below).
[0222] To prepare anti-EE Sepharose, a 100 ml bed volume of protein
G-Sepharose (Pharmacia, Piscataway, N.J.) washed 3 times with 100
ml of PBS containing 0.02% sodium azide using a 500 ml Nalgene 0.45
micron filter unit. The gel washed with 6.0 volumes of 200 mM
triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.), and an equal
volume of EE antibody solution containing 900 mg of antibody was
added. After an overnight incubation at 4.degree. C., unbound
antibody was removed by washing the resin with 5 volumes of 200 mM
TEA as described above. The resin was resuspended in 2 volumes of
TEA, transferred to a suitable container, and
dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in
TEA, was added to a final concentration of 36 mg/ml of protein
G-Sepharose gel. The gel was rocked at room temperature for 45 min
and the liquid was removed using the filter unit as described
above. Nonspecific sites on the gel were then blocked by incubating
for 10 min. at room temperature with 5 volumes of 20 mM
ethanolamine in 200 mM TEA. The gel was then washed with 5 volumes
of PBS containing 0.02% sodium azide and stored in this solution at
4.degree. C.
B. Purification of zalpha11CFLAG Polypeptide from BHK 570
[0223] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
zalpha11 polypeptide containing C-terminal FLAG.RTM. (FLG)
(Sigma-Aldrich Co.) tags. Thirty liters of cell factory conditioned
media was concentrated to 1.7 liters with an Amicon S10Y3 spiral
cartridge on a ProFlux A30. A Protease inhibitor solution was added
to the 1.7 liters of concentrated cell factory conditioned media
from transfected BHK 570 cells (see, Example 5) to final
concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA,
Sigma Chemical Co. St. Louis, Mo.), 0.003 mM leupeptin
(Boehringer-Mannheim, Indianapolis, Ind.), 0.001 mM pepstatin
(Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim).
Samples were removed for analysis and the bulk volume was frozen at
-80.degree. C. until the purification was started. Total target
protein concentrations of the cell factory conditioned media was
determined via SDS-PAGE and Western blot analysis with the
anti-FLAG (Kodak) HRP conjugated antibody. A 125 ml column of
anti-FLAG.RTM. M2-Agarose affinity gel (Sigma-Aldrich Co.) was
poured in a Waters AP-5, 5 cm.times.10 cm glass column. The column
was flow packed and equilibrated on a BioCad Sprint (PerSeptive
BioSystems, Framingham, Mass.) with phosphate buffered saline (PBS)
pH 7.4. The concentrated cell factory conditioned media was thawed,
0.2 micron sterile filtered, pH adjusted to 7.4, then loaded on the
column overnight with 1 ml/minute flow rate. The column washed with
10 column volumes (CVs) of phosphate buffered saline (PBS, pH 7.4),
then plug eluted with 250 ml of PBS (pH 6.0) containing 0.5 mg/ml
FLAG.RTM. (Sigma-Aldrich Co.) peptide at 5 ml/minute. The FLAG.RTM.
peptide used has the sequence DYKDDDDK (SEQ ID NO:23). The column
washed for 10 CVs with PBS, then eluted with 5 CVs of 0.2M glycine,
pH 3.0. The pH of the glycine-eluted column was adjusted to 7.0
with 2 CVs of 5.times.PBS, then equilibrated in PBS (pH 7.4). Five
ml fractions were collected over the entire elution chromatography
and absorbence at 280 and 215 nM were monitored; the pass through
and wash pools were also saved and analyzed. The
FLAG.RTM.-polypeptide elution peak fractions were analyzed for the
target protein via SDS-PAGE Silver staining and Western Blotting
with the anti-FLAG HRP conjugated antibody. The polypeptide elution
fractions of interest were pooled and concentrated from 80 ml to 12
ml using a 10,000 Dalton molecular weight cutoff membrane spin
concentrator (Millipore, Bedford, Mass.) according to the
manufacturer's instructions.
[0224] To separate zalpha11 CFLG from other co-purifying proteins,
the polypeptide elution pooled fractions were subjected to a POROS
HQ-50 (strong anion exchange resin from PerSeptive BioSystems,
Framingham, Mass.) at pH 8.0. A 1.0.times.6.0 cm column was poured
and flow packed on a BioCad Sprint. The column was counter ion
charged then equilibrated in 20 mM TRIS pH 8.0 (Tris (Hydroxymethyl
Aminomethane)). The sample was diluted 1:13 (to reduce the ionic
strength of PBS) then loaded on the Poros HQ-50 column at 5
ml/minute. The column washed for 10 column volumes (CVs) with 20 mM
Tris pH 8.0 then eluted with a 40 CV gradient of 20 mM Tris/1 M
sodium chloride (NaCl) at 10 ml/minute. 1.5 ml fractions were
collected over the entire chromatography and absorbance at 280 and
215 nM were monitored. The elution peak fractions were analyzed via
SDS-PAGE Silver staining. Fractions of interest were pooled and
concentrated to 1.5-2 ml using a 10,000 Dalton molecular weight
cutoff membrane spin concentrator (Millipore, Bedford, Mass.)
according to the manufacturer's instructions.
[0225] To separate zalpha11CFLG polypeptide from free FLAG.RTM.
peptide and any contaminating co-purifying proteins, the pooled
concentrated fractions were subjected to chromatography on a
1.5.times.90 cm Sephacryl S200 (Pharmacia, Piscataway, N.J.) column
equilibrated and loaded in PBS at a flow rate of 1.0 ml/min using a
BioCad Sprint. 1.5 ml fractions were collected across the entire
chromatography and the absorbance at 280 and 215 nM were monitored.
The peak fractions were characterized via SDS-PAGE Silver staining,
and only the most pure fractions were pooled. This material
represented purified zalpha11CFLG polypeptide.
[0226] This purified material was finally subjected to a 4 ml
ActiClean Etox (Sterogene) column to remove any remaining
endotoxins. The sample was passed over the PBS equilibrated gravity
column four times then the column washed with a single 3 ml volume
of PBS, which was pooled with the "cleaned" sample. The material
was then 0.2 micron sterile filtered and stored at -80.degree. C.
until it was aliquoted.
[0227] On Western blotted, Coomassie Blue and Silver stained
SDS-PAGE gels, the zalpha11CFLG polypeptide was one major band of
an apparent molecular weight of 50,000 Daltons. The mobility of
this band was the same on reducing and non-reducing gels.
[0228] The protein concentration of the purified material was
performed by BCA analysis (Pierce, Rockford, Ill.) and the protein
was aliquoted, and stored at -80.degree. C. according to our
standard procedures. On IEF (isoelectric focusing) gels the protein
runs with a PI of less than 4.5. The concentration of zalpha11 CFLG
polypeptide was 1.2 mg/ml.
C. Purification of zalpha11-Fc4 Polypeptide from Transfected BHK
570 Cells
[0229] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
zalpha11 polypeptide containing C-terminal fusion to human IgG/Fc
(zalpha11-Fc4; Examples 4 and 5). 12,000 ml of conditioned media
from BHK 570 cells transfected with zalpha11-Fc4 (Example 5) was
filtered through a 0.2 mm sterilizing filter and then supplemented
with a solution of protease inhibitors, to final concentrations of
0.001 mM leupeptin (Boerhinger-Mannheim, Indianapolis, Ind.), 0.001
mM pepstatin (Boerhinger-Mannheim) and 0.4 mM Pefabloc
(Boerhinger-Mannheim). A protein G sepharose (6 ml bed volume,
Pharmacia Biotech) was packed and washed with 500 ml PBS
(Gibco/BRL) The supplemented conditioned media was passed over the
column with a flow rate of 10 ml/minute, followed by washing with
1000 ml PBS (BRL/Gibco). zalpha11-Fc4 was eluted from the column
with 0.1 M Glycine pH 3.5 and 2 ml fractions were collected
directly into 0.2 ml 2M Tris pH 8.0, to adjust the final pH to 7.0
in the fractions. The eluted fractions were characterized by
SDS-PAGE and western blotting with anti-human Fc (Amersham)
antibodies. Western blot analysis of reducing SDS-PAGE gels reveal
an immunoreactive protein of 80,000 KDa in fractions 2-10. Silver
stained SDS-PAGE gels also revealed an 80,000 KDa zalpha11:Fc
polypeptide in fractions 2-10. Fractions 2-10 were pooled.
[0230] The protein concentration of the pooled fractions was
performed by BCA analysis (Pierce, Rockford, Ill.) and the material
was aliquoted, and stored at -80.degree. C. according to our
standard procedures. The concentration of the pooled fractions was
0.26 mg/ml.
Example 7
Assay Using zalpha11 Soluble Receptor zalpha11CEE, zalpha11CFLG and
zalpha11-Fc4 Soluble Receptors in Competitive Inhibition Assay
[0231] BaF3/Zalpha11 cells were spun down and washed in mIL-3 free
media. The cells were spun and washed 3 times to ensure the removal
of the mIL-3. Cells were then counted in a hemacytometer. Cells
were plated in a 96-well format at 5000 cells per well in a volume
of 100 .mu.l per well using the mIL-3 free media.
[0232] Both media from the monkey spleen cell activation and the
CD3+ selected cells, described in Example 3, were added in separate
experiments at 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and
0.375% concentrations, with or without zalpha11 soluble receptors
(CEE, C-flag, and Fc4 constructs; See, Example 6) at 10 g/ml. The
total assay volume was 200 .mu.l.
[0233] The assay plates were incubated 37.degree. C., 5% CO.sub.2
for 3 days at which time Alamar Blue (Accumed) was added at 20
.mu.l/well. Plates were again incubated at 37.degree. C., 5%
CO.sub.2 for 24 hours. Plates were read on the Fmax.TM. plate
reader (Molecular Devices) as described above (Example 2). Results
demonstrated complete inhibition of cell growth from each of the
different zalpha11 soluble receptor constructs at 10 g/ml,
confirming that the factor in each sample was specific for the
zalpha11 receptor.
[0234] Titration curves, diluting out the soluble receptors, were
also run using the above stated assay. Both the zalpha11CEE and
zalpha11CFLG soluble zalpha11 receptors were able to completely
inhibit growth as low as 20 ng/ml. The zalpha11-Fc4 soluble
zalpha11 receptor was only as effective at 1.5 .mu.g/ml.
Example 8
Expression of Human zalpha11 Soluble Receptor in E. coli
[0235] A. Construction of Expression Vector pCZR225 that Expresses
huzalpha11/MBP-6H Fusion Polypeptide
[0236] An expression plasmid containing a polynucleotide encoding a
human zalpha11 soluble receptor fused C-terminally to maltose
binding protein (MBP) was constructed via homologous recombination.
The polynucleotide sequence for the MBP-zalpha11 soluble receptor
fusion polypeptide is shown in SEQ ID NO:29, with the corresponding
protein sequence shown in SEQ ID NO:30. The fusion polypeptide,
designated huzalpha11/MBP-6H, in Example 9, contains an MBP portion
(amino acid 1 (Met) to amino acid 388 (Ser) of SEQ ID NO:30) fused
to the human zalpha11 soluble receptor (amino acid 389 (Cys) to
amino acid 606 (His) of SEQ ID NO:30). A fragment of human zalpha11
cDNA (SEQ ID NO:31) was isolated using PCR. Two primers were used
in the production of the human zalpha11 fragment in a PCR reaction:
(1) Primer ZC20,187 (SEQ ID NO:32), containing 40 bp of the vector
flanking sequence and 25 bp corresponding to the amino terminus of
the human zalpha11, and (2) primer ZC20,185 (SEQ ID NO:33),
containing 40 bp of the 3' end corresponding to the flanking vector
sequence and 25 bp corresponding to the carboxyl terminus of the
human zalpha11. The PCR Reaction conditions were as follows: 25
cycles of 94.degree. C. for 30 seconds, 50.degree. C. for 30
seconds, and 72.degree. C. for 1 minute; followed by 4.degree. C.
soak, run in duplicate. Two .mu.l of the 100 .mu.l PCR reaction was
run on a 1.0% agarose gel with 1.times.TBE buffer for analysis, and
the expected approximately 660 bp fragment was seen. The remaining
90 .mu.l of PCR reaction was combined with the second PCR tube
precipitated with 400 .mu.l of absolute ethanol. The precipitated
DNA used for recombining into the Sma1 cut recipient vector pTAP98
to produce the construct encoding the MBP-zalpha11 fusion, as
described below.
[0237] Plasmid pTAP98 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 pTAP98 was constructed using yeast homologous
recombination. 10 ng of EcoR1 cut pMAL-c2 was recombined with 1
.mu.g Pvu1 cut pRS316, 1 .mu.g linker, and 1 .mu.g Sca1/EcoR1 cut
pRS316. The linker consisted of oligos ZC19,372 (SEQ ID NO:34) (100
.mu.mol): ZC19,351 (SEQ ID NO:35) (1 .mu.mol): ZC19,352 (SEQ ID
NO:36) (1 .mu.mol), and ZC19,371 (SEQ ID NO:37) (100 .mu.mol)
combined in a PCR reaction. 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.
[0238] 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 zalpha11 receptor PCR product
above, and 100 ng of SmaI digested pTAP98 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 and the yeast
was then plated in two 300 .mu.l aliquots onto two -URA D plates
and incubated at 30.degree. C.
[0239] 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 200 .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.
[0240] 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% Bacto.TM. Tryptone (Difco, Detroit,
Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl.sub.2, 10 mM MgSO4, 20 mM glucose) was plated in one aliquot
on MM/CA +AMP 100 mg/L plates (Pryor and Leiting, Protein
Expression and Purification 10:309-319, 1997).
[0241] Cells harboring the correct expression construct for human
zalpha11 receptor were identified by expression. Cells were grown
in MM/CA with 100 .mu.g/ml Ampicillin for two hours, shaking, at
37.degree. C. 1 ml of the culture was induced with 1 mM IPTG. 2-4
hours later the 250 .mu.l of each culture was mixed with 250 .mu.l
acid washed glass beads and 250 .mu.l Thorner buffer with 5% OME
and dye (8M urea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5%
SDS). Samples were vortexed for one minute and heated to 65.degree.
C. for 10 minutes. 20 .mu.l were loaded per lane on a 4%-12% PAGE
gel (NOVEX). Gels were run in 1XMES buffer. The positive clones
were designated pCZR225 and subjected to sequence analysis. The
polynucleotide sequence of MBP-zalpha11 fusion is shown in SEQ ID
NO:50.
B. Bacterial Expression of Human huzalpha11/MBP-6H Fusion
Polypeptide
[0242] One microliter of sequencing DNA was used to transform
strain BL21. The cells were electropulsed at 2.0 kV, 25.degree. F.
and 400 ohms. Following electroporation, 0.6 ml MM/CA with 100 mg/L
Ampicillin.
[0243] Cells were grown in MM/CA with 100 .mu.g/ml Ampicillin for
two hours, shaking, at 37.degree. C. 1 ml of the culture was
induced with 1 mM IPTG. 2-4 hours later the 250 .mu.l of each
culture was mixed with 250 .mu.l acid washed glass beads and 250
.mu.l Thorner buffer with 5% .beta.ME and dye (8M urea, 100 mM Tris
pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples were vortexed for
one minute and heated to 65.degree. C. for 10 minutes. 20 .mu.l
were loaded per lane on a 4%-12% PAGE gel (OVEX). Gels were run in
1XMES buffer. The positive clones were used to grow up for protein
purification of the huzalpha11/MBP-6H fusion protein (Example 9,
below).
Example 9
Purification of huzalpha11/MBP-6H Soluble Receptor From E. coli
Fermentation
[0244] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
huzalpha11/MBP-6H soluble receptor polypeptide. E. coli cells
containing the pCZR225 construct and expressing huzalpha11/MBP-6H
soluble receptor (Example 8) were grown up in SuperBroth II (12 g/L
Casien, 24 g/L Yeast Extract, 11.4 g/L di-potassium phosphate, 1.7
g/L Mono-potassium phosphate; Becton Dickenson, Cockeysville, Md.),
and frozen in 0.5% glycerol. Twenty grams of the frozen cells in
SuperBroth II+Glycerol were used to purify the protein. The frozen
cells were thawed and diluted 1:10 in a protease inhibitor solution
(Extraction buffer) prior to lysing the cells and releasing the
huzalpha11/MBP-6H soluble receptor protein. The diluted cells
contained final concentrations of 20 mM Tris (JT Baker,
Philipsburg, N.J.) 100 mM Sodium Chloride (NaCl, Mallinkrodt,
Paris, Ky.), 0.5 mM phenylmethylsulfonyl fluoride (PMSF, Sigma
Chemical Co., St. Louis, Mo.), 2 .mu.g/ml Leupeptin (Fluka,
Switzerland), and 2 .mu.g/ml Aprotinin (Sigma). A French Press cell
breaking system (Constant Systems Ltd., Warwick, UK) with
temperature of -7 to -10.degree. C. and 30K PSI was used to lyse
the cells. The diluted cells were checked for breakage by A.sub.600
readings before and after the French Press. The lysed cells were
centrifuged @18,000G for 45 minutes to remove the broken cell
debris, and the supernatant used to purify the protein. Total
target protein concentrations of the supernatant was determined via
BCA Protein Assay (Pierce, Rockford, Ill.), according to
manufacturer's instructions.
[0245] A 25 ml column of Talon Metal Affinity resin (Clontech, Palo
Alto, Calif.) (prepared as described below) was poured in a
Bio-Rad, 2.5 cm D.times.10 cm H glass column. The column was packed
and equilibrated by gravity with 10 column volumes (CVs) of Talon
Equilibration buffer (20 mM Tris, 100 mM NaCl, pH 8.0). The
supernatant was batch loaded to Talon metal affinity resin and was
rocked overnight. The resin was poured back into the column and
washed with 10 CV's of Talon Equilibration buffer by gravity, then
gravity eluted with 140 ml of Elution buffer (Talon Equilibration
buffer+200 mM Imidazole-Fluka Chemical). The talon column was
cleaned with 5 CVs of 20 mM 2-(N-Morpholino) ethanesulfonic acid pH
5.0 (MES, Sigma), 5 CVs of distilled H.sub.2O, then stored in 20%
Ethanol/0.1% Sodium Azide. Fourteen ml fractions were collected
over the entire elution chromatography and the fractions were read
with absorbance at 280 and 320 nM and BCA protein assay; the pass
through and wash pools were also saved and analyzed. The protein
elution fractions of interest were pooled and loaded straight to
Amylose resin (New England Biolabs, Beverly, Mass.).
[0246] To obtain more pure huzalpha11/MBP-6H polypeptide, the talon
affinity elution pooled fractions were subjected to Amylose resin
(22 mls) at pH 7.4. A 2.5 cm D.times.10 cm H Bio-Rad column was
poured, packed and equilibrated in 10 CVs of Amylose equilibration
buffer-20 mM Tris (JT Baker), 100 mM NaCl (Mallinkrodt), 1 mM PMSF
(Sigma), 10 mM beta-Mercaptoethanol (BME, ICN Biomedicals Inc.,
Aurora, Ohio) pH 7.4. The sample was loaded by gravity flow rate of
0.5 ml/min. The column washed for 10 CVs with Amylose equilibration
buffer, then eluted with .about.2 CV of Amylose equilibration
buffer+10 mM Maltose (Fluka Biochemical, Switzerland) by gravity. 5
ml fractions were collected over the entire chromatography and
absorbance at 280 and 320 nM were read. The Amylose column was
regenerated with 1 CV of distilled H.sub.2O, 5 CVs of 0.1% (w/v)
SDS (Sigma), 5 CVs of distilled H.sub.2O, and then 5 CVs of Amylose
equilibration buffer.
[0247] Fractions of interest were pooled and dialyzed in a
Slide-A-Lyzer (Pierce) with 4.times.4 L PBS pH 7.4 (Sigma) to
remove low molecular weight contaminants, buffer exchange and
desalt. After the changes of PBS, the material harvested
represented the purified huzalpha11/MBP-6H polypeptide. The
purified huzalpha11/MBP-6H polypeptide was analyzed via SDS-PAGE
Coomassie staining and Western blot analysis with the anti-rabbit
HRP conjugated antibody (Rockland, Gilbertsville, Pa.). The
concentration of the huzalpha11/MBP-6H polypeptide was 1.92 mg/ml
as determined by BCA analysis.
[0248] Purified huzalpha11/MBP-6H polypeptide was prepared for
injection into rabbits and sent to R & R Research and
Development (Stanwood, Wash.) for antibody production. Rabbits were
injected to produce anti anti-huzalpha11/MBP-6H serum (Example 10,
below).
Example 10
Zalpha11 Soluble Receptor Polyclonal Antibodies
[0249] Polyclonal antibodies were prepared by immunizing two female
New Zealand white rabbits with the purified huzalpha11/MBP-6H
polypeptide (Example 9), or the purified recombinant zalpha11CEE
soluble receptor (Example 6A). Corresponding polyclonal antibodies
were designated rabbit anti-huzalpha11/MBP-6H and rabbit
anti-huzalpha11-CEE-BHK respectively. The rabbits were each given
an initial intraperitoneal (IP) injection of 200 mg of purified
protein in Complete Freund's Adjuvant (Pierce, Rockford, Ill.)
followed by booster IP injections of 100 mg purified protein in
Incomplete Freund's Adjuvant every three weeks. Seven to ten days
after the administration of the third booster injection, the
animals were bled and the serum was collected. The rabbits were
then boosted and bled every three weeks.
[0250] The zalpha11-specific polyclonal antibodies were affinity
purified from the rabbit serum using an CNBr--SEPHAROSE 4B protein
column (Pharmacia LKB) that was prepared using 10 mg of the
purified huzalpha11/MBP-6H polypeptide (Example 9) per gram
CNBr--SEPHAROSE, followed by 20.times. dialysis in PBS overnight.
Zalpha11-specific antibodies were characterized by an ELISA titer
check using 1 mg/ml of the appropriate protein antigen as an
antibody target. The lower limit of detection (LLD) of the rabbit
anti-huzalpha11/MBP-6H affinity purified antibody is a dilution of
500 .mu.g/ml. The LLD of the rabbit anti-huzalpha11-CEE-BHK
affinity purified antibody is a dilution of 50 .mu.g/ml.
Example 11
Identification of Cells Expressing zalpha11 Receptor Using
RT-PCR
[0251] Specific human cell types were isolated and screened for
zalpha11 expression by RT-PCR. B-cells were isolated from fresh
human tonsils by mechanical disruption through 100 .mu.m nylon cell
strainers (Falcon.TM.; Bectin Dickenson, Franklin Lakes, N.J.). The
B-cell suspensions were enriched for CD19+ B-cells by positive
selection with VarioMACS VS+ magnetic column and CD19 microbeads
(Miltenyi Biotec, Auburn, Calif.) as per manufacturer's
instructions. T-cells and monocytes were isolated from human
apheresed blood samples. CD3+ T-cells were purified by CD3
microbead VarioMACS positive selection and monocytes were purified
by VarioMACS negative selection columns (Miltenyi) as per
manufacturer's instructions. Samples from each population were
stained and analyzed by fluorescent antibody cell sorting (FACS)
(Bectin Dickinson, San Jose, Calif.) analysis to determine the
percent enrichment and resulting yields. CD19+B-cells were
approximately 96%, purified CD3+ T-cells were approximately 95%
purified, and monocytes were approximately 96% purified.
[0252] RNA was prepared, using a standard method in the art, from
all three cell types that were either resting or activated. RNA was
isolated from resting cells directly from the column preparations
above. The CD19+ and CD3+ cells were activated by culturing at
500,000 cells/ml in RPMI+10% FBS containing PMA 5 ng/ml
(Calbiochem, La Jolla, Calif.) and Ionomycin 0.5 .mu.g/ml
(Calbiochem) for 4 and 24 hours. The monocytes were activated by
culturing in RPMI+10% FBS containing LPS10 ng/ml (Sigma St. Louis
Mo.) and rhIFN-.gamma. 10 ng/ml (R&D, Minneapolis, Minn.) for
24 hours. Cells were harvested and washed in PBS. RNA was prepared
from the cell pellets using RNeasy Midiprep.TM. Kit (Qiagen,
Valencia, Calif.) as per manufacturer's instructions and first
strand cDNA synthesis was generated with Superscript II.TM. Kit
(GIBCO BRL, Grand Island, N.Y.) as per manufacturer's protocol.
[0253] Oligos ZC19907 (SEQ ID NO:38) and ZC19908 (SEQ ID NO:39)
were used in a PCR reaction to screen the above described samples
for a 1.2 kb fragment corresponding to zalpha11 message. PCR
amplification was performed with Taq Polymerase (BRL Grand Island
N.Y.), and conditions as follows: 35 cycles of 95.degree. C. for 1
min., 60.degree. C. for 1 min., 72.degree. C. for 30 sec.; 1 cycle
at 72.degree. C. for 10 min.; and 4.degree. C. soak. 10 .mu.l of
each 50 .mu.l reaction volume was run on a 2% agarose 1.times.TAE
gel to identify resultant products. PCR products were scored as (-)
for no product, (+) for band visible, (++) increased presence of
band and (+++) being the most predominant band, with results shown
in Table 5 below.
TABLE-US-00005 TABLE 5 cDNA Source Activation PCR Product CD19+
cells 0 hr resting + 4 hr activated ++ 24 hr activated +++ CD3+
cells 0 hr resting - 4 hr activated ++ 24 hr activated - monocytes
0 hr resting - 24 hr activated -
[0254] These results indicated that zalpha11 message is present in
resting human CD19+ B-cells and increases with mitogenic
activation. It also appears to be expressed by human CD3+ T-cells
only after 4 hour activation. There was no apparent message in
either resting or activated human monocytes.
Example 12
Zalpha11 Immunohistochemistry
A. Cell and Tissue Preparations
[0255] Positive controls consisted of BaF3 cells transfected with
zalpha11 receptor (Example 2) and lymphoid tissues known to express
zalpha11 receptor including mouse lymph node, spleen and thymus
received from HSD (Harlan Sprague Dawley, Indianapolis, Ind.),
monkey lymph node and spleen received from Regional Primate
Research Center (University of Washington, Seattle, Wash.), human
lymph node and spleen received from CHTN (Cleveland, Ohio).
Negative controls performed on each sample included: (1)
untransfected BaF3 cells, (2) liver and brain tissue from mouse and
human known not to express zalpha11 receptor, (3) staining with
antibody dilution buffer (Ventann Bioteck Systems, Tucson Ariz.) in
the absence of primary antibody, and (4) using zalpha11 soluble
receptor protein in competition experiments.
[0256] Other cell samples were examined. Both non-stimulated and
stimulated HL60 cells were assayed. HL60 cells are a promyelocytic
cell line, which can be differentiated into myeloid or granulocyte
lineages with different reagents. Stimulated HL60 samples were
prepared as follows: (1) HL60 cells were treated with 10 ng/ml of
phorbol-myristate-acetate (PMA) (Sigma, St. Louis, Mo.) for 48
hours to differentiate into monocyte lineage cells; and (2) HL60
cells treated with 1.25% DMSO (Sigma) for 4 days to differentiate
into neutrophil-like cells. In addition, human polymorphonuclear
(PMN) cells, human granulocytes, human peripheral blood lymphocytes
(PBL) and human monocytes from fresh human blood were examined
(prepared in house using routine methods in the art). The cells and
tissues described above were fixed overnight in 10% NBF (Surgipath,
Richmond, Ill.), and embedded in paraplast X-tra (Oxford
Scientific, St. Louis, Mo.), and sectioned at 5 .mu.m with a
Reichart-Jung 2050 microme (Leica Instruments GmbH, Nussloch,
Germany).
B. Immunohistochemistry
[0257] Tissue slides were deparaffinized, hydrated to buffer
(water), and subjected to steam HIER treatment in Antigen Retrieval
Citra buffer (BioGenex, San Roman, Calif.) for 20 minutes. 5%
normal goat serum (Vector, Burlingame, Calif.) was used to block
non-specific binding for 10 minutes. Immunocytochemical screening
analyses were performed using polyclonal antibodies to zalpha11
soluble receptor protein (rabbit anti-huzalpha11-MBP-6H and rabbit
anti-huzalpha11-CEE-BHK; Example 10) as the primary antibodies, at
dilutions of 1:200 and 1:400 respectively. Biotin conjugated goat
anti-rabbit IgG (Vector; Cat. No. BA-1000, 1.5 mg/ml) was used as
the secondary antibody at dilution of 1:200. In separate samples,
protein competition was performed by using additional zalpha11 CEE
soluble receptor protein (in 10X fold excess) (Example 6A) to the
primary antibody to pre-block primary antibody immunoreaction. This
competition was used as a control for the rabbit polyclonal
antibody specificity to zalpha11. Detection was performed on the
Ventana ChemMate 500 instrument using a ChemMate DAB Kit (labeled
Streptavidin-Biotin Kit with application of a
streptavidin-horseradish peroxidase conjugate, and DAB substrate)
according to manufacturer's instruction and using the
manufacturer's hematoxylin counterstain for 30 seconds (Ventana
Biotek Systems, Tucson, Ariz.).
[0258] High expression of zalpha11 was observed in the
PMA-activated HL60 cells. Low level expression was observed in PBL
and HL60 cells without stimulation. A subset of cells in the
spleen, thymus and lymph node of mouse showed positive staining.
Lymph node and spleen of both human and monkey, and HL60 cells with
DMSO stimulation showed minimal or no staining. The signal seen in
the cells and tissues was mostly competed out by using the excess
zalpha11 soluble receptor protein. The negative control tissues of
brain and liver showed no staining.
Example 13
Identifying Peripheral Blood Mononuclear Cells (PBMNC's) That
Express zalpha11 Receptor Using Polyclonal Rabbit Anti-sera to
zalpha11 Soluble Receptor
[0259] 200 ml fresh heparinized blood was obtained from a normal
donor. Blood was diluted 1:1 in PBS, and separated using a
Ficoll-Paque PLUS gradient (Pharmacia Biotech, Uppsala, Sweden),
and the lymphocyte interface collected. Cells were washed 2.times.
in PBS and resuspended in RPMI+5% FBS media at a concentration of
2.times.10.sup.6 cells/ml.
[0260] In order to determine whether expression of zalpha11
receptor is affected by the activation state of the lymphocyte
cells, i.e., between resting and activated cells several
stimulation conditions were used: 1) unstimulated, i.e., media
alone (RPMI+5% FBS media); 2) stimulated with PMA 10
ng/ml+Ionomycin 0.5 .mu.g/ml (both from Calbiochem); and 3) PHA
activation (phytohemagglutinin-P, Difco/VWR). The cells were
incubated at 37.degree. C. for 17 hours then collected for staining
to detect expression of zalpha11 receptor.
[0261] An indirect staining protocol was used. Briefly, the human
lymphocyte cells were suspended in staining buffer (PBS+0.02%
NaN.sub.3+BSA 1% normal human serum 2%) and plated at
2.times.10.sup.5 cells in 50 .mu.l/well in a 96 well plate.
Antibodies to the zalpha11CEE soluble receptor (Example 15) were
used to determine whether they co-stained with a B-cell (CD19),
T-cell (CD3) or monocyte marker (CD14) on the isolated human
lymphocytes. A rabbit polyclonal sera to zalpha11 soluble receptor
(Rb anti-huzalpha11-CEE-BHK) (Example 10) at 10 .mu.g/ml was used
as the antibody to identify zalpha11 on the lymphocytes. A
secondary antibody, goat anti-rabbit Ig-FITC (Biosource, Camarillo,
Calif.), was used to visualize the Rb anti-huzalpha11-CEE-BHK
antibody binding to the zalpha11 receptors. Other antibodies were
simultaneously used to stain T cells (CD3-PE; PharMingen, San
Diego, Calif.), B cells (CD19-PE) (PharMingen), and monocytes
(CD14-PE) (PharMingen) in order to identify co-staining of the
anti-zalpha11 receptor antibody on these cell types. Various
controls were used to determine non-specific binding and background
levels of staining: (1) an irrelevant rabbit polyclonal sera was
used as a non-specific control; and (2) secondary antibody alone
was used to determine background binding of that reagent. Purified,
zalpha11CEE soluble receptor (Example 6) was used in about a
10-fold excess as a competitive inhibitor to verify the specificity
of the rabbit anti-huzalpha11-CEE-BHK antibody to zalpha11 soluble
receptor.
[0262] After plating the cells and adding the primary and
co-staining antibodies, the cells were incubated on ice for 30
minutes, washed 2.times. with staining buffer, and stained with the
secondary antibody, goat anti-rabbit Ig-FITC (Biosource), for 30
minutes on ice. Cells were washed 2.times. staining buffer, and
resuspended at 200 .mu.l per well in staining buffer containing the
viability stain 7AAD at about 1 .mu.g/ml final concentration
(Sigma, St. Louis, Mo.). Samples were read on the FACS-Caliber
(Becton-Dickinson, San Jose, Calif.) and viable cells analyzed.
[0263] The rabbit polyclonal to zalpha11 receptor stained resting B
cells. The signal on resting B cells was brighter than the signal
achieved using the irrelevant rabbit sera, and the signal was
diminished to a greater extent on B cells than on T cells with the
addition of excess zalpha11-CEE soluble receptor. This experiment
was repeated using separated B and T cells, and the results were
very similar. Again the staining with the polyclonal rabbit
anti-huzalpha11-CEE-BHK antibody to zalpha11 receptor was highest
on resting B cells.
Example 14
Zalpha11 Receptor Expression in Various Tissues Using Real-Time
Quantitative RT/PCR
A. Primers and Probes for Quantitative RT-PCR--
[0264] Real-time quantitative RT-PCR using the ABI PRISM 7700
Sequence Detection System (PE Applied Biosystems, Inc., Foster
City, Calif.) has been previously described (See, Heid, C. A. et
al., Genome Research 6:986-994, 1996; Gibson, U. E. M. et al.,
Genome Research 6:995-1001, 1996; Sundaresan, S. et al.,
Endocrinology 139:4756-4764, 1998. This method incorporates use of
a gene specific probe containing both reporter and quencher
fluorescent dyes. When the probe is intact the reporter dye
emission is negated due to the close proximity of the quencher dye.
During PCR extension using additional gene-specific forward and
reverse primers, the probe is cleaved by 5' nuclease activity of
Taq polymerase which releases the reporter dye from the probe
resulting in an increase in fluorescent emission.
[0265] The primers and probes used for real-time quantitative
RT-PCR analyses of zalpha11 receptor expression were designed using
the primer design software Primer Express.TM. (PE Applied
Biosystems, Foster City, Calif.). Primers for human zalpha11
receptor were designed spanning an intron-exon junction to
eliminate amplification of genomic DNA. The forward primer,
ZC22,277 (SEQ ID NO:40) and the reverse primer, ZC22,276 (SEQ ID
NO:41) were used in a PCR reaction (below) at about 300 nM
concentration to synthesize a 143 bp product. The corresponding
zalpha11 TaqMan.RTM. probe, designated ZG31 (SEQ ID NO:42) was
synthesized and labeled by PE Applied Biosystems. The ZG31 probe
was labeled at the 5' end with a reporter fluorescent dye
(6-carboxy-fluorescein) (FAM) (PE Applied Biosystems) and at the 3'
end with a quencher fluorescent dye
(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied
Biosystems).
[0266] As a control to test the integrity and quality of RNA
samples tested, all RNA samples (below) were screened for rRNA
using a primer and probe set ordered from PE Applied Biosystems
(cat No. 430-4483). The kit contains an rRNA forward primer (SEQ ID
NO:43) and the rRNA reverse primer (SEQ ID NO:44), rRNA TaqMan.RTM.
probe (SEQ ID NO:45) The rRNA probe was labeled at the 5' end with
a reporter fluorescent dye VIC (PE Applied Biosystems) and at the
3' end with the quencher fluorescent dye TAMRA (PE Applied
Biosystems). The rRNA results also serve as an internal control and
allow for the normalization of the zalpha11 mRNA expression results
seen in the test samples.
[0267] RNA samples from human CD3, CD19 and monocyte cell types
were prepared and described as per Example 11 above. Control RNA
was prepared, using RNeasy Miniprep.TM. Kit (Qiagen, Valencia,
Calif.) as per manufacturer's instructions, from approximately 10
million BaF3 cells expressing human zalpha11 receptor (Example
2A).
B. Real-time Quantitative RT-PCR
[0268] Relative levels of zalpha11 mRNA were determined by
analyzing total RNA samples using the one-step RT-PCR method (PE
Applied Biosystems). Total RNA from BaF3 cells expressing human
zalpha11 receptor was isolated by standard methods and used to
generate a standard curve used for quantitation. The curve
consisted of 10-fold serial dilutions ranging from
2.5-2.5.times.10.sup.-4 ng/.mu.l for the rRNA screen and 250-0.025
ng/.mu.l for the zalpha11 screen with each standard curve point
analyzed in triplicate. The total RNA samples from the cells were
also analyzed in triplicate for human zalpha11 receptor transcript
levels and for levels of rRNA as an endogenous control. In a total
volume of 25 .mu.l, each RNA sample was subjected to a One-Step
RT-PCR reaction containing: approximately 25 ng of total RNA in
buffer A (50 mM KCL, 10 mM Tris-HCL); the internal standard dye,
carboxy-x-rhodamine (ROX); appropriate primers (approximately 50 nM
rRNA primers (SEQ ID NO:43 and SEQ ID NO:44) for the rRNA samples;
and approximately 300 nM ZC22,277 (SEQ ID NO:40) and ZC22,276 (SEQ
ID NO:41) primers for zalpha11 samples); the appropriate probe
(approximately 50 nM rRNA TaqMan.RTM. probe (SEQ ID NO:45) for rRNA
samples, approximately 100 nM ZG31 (SEQ ID NO:42) probe for
zalpha11 samples); 5.5 mM MgCl.sub.2; 300 .mu.M each d-CTP, d-ATP,
and d-GTP and 600 .mu.M of d-UTP; MuLV reverse transcriptase (0.25
U/.mu.l); AmpliTaq.TM. Gold DNA polymerase (0.025 U/.mu.l) (PE
Applied Biosystems); and RNase Inhibitor (0.4 U/.mu.l) (PE Applied
Biosystems). PCR thermal cycling conditions were as follows: an
initial reverse transcription (RT) step of one cycle at 48.degree.
C. for 30 minutes; followed by an AmpliTaq Gold.TM. (PE Applied
Biosystems) activation step of one cycle at 95.degree. C. for 10
minutes; followed by 40 cycles of amplification at 95.degree. C.
for 15 seconds and 60.degree. C. for 1 minute.
[0269] Relative zalpha11 RNA levels were determined by using the
Standard Curve Method as described by the manufacturer, PE
Biosystems (User Bulletin No. 2: ABI Prism 7700 Sequence Detection
System, Relative Quantitation of Gene Expression, Dec. 11, 1997).
The rRNA measurements were used to normalize the zalpha11 levels
and the resting CD3+RNA sample was used as a calibrator. Resting
CD3 was arbitrarily chosen as the calibrator and given a value of
1.00. The rest of the samples were compared relative to the
calibrator. Data are shown in Table 6 below.
TABLE-US-00006 TABLE 6 Sample Resting 4 hr Stimulation 24 hr
Stimulation CD3 1.00 15.27 16.70 CD19 20.14 65.08 25.42 Monocytes
0.05 no data 0.26
[0270] There was a 15-fold increase in zalpha11 receptor expression
in CD3+ at 4 and 24 hrs. Resting CD19 had 20 fold increase in
receptor expression relative to resting CD3+. There was a 3 fold
increase with 4 hr stimulation that fell back to resting levels by
24 hrs.
[0271] Monocytes showed no detectable zalpha11 receptor expression
in this assay.
C. Purified Human T, NK, and B Cells as a Primary Source used to
Assess Human zalpha11Receptor Expression
[0272] Whole blood (150 ml) was collected from a healthy human
donor and mixed 1:1 with PBS in 50 ml conical tubes. Thirty ml of
diluted blood was then underlayed with 15 ml of Ficoll Paque Plus
(Amersham Pharmacia Biotech, Uppsala, Sweden). These gradients were
centrifuged 30 min at 500 g and allowed to stop without braking.
The RBC-depleted cells at the interface (PBMC) were collected and
washed 3 times with PBS. The isolated human PBMC yield was
200.times.10e6 prior to selection described below. The PBMCs were
suspended in 1.5 ml MACS buffer (PBS, 0.5% EDTA, 2 mM EDTA) and
3.times.10 e6 cells were set aside for control RNA and for flow
cytometric analysis. The 0.25 ml anti-human CD8 microbeads
(Miltenyi Biotec) were added and the mixture was incubated for 15
min at 4 degrees C. These cells labeled with CD8 beads were washed
with 30 ml MACS buffer, and then resuspended in 2 ml MACS
buffer.
[0273] A VS+ column (Miltenyi) was prepared according to the
manufacturer's instructions. The VS+ column was then placed in a
VarioMACS magnetic field (Miltenyi).
[0274] The column was equilibrated with 5 ml MACS buffer. The
isolated primary mouse cells were then applied to the column. The
CD8 negative cells were allowed to pass through. The column was
rinsed with 9 ml (3.times.3 ml) MACS buffer. The column was then
removed from the magnet and placed over a 15 ml falcon tube. CD8+
cells were eluted by adding 5 ml MACS buffer to the column and
bound cells flushed out using the plunger provided by the
manufacturer. The yield of CD8+ selected human peripheral T cells
was 51.times.10e6 total cells. The CD8-negative flow through cells
were collected, counted, stained with anti-human CD4 coated beads,
then incubated and passed over a new VS+ column at the same
concentrations as described above. The yield of CD4+ selected human
peripheral T cells was 42.times.10e6 total cells.
[0275] A sample of each of the CD8+ and CD4+ selected human T cells
was removed for staining and sorting on a fluorescence activated
cell sorter (FACS) to assess their purity. A PE-conjugated
anti-human CD4 antibody, an anti-human CD8-FITC Ab, and an
anti-human CD19-CyChrome Ab (all from PharMingen) were used for
staining the CD8+ and CD4+ selected cells. The CD8-selected cells
in this first experiment were 80% CD8+, and the CD4-selected cells
were 85% CD4+. In 2 subsequent experiments (Example 14B), the CD8+
purified cells were 84% and 81% pure, and the CD4+ cells were 85%
and 97% pure, respectively. In one experiment, we stained the
non-binding (flow-through) cells with anti-human CD19-coated beads
(Miltenyi) and ran them over a third magnetic bead column to
isolate CD 19+B cells (these were 92% pure).
[0276] The human CD8+, CD4+ and CD19+ selected cells were activated
by incubating 0.5.times.10.sup.6 cells/ml in RPMI+5% human
ultraserum (Gemini Bioproducts, Calabasas, Calif.)+PMA 10 ng/ml and
Ionomycin 0.5 .mu.g/ml (Calbiochem) for 4, 16, or 24 hours at
37.degree. C. The T-cells (2.5.times.10 e6/well) were alternately
stimulated in 24-well plates pre-coated overnight with 0.5 .mu.g/ml
plate-bound anti-CD3 mAb UCHT1 (PharMingen) with or without soluble
anti-CD28 mAb (PharMingen) at 5 .mu.g/ml. At each timepoint, the
cells were harvested, pelleted, washed once with PBS, and pelleted
again. The supernatant was removed and the pellets were snap-frozen
in a dry ice/ethanol bath, then stored at -80.degree. C. for RNA
preparation at a later date.
[0277] In a separate experiment, human NK cells were enriched from
Ficolled PBMC by negative selection using the human NK enrichment
system (consisting of antibodies to CD3, CD4, CD14, CD19, CD66b,
and glycophorin A) from Stem Cell Technologies (Vancouver, B.C.,
Canada). Cell pellets were prepared from freshly isolated NK cells
from 2 different donors, or from NK cells cultured 24 hours in
media only or in media supplemented with 20 ng/ml IL-15. RNA from a
human NK cell line derived from a malignant non-Hodgkin's lymphoma
and designated NK-92 (ATCC No. CRL-2407) was also tested. As
positive controls, RNA was isolated from the human B cell lines
CESS (ATCC No. TIB-190), IM-9 (ATCC No. CCL-159), and HS-Sultan
(CRL-1484).
[0278] Real Time-PCR was performed on these human NK, CD8+, CD4+
and CD 19+ selected cells as described above for assessing human
zalpha11 receptor expression. Relative levels of zalpha11 receptor
RNA were determined by analysis of total RNA samples using the
One-Step RT-PCR method (PE Applied Biosystems). RNA from BaF3 cells
expressing human zalpha11 receptor was used to generate appropriate
control for standard curves for the real-time PCR described in
Example 14C above. Results of the experiments analyzing the
expression of the zalpha11 Ligand and zalpha receptor in stimulated
and unstimulated cells are as described in Example 14D-E below.
D. Expression of Human zalpha11 Receptor and Ligand in CD4+, CD8+
and CD19+ Cells
[0279] The first experiment used RT-PCR, described above, to assess
zalpha11 receptor expression in unstimulated and anti-CD3
stimulated CD4+ and CD8+ samples at timepoints of 0 h (unstimulated
("resting") cells), and at 4 h, 15.5 h and 24 h, after stimulation.
The resting CD4+ sample was arbitrarily chosen as the calibrator
and given a value of 1.00. There was approximately a 4-fold
increase in receptor expression in unstimulated CD4+ cells from 4 h
to 24 h of culture and about an 8-fold increase over the same time
period in anti-CD3 stimulated CD4+ cells. The CD8+ cells showed a
7-fold increase in zalpha11 receptor expression that peaked at 4
hrs and decreased over time. With anti-CD3 stimulation, the CD8+
cells had a constant 8-fold increase in receptor expression.
[0280] The second experiment used RT-PCR to assess zalpha11
receptor expression in anti-CD3-stimulated,
PMA+Ionomycin-stimulated and unstimulated CD4+ and CD8+ samples at
timepoints of 0 h, and at 3.5 h, 16 h and 24 h after activation.
The resting CD8+ sample was arbitrarily chosen as the calibrator
and given a value of 1.00. The resting CD4+ and CD8+ cells did not
have significant amounts of receptor expression. The expression was
about 3 fold higher in the PMA+Ionomycin-stimulated CD4+ samples at
3.5 h, 16 h and 24 h after stimulation. The expression in anti-CD3
activated CD4+ cells peaked at 10-fold above background levels at
3.5 h after stimulation, then fell back to levels 4-fold above
background at 16 h after stimulation. The CD8+ cells showed a
4-fold expression increase at 3.5 h after PMA+Ionomycin
stimulation, with expression decreasing at subsequent timepoints.
As in the first experiment, the anti-CD3 stimulated CD8+ cells
again exhibited an 8-fold above background induction of receptor
expression.
[0281] The final experiment used RT-PCR to assess zalpha11 receptor
expression in anti-CD3- and anti-CD3/anti-CD28-stimulated and
unstimulated CD4+ and CD8+ samples at timepoints of 0 h, and at 2
h, 4 h, and 16 h after stimulation. CD 19+ cells activated with
PMA+Ionomycin were also screened for receptor expression at the
same time intervals. The resting CD4+ sample was arbitrarily chosen
as the calibrator and given a value of 1.00. The 2 h anti-CD3
stimulated CD4+ cells only had a 4-fold induction of receptor,
compared to the 10-fold induction seen at 3.5 h in the previous
experiment. The combination of anti-CD3 and anti-CD28 increased
expression to 8-fold above background. The 16 h anti-CD3/anti-CD28
stimulated CD8+ cells had very low receptor expression levels, as
seen in the CD8+ cells in previous experiments (above). The CD19+
cells stimulated with PMA+Ionomycin had the most significant
receptor expression with a 19-fold increase at 2 h, but the
expression levels decreased back to those of resting cells by 16
h.
[0282] A certain amount of variation was expected between blood
draws (i.e. multiple samples at different times from the same
patient and between multiple patients). Therefore, data trends were
analyzed within each study or from a single blood sample and the
three experiments above were compared for an overall conclusion.
The trend from the Real Time PCR experiments described above is
that of all the cell types tested, CD19+ B cells activated with
PMA+ ionomycin expressed the highest levels of zalpha11 receptor
RNA. CD4+ and CD8+ cells can also be stimulated to express
receptor, but at lower levels than in B cells.
E. Expression of Human zalpha11 Receptor in Human NK cells
[0283] Real Time PCR was also performed on human NK cells, purified
as described in Example 14C, above. The NK-92 sample was
arbitrarily chosen as the calibrator and given a value of 1.00.
There was approximately a 4.5-fold increase in receptor expression
in the positive control CESS cells, a 1.5-fold increase in IM-9
cells, and no increase in the HS-Sultan cells (0.9-fold relative to
NK-92). The NK cells, either fresh or cultured overnight with or
without IL-15, expressed very similar levels of zalpha11 Receptor
as NK-92 (with values ranging from 0.9-1,2-fold different relative
to NK-92).
Example 15
Identification of Cells Expressing zalpha11 Receptor Using In Situ
Hybridization
[0284] Specific human tissues were isolated and screened for
zalpha11 expression by in situ hybridization. Various human tissues
prepared, sectioned and subjected to in situ hybridization included
thymus, spleen, tonsil, lymph node and lung. The tissues were fixed
in 10% buffered formalin and blocked in paraffin using standard
techniques. Tissues were sectioned at 4 to 8 microns. Tissues were
prepared using a standard protocol ("Development of non-isotopic in
situ hybridization" at http://dir.niehs.nih.gov/dirlep/ish.html).
Briefly, tissue sections were deparaffinized with HistoClear
(National Diagnostics, Atlanta, Ga.) and then dehydrated with
ethanol. Next they were digested with Proteinase K (50 .mu.g/ml)
(Boehringer Diagnostics, Indianapolis, Ind.) at 37.degree. C. for 2
to 20 minutes. This step was followed by acetylation and
re-hydration of the tissues.
[0285] Two in situ probes generated by PCR were designed against
the human zalpha11 sequence. Two sets of oligos were designed to
generate probes for separate regions of the zalpha11cDNA: (1)
Oligos ZC23,684 (SEQ ID NO:60) and ZC23,656 (SEQ ID NO:61) were
used to generate a 413 bp probe for zalpha11; and (2) Oligos
ZC23,685 (SEQ ID NO:62) and ZC23,657 (SEQ ID NO:63) were used to
generate a 430 bp probe for zalpha11. The second probe is 1500 bp
3' of the first zalpha11 probe. The antisense oligo from each set
also contained the working sequence for the T7 RNA polymerase
promoter to allow for easy transcription of antisense RNA probes
from these PCR products. The PCR reaction conditions were as
follows: 30 cycles at 94.degree. C. for 30 sec, 60.degree. C. for 1
min., 72.degree. C. for 1.5 min. The PCR products were purified by
Qiagen spin columns followed by phenol/chloroform extraction and
ethanol precipitation. Probes were subsequently labeled with
digoxigenin (Boehringer) or biotin (Boehringer) using an In Vitro
transcription System (Promega, Madison, Wis.) as per manufacturer's
instruction.
[0286] In situ hybridization was performed with a digoxigenin- or
biotin-labeled zalpha11 probe (above). The probe was added to the
slides at a concentration of 1 to 5 pmol/ml for 12 to 16 hours at
55-60.degree. C. Slides were subsequently washed in 2.times.SSC and
0.1.times.SSC at 50.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.).
[0287] A signal was seen in the thymus, tonsil, lung, and lymph
node. The positive-staining cells appeared to be lymphocytes.
Example 16
Secretion Trap Assay
[0288] A secretion trap assay was used to identify the cDNA for the
zalpha11 Ligand. The positive DNA pools obtained from the
expression cloning effort were described in commonly owned U.S.
patent application Ser. No. 09/522,217.
[0289] Conditioned medium from DNA clones transfected into BHK
cells in 96-well format, were put into the proliferation assay
using BaF3/zalpha11 cells described in Example 2. Several DNA pools
gave positive activities that were repeated and neutralized with
zalpha11 soluble receptors (Example 6). One positive DNA pool was
transfected into COS cells in 12-well format, using the
Lipofectamine.TM. method described below.
[0290] A secretion trap assay was then performed using zalpha11
soluble receptors (C-terminal Glu-Glu tagged either with or without
biotinylation; C-terminal Flag tagged; or Fc4 zalpha11 soluble
receptor fusions) (Example 6) to test the direct binding between
the zalpha11 Ligand in the positive pool and zalpha11 soluble
receptors (see below). The result was positive, enabling the
detection and isolation of clones expressing the zalpha11 Ligand.
Plates were shaken at 37.degree. C. for 24 hours, and then DNA
minipreps (QiaPrep.TM. 96 Turbo Miniprep Kit; Qiagen) were prepared
in 96-well format using a TomTech Quadra 9600. The plasmid DNA was
then pooled in the format of rows and columns, transfected into COS
cells, and then the positive pools were determined by secretion
trap as described below.
COS Cell Transfections
[0291] The COS cell transfection was performed as follows: Mix 3 ul
pooled DNA and 5 ul Lipofectamine.TM. in 92 ul serum free DMEM
media (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin,
2.5 mg insulin, 1 .mu.g selenium and 5 mg fetuin in 500 ml DMEM),
incubate at room temperature for 30 minutes and then add 400 ul
serum free DMEM media. Add this 500 ul mixture onto
1.5.times.10.sup.5 COS cells/well plated on 12-well tissue culture
plate and incubate for 5 hours at 37.degree. C. Add 500 ul 20% FBS
DMEM media (100 ml FBS, 55 mg sodium pyruvate and 146 mg
L-glutamine in 500 ml DMEM) and incubate overnight.
Secretion Trap Assay
[0292] The secretion trap was performed as follows: Media was
rinsed off cells with PBS and then fixed for 15 minutes with 1.8%
Formaldehyde in PBS. Cells were then washed with TNT (0.1M
Tris-HCL, 0.15M NaCl, and 0.05% Tween-20 in H.sub.2O), and
permeated with 0.1% Triton-X in PBS for 15 minutes, and again
washed with TNT. Cells were blocked for 1 hour with TNB (0.1M
Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NEN Renaissance
TSA-Direct Kit) in H.sub.2O), and washed again with TNT. If using
the biotinylated protein, the cells were blocked for 15 minute
incubations with Avidin and then Biotin (Vector Labs), washing
in-between with TNT. Depending on which soluble receptor was used,
the cells were incubated for 1 hour with: (A) 1-3 .mu.g/ml zalpha11
soluble receptor zalpha11-Fc4 fusion protein (Example 6); (B) 3
.mu.g/ml zalpha11 soluble receptor C-terminal FLAG tagged,
zalpha11CFLG (Example 6); (C) 3 .mu.g/ml zalpha11 soluble receptor
C-terminal GluGlu tagged, zalpha11 CEE (Example 6); or (D) 3
.mu.g/ml biotinylated zalpha11 soluble receptor zalpha11CEE
(Example 6) in TNB. Cells were then washed with TNT. Depending on
which soluble receptor was used, cells were incubated for another
hour with: (A) 1:200 diluted goat-anti-human Ig-HRP (Fc specific);
(B) 1:1000 diluted M2-HRP; (C) 1:1000 diluted anti-GluGlu
antibody-HRP; or (D) 1:300 diluted streptavidin-HRP (NEN kit) in
TNB. Again cells were washed with TNT.
[0293] Positive binding was detected with fluorescein tyramide
reagent diluted 1:50 in dilution buffer (NEN kit) and incubated for
4-6 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.
Example 17
Mouse zalpha11 Ligand Binds to Human zalpha11 Soluble Receptor in
Secretion Trap Assay
[0294] A plasmid containing DNA encoding the mouse zalpha11 Ligand
(SEQ ID NO:47) was transfected into COS cells, and the binding of
human zalpha11 soluble receptor zalpha11-Fc4 (Example 6C) to the
transfected COS cells was tested by a secretion trap assay (Example
16). The assay confirmed that the mouse zalpha11 Ligand binds to
human zalpha11 soluble receptor.
[0295] The COS cell transfection was performed as per Example 16
using 0.7 .mu.g of the plasmid in 3 .mu.l. The secretion trap was
performed as per Example 16 using 1 .mu.g/ml zalpha11 soluble
receptor Fc4 fusion protein (Example 6C) in TNB, and 1:200 diluted
goat-anti-human Ig-HRP (Fc specific) in TNB for the detectable
antibody. Positive binding of the soluble human zalpha11 receptor
to the prepared fixed cells was detected with fluorescein tyramide
reagent, preserved and visualized according to Example 16. The
positive result indicated the mouse zalpha11 Ligand binds to human
zalpha11 soluble receptor.
Example 18
Mouse zalpha11 Ligand Activates Human zalpha11 Receptor in BaF3
Assay Using Alamar Blue
[0296] BaF3/Zalpha11 cells were spun down, washed and plated in
mIL-3 free media as described in Example 2. Proliferation of the
BaF3/Zalpha11 cells was assessed using serum-free conditioned media
from BHK cells expressing mouse zalpha11 Ligand (SEQ ID NO:47).
Conditioned media was diluted with mIL-3 free media to: 50%, 25%,
12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations. The
proliferation assay was performed as per Example 2. Results
confirmed the proliferative response of the BaF3/Zalpha11 cells to
mouse zalpha11 Ligand. The response, as measured, was approximately
5-fold over background at the 50% concentration.
Example 19
Zalpha11 Ligand Activates Human zalpha11 Receptor in Luciferase
Assay
[0297] A. Construction of BaF3/KZ134/zalpha11 Cell Line
[0298] The KZ134 plasmid was constructed with complementary
oligonucleotides ZC12,749 (SEQ ID NO:48) and ZC12,748 (SEQ ID
NO:49) 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 SIEI
from the p21 WAFI gene (Chin, Y. et al., Science 272:719-722,
1996), the mammary gland response element of the .beta.-casein gene
(Schmitt-Ney, M. et al., Mol. Cell. Biol. 11:3745-3755, 1991), and
a STAT inducible element of the Fcg RI gene, (Seidel, H. et al.,
Proc. Natl. Acad. Sci. 92:3041-3045, 1995). These oligonucleotides
contain Asp718-XhoI compatible ends and were ligated, using
standard methods, into a recipient firefly luciferase reporter
vector with a c-fos promoter (Poulsen, L. K. et al., J. Biol. Chem.
273:6229-6232, 1998) digested with the same enzymes and containing
a neomycin selectable marker. The KZ134 plasmid was used to stably
transfect BaF3 cells, using standard transfection and selection
methods, to make the BaF3/KZ134 cell line.
[0299] A stable BaF3/KZ134 indicator cell line, expressing the
full-length zalpha11 receptor was constructed as per Example 1,
using about 30 .mu.g of the zalpha11 expression vector. Clones were
diluted, plated and selected using standard techniques. Clones were
screened by luciferase assay (see Example 19B, below) using the
human zalpha11 Ligand conditioned media as an inducer. Clones with
the highest luciferase response (via STAT luciferase) and the
lowest background were selected. A stable transfectant cell line
was selected. The cell line was called BaF3/KZ134/zalpha11.
B. Human and Mouse Zalpha11 Ligand Activates Human zalpha11
receptor in BaF3/KZ134/Zalpha11 Luciferase Assay
[0300] BaF3/KZ134/Zalpha11 cells were spun down and washed in mIL-3
free media. The cells were spun and washed 3 times to ensure
removal of mIL-3. Cells were then counted in a hemacytometer. Cells
were plated in a 96-well format at about 30,000 cells per well in a
volume of 100 .mu.l per well using the mIL-3 free media. The same
procedure was used for untransfected BaF3/KZ134 cells for use as a
control in the subsequent assay.
[0301] STAT activation of the BaF3/KZ134/Zalpha11 cells was
assessed using conditioned media from (1) BHK570 cells transfected
with an expression vector encoding the human zalpha11 Ligand (SEQ
ID NO:10) or (2) BHK570 cells transfected with an expression vector
encoding the mouse zalpha11 Ligand (SEQ ID NO:47), or (3) mIL-3
free media to measure media-only control response. Conditioned
media was diluted with RPMI mIL-3 free media to 50%, 25%, 12.5%,
6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations. 100 .mu.l of
the diluted conditioned media was added to the BaF3/KZ134/Zalpha11
cells. The assay using the conditioned media was done in parallel
on untransfected BaF3/KZ134 cells as a control. The total assay
volume was 200 .mu.l. The assay plates were incubated at 37.degree.
C., 5% CO.sub.2 for 24 hours at which time the cells were pelleted
by centrifugation at 2000 rpm for 10 min., and the media was
aspirated and 25 .mu.l of lysis buffer (Promega) was added. After
10 minutes at room temperature, the plates were measured for
activation of the STAT reporter construct by reading them on a
luminometer (Labsystems Luminoskan, model RS) which added 40 .mu.l
of luciferase assay substrate (Promega) at a five second
integration.
[0302] Results confirmed the STAT reporter response of the
BaF3/KZ134/Zalpha11 cells to the human zalpha11 Ligand. The
response, as measured, was approximately 50 fold over media-only
control at the 50% concentration. STAT activation in response to
human zalpha11 Ligand was absent in the untransfected BaF3/KZ134
control cells, showing that the response is mediated through the
Zalpha11 receptor.
[0303] Results also confirmed the STAT reporter response of the
BaF3/KZ134/Zalpha11 cells to the mouse zalpha11 Ligand. The
response, as measured, was approximately 40 fold over media-only
control at the 50% concentration. Moreover, STAT activation in
response to mouse zalpha11 Ligand was evident (about 5-fold) on the
untransfected BaF/KZ134 control cells, suggesting that the murine
BaF3 cells may have endogenous mouse receptor.
Example 20
Mouse zalpha11 Ligand is Active in Mouse Bone Marrow Assay
A. Isolation of Non-adherent Low Density Marrow Cells:
[0304] Fresh mouse femur aspirate (marrow) was obtained from 6-10
week old male Balb/C or C57BL/6 mice. The marrow was then washed
with RPMI+10% FBS (JRH, Lenexa Kans.; Hyclone, Logan Utah) and
suspended in RPMI+10% FBS as a whole marrow cell suspension. The
whole marrow cell suspension was then subjected to a density
gradient (Nycoprep, 1.077, Animal; Gibco BRL) to enrich for low
density, mostly mononuclear, cells as follows: The whole marrow
cell suspension (About 8 ml) was carefully pipeted on top of about
5 ml Nycoprep gradient solution in a 15 ml conical tube, and then
centrifuged at 600.times.g for 20 minutes. The interface layer,
containing the low density mononuclear cells, was then removed,
washed with excess RPMI+10% FBS, and pelleted by centrifugation at
400.times.g for 5-10 minutes. This pellet was resuspended in
RPMI+10% FBS and plated in a T-75 flask at approximately 10.sup.6
cells/ml, and incubated at 37.degree. C. 5% CO.sub.2 for
approximately 2 hours. The resulting cells in suspension were
Non-Adherent Low Density (NA LD) Marrow Cells.
B. 96-Well Assay
[0305] NA LD Mouse Marrow Cells were plated at 25,000 to 45,000
cells/well in 96 well tissue culture plates in RPMI+10% FBS+1 ng/mL
mouse Stem Cell Factor (mSCF) (R&D Systems, Minneapolis,
Minn.), plus 5% conditioned medium from one of the following: (1)
BHK 570 cells expressing mouse zalpha11 Ligand (SEQ ID NO:47), (2)
BHK 570 cells expressing human zalpha11 Ligand (SEQ ID NO:10), or
(3) control BHK 570 cells containing vector and not expressing
either Ligand. These cells were then subjected to a variety of
cytokine treatments to test for expansion or differentiation of
hematopoietic cells from the marrow. To test, the plated NA LD
mouse marrow cells were subjected to human Interleukin-15 (hIL-15)
(R&D Systems), or one of a panel of other cytokines (R&D
Systems). Serial dilution of hIl-15, or the other cytokines, were
tested, with 2-fold serial dilution from about 50 ng/ml down to
about 6025 ng/ml concentration. After 8 to 12 days the 96-well
assays were scored for cell proliferation by Alamar blue assay as
described in Example 2.
C. Results from the 96-well NA LD Mouse Marrow Assay
[0306] Conditioned media from the BHK cells expressing both mouse
and human zalpha11 Ligand acted in synergy with hIL-15 to promote
the expansion of a population of hematopoietic cells in the NA LD
mouse marrow. This expansion of hematopoietic cells was not shown
with control BHK conditioned medium plus IL-15. The population
hematopoietic cells expanded by the mouse zalpha11 Ligand with
hIL-15, and those hematopoietic cells expanded by the human
zalpha11 Ligand with hIL-15, were further propagated in cell
culture. These hematopoietic cells were stained with a
Phycoerythrin labeled anti-Pan NK cell antibody (Pharmingen) and
subjected to flow cytometry analysis, which demonstrated that the
expanded cells stained positively for this natural killer (NK) cell
marker.
[0307] The same 96-well assay was run, using fresh human marrow
cells bought from Poietic Technologies, Gaithersburg, Md. Again, in
conjunction with IL-15, the mouse and human zalpha11 Ligand
expanded a hematopoietic cell population that stained positively
for the NK cell marker using the antibody disclosed above.
[0308] The soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
can be used in this assay to measure binding, antagonist or
inhibitory effects of the soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. on the zalpha11 Ligand.
Example 21
Purification of zalpha11-MBP Receptor
[0309] Unless otherwise stated, all operations were carried out at
4.degree. C. The following procedure was used for purifying human
(or mouse) zalpha11-MBP soluble receptor fusions from E. Coli
(Example 8). Pre-spun frozen E. Coli paste was thawed and diluted
into 2 liters of Buffer B (0.02 M TRIS (EM Science); 0.2 M NaCl
(Mallincrodt); 0.01 M 2-mercapto-ethanol (EM Science); pH 8.0; with
5 mg/l Pepstatin A (Boehringer Mannheim); 5 mg/l Aprotinin
(Boerhinger Mannheim); and 1 mg/l PMSF (Fluka)) plus 1-2 ml of an
anti-foaming reagent AF289 antifoam (Sigma). The mixture was
processed in a pre-chilled French Press cell disrupter (Constant
Systems LTD) with 20-30 kPSI.
[0310] The lysate was then centrifuged at 18,000.times.g for 45
minutes at 4.degree. C. and the supernatant retained. A 200 ml
slurry of Amylose resin (New England BioLabs), pre-equilibrated in
Buffer A (0.02 M TRIS (EM Science); 0.2 M NaCl (Mallincrodt); 0.01
M 2-mercapto-ethanol (EM Science); pH 8.0), was added to the lysate
supernatant and incubated overnight in 21 roller bottles to allow
for maximum batch absorption of the MBP fusion protein.
[0311] The resin washed in batch column format for .gtoreq.5 column
volumes with Buffer A, then batch eluted with Buffer C (Buffer A
with 0.02 M Maltose (Sigma). Crude fractions were collected and
monitored by absorbance 280 nm.
[0312] The eluted protein was analyzed by SDS NuPAGE (NOVEX)
Coomassie (Sigma) staining. Sample and bulk protein were stored at
-80.degree. C.
Example 22
Activity of Human and Mouse zalpha11 Ligand Expanded Cells and
Mature Murine NK Cells in NK Cell Cytotoxicity Assays
[0313] A. NK cell Assay
[0314] NK cell-mediated target cytolysis was examined by a standard
.sup.51Cr-release assay. Target cells (K562 cells (ATCC No.
CCL-243) in human assays, and YAC-1 cells (ATCC No. TIB-160) in
mouse assays) lack expression of major histocompatability complex
(MHC) molecules, rendering them susceptible to NK cell-mediated
lysis. A negative control target cell line in mouse assays is the
MHC.sup.+ thymoma EL4 (ATCC No. TIB-39). We grew K562, EL4, and
YAC-1 cells in RP10 medium (standard RPMI 1640 (Gibco/BRL, Grand
Island, N.Y.) supplemented with 10% FBS (Hyclone, Logan, Utah), as
well as 4 mM glutamine (Gibco/BRL), 100 I.U./ml penicillin+100
MCG/ml streptomycin (Gibco/BRL), 50 .mu.M .beta.-mercaptoethanol
(Gibco/BRL) and 10 mM HEPES buffer (Gibco/BRL). On the day of
assay, 1-2.times.10.sup.6 target cells were harvested and
resuspended at 2.5-5.times.10.sup.6 cells/ml in RP10 medium. We
added 50-100 .mu.l of 5 mCi/ml .sup.51Cr-sodium chromate SEN,
Boston, Mass.) directly to the cells and incubated them for 1 hour
at 37.degree. C., then washed them twice with 12 ml of PBS and
resuspended them in 2 ml of RP10 medium. After counting the cells
on a hemacytometer, the target cells were diluted to
0.5-1.times.10.sup.5 cells/ml and 100 .mu.l (0.5-1.times.10.sup.4
cells) were mixed with effector cells as described below.
[0315] In human assays, effector cells were prepared from selected
and expanded human CD34.sup.+BM cells which were harvested, washed,
counted, mixed at various concentrations with .sup.51Cr-labeled
target cells in 96-well round bottomed plates, and incubated for 4
hours at 37.degree. C. After co-incubation of effector cells and
the labeled target cells, half of the supernatant from each well
was collected and counted in a gamma counter for 1 min/sample. The
percentage of specific .sup.51Cr release was calculated from the
formula 100.times. (X-Y)/(Z-Y), where X is .sup.51Cr release in the
presence of effector cells, Y is the spontaneous release in the
absence of effectors, and Z is the total .sup.51Cr release from
target cells incubated with 0.5% Triton X-100. Data were plotted as
the % specific lysis versus the effector-to-target ratio in each
well.
B. Activity of Human zalpha11 Ligand Expanded Cells
[0316] Isolated CD34+ human HPCs cultured with flt3+/-zalpha11
Ligand and flt3+IL-15+/-zalpha11 Ligand, were harvested the cells
on day 15 to assess their capacity to lyse MHC.sup.- K562 cells in
a standard .sup.51Cr-release assay as described above, and to
analyze their surface phenotype by flow cytometry. As expected from
previous reports (Mrozek, E et al., Blood 87:2632-2640, 1996; and
Yu, H et al., Blood 92:3647-3657, 1998), simultaneous addition of
IL-15 and flt3L did induce the outgrowth of a small population of
CD56.sup.+ cells. Interestingly, although BM cells cultured
simultaneously with zalpha11 Ligand and flt3L did not expand
significantly, there was a significant increase in total cell
numbers in cultures containing a combination of flt3L, zalpha11
Ligand and IL-15.
[0317] For an assessment of the surface phenotype of these human BM
cultures, we stained small aliquots of the cells for 3-color flow
cytometric analysis with anti-CD3-FITC, anti-CD56-PE and
anti-CD16-CyChrome mAbs (all from PharMingen, San Diego, Calif.)
and analyzed them on a FACSCalibur using CellQuest software (Becton
Dickinson, Mountain View, Calif.). This flow cytometric analysis
confirmed that the cells growing out of these cultures were
differentiated NK cells, as they were large and granular and
expressed both CD56 and CD16, and were CD3.sup.- (Lanier, L L Annu.
Rev. Immunol. 16:359-393, 1998). Furthermore, these cells exhibited
significantly higher effector function than those cells grown with
IL-15 and flt3. More specifically, cells grown in all three
cytokines lysed more than 40% of the K562 targets at an
effector-to-target ratio (E:T) of 1.5, whereas cells grown in
IL-15+ flt3L lysed fewer than 5% of the targets at an E:T of 2.
These data demonstrate that, in combination with IL-15, zalpha11
Ligand (commonly owned U.S. patent application Ser. No. 09/522,217)
stimulates the differentiation of NK cells from CD34.sup.+ BM
cells.
C. Activity of Mouse zalpha11 Ligand Expanded Cells
[0318] To test the effects of mouse zalpha11 Ligand (commonly owned
U.S. patent application Ser. No. 09/522,217) on murine
hematopoietic progenitor cells, purified Lineage-negative (Lin-)
bone marrow cells from C57Bl/6 mice were expanded in
flt3+IL-15+/-zalpha11 Ligand. On day 6 of culture, the cells
("effectors") were harvested and counted, then resuspended in 0.4
ml of RP10 medium (Example 22A). Two aliquots (0.15 ml each) of
each sample expanded with or without zalpha11 Ligand (Example 22A)
were diluted serially 3-fold in duplicate in 96-well round bottomed
plates, for a total of 6 wells of 100 .mu.l each. The remaining 100
.mu.l of cells were stained for NK cell surface markers with
FITC-anti-2B4 and PE-anti-DX5 mAbs (PharMingen) and analyzed by
flow cytometry. Each group of cells exposed to flt3+IL-15 with or
without the presence of mouse zalpha11 Ligand had similar fractions
of 2B4+DX5+ cells, ranging from 65-75% positive for both NK
markers.
[0319] For the NK lysis assay, target cells (YAC-1 and EL4) were
labeled with .sup.51Cr as described above. After counting the
target cells on a hemacytometer, the target cells were diluted to
0.5-1.times.10.sup.5 cells/ml and 100 .mu.l of YAC-1 or EL4
(0.5-10.sup.4 cells) were mixed with 100 .mu.l effector cells and
incubated for 4 hours at 37.degree. C. Specific lysis was
determined for each well as described above.
[0320] We found that cells grown in the presence of flt3+IL-15+
zalpha11 Ligand exhibited enhanced lytic activity (roughly 2-fold)
against the YAC-1 targets (but did not kill the MHC+control cell
line EL4). At an effector-to-target ratio (E:T) of 5, NK cells
generated in the presence of all 3 cytokines (zalpha11
Ligand+flt3+IL-15) lysed 12% of the YAC-1 cells, whereas those NK
cells expanded with flt3+IL-15 lysed 6% of the YAC-1 targets.
Subsequent experiments confirmed this trend.
[0321] In a second approach to determine the biological activity of
zalpha11 Ligand on murine NK cells, we isolated immature CD4 CD8
("double negative", DN) mouse thymocytes using routine methods and
cultured them with IL-15+ flt3+IL-7 or IL-15+ flt3+IL-2, with or
without zalpha11 Ligand. On day 6 of culture, the cells were
harvested and assayed for NK lytic activity on YAC-1 and EL4 cells
as described above. We found that cells cultured in the presence of
zalpha11 Ligand had the greatest lytic activity in this assay, with
enhanced lytic activity over those cells cultured in the presence
of the other cytokines. Specifically, DN thymocytes grown with
IL-15+ flt3+IL-7 killed 18% of the YAC-1 cells at E:T of 24 while
cells grown in the presence of IL-15+ flt3+IL-7 plus zalpha11
Ligand killed 48% of the targets at the same E:T. DN thymocytes
grown in IL-15+ flt3+IL-2 killed 15% of the YAC-1 targets at an E:T
of 6, whereas cells grown with these 3 cytokines and zalpha11
Ligand killed 35% of the YAC-1 cells at an E:T of 9. Flow cytometry
was performed on the cultured cells one day before the NK lysis
assay. As was true for the bone marrow cultures, despite the
proliferative effect of zalpha11 Ligand (cell numbers increase
approximately 2-fold when zalpha11 Ligand is added), it did not
significantly enhance the fraction of DX5.sup.+ cells (17-20% of
total cells in the cultures with IL-7, and 35-46% of total in
cultures with IL-2). These data imply that zalpha11 Ligand, in
combination with IL-15 and flt3, enhances the lytic activity of NK
cells generated from murine bone marrow or thymus.
D. Activity of Mouse zalpha11 Ligand on Mature Murine NK Cells
[0322] In order to test the effects of mouse zalpha11 Ligand on
mature NK cells, we isolated spleens from four 5-week old C57Bl/6
mice (Jackson Laboratories, Bar Harbor, Me.) and mashed them with
frosted-end glass slides to create a cell suspension. Red blood
cells were removed by hypotonic lysis as follows: cells were
pelleted and the supernatant removed by aspiration. We disrupted
the pellet with gentle vortexing, then added 900 .mu.l of sterile
water while shaking, followed quickly (less than 5 sec later) by
100 .mu.l of 10.times. HBSS (Gibco/BRL). The cells were then
resuspended in 10 ml of 1.times. HBSS and debris was removed by
passing the cells over a nylon mesh-lined cell strainer (Falcon).
These RBC-depleted spleen cells were then pelleted and resuspended
in MACS buffer (PBS+1% BSA+2 mM EDTA) and counted. We stained
300.times.10.sup.6 of the cells with anti-DX5-coated magnetic beads
(Miltenyi Biotec) and positively selected DX5+NK cells over a MACS
VS+separation column, according to the manufacturer's instructions,
leading to the recovery of 8.4.times.10.sup.6 DX5+ cells and
251.times.10.sup.6 DX5-cells. Each of these groups of cells were
cultured in 24-well plates (0.67.times.10.sup.6 cells/well, 2 wells
per treatment condition) in RP10 medium (Example 22A) alone or with
1) 30 ng/ml mouse zalpha11 Ligand (commonly owned U.S. patent
application Ser. No. 09/522,217), 2) 30 ng/ml recombinant mouse
IL-2 (R&D Systems, Inc., Minneapolis, Minn.), 3) 30 ng/ml
recombinant human IL-15 (R&D), 4) 30 ng/ml each of mouse
zalpha11 Ligand and hIL-15, or 5) 30 ng/ml each of mIL-2 and
hIL-15. The cells were harvested after 21 hours, washed, and
resuspended in RP10 medium and counted. The cells were then assayed
for their ability to lyse .sup.51Cr-labeled YAC-1 or EL4 targets
cells, as described in Example 22A.
[0323] In general, there was little NK activity from the
DX5-(non-NK cells) groups, but the DX5.sup.- cells cultured with
zalpha11 Ligand and hIL-15 did lyse 25% of the YAC-1 target cells
at an E:T of 82. By comparison, DX5.sup.- cells cultured with
hIL-15 alone lysed 14% of the YAC-1 targets at an E:T of 110. This
suggests that zalpha11 Ligand and IL-15 are acting together on the
residual NK1.1.sup.+ NK cells in this cell preparation. As for the
DX5+ cell preparation, treatment with mouse zalpha11 Ligand alone
did not significantly increase their effector function (their lysis
of YAC-1 cells was similar to the untreated group). As expected,
both IL-2 and IL-15 significantly improved NK activity. The highest
level of lysis, however, was detected in the group treated with
zalpha11 Ligand and hIL-15 (65% lysis of YAC-1 cells at an E:T of
3.3, vs. 45% lysis at an E:T of 4 for the hIL-15 treatment group).
Taken together, these results suggest that although zalpha11 Ligand
alone may not increase NK cell lysis activity, it does enhance NK
lysis activity of mature NK cells, when administered with
IL-15.
[0324] The soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
can be used in this assay to measure binding, antagonist or
inhibitory effects of the soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. on the zalpha11 Ligand.
Example 23
Zalpha11 Ligand Proliferation of Human and Mouse T-cells in a
T-cell Proliferation Assay
A. Murine Zalpha11 Ligand Proliferation of Mouse T-Cells
[0325] T cells from C57Bl/6 mice (Jackson Laboratories, Bar Harbor,
Me.) were isolated from pooled splenocytes and lymphocytes from
axillary, brachial, inguinal, cervical, and mesenteric lymph nodes
(LNs). Spleens were mashed with frosted-end glass slides to create
a cell suspension. LNs were teased apart with forceps and passed
through a cell strainer to remove debris. Pooled splenocytes and LN
cells were separated into CD8+ and CD4+ subsets using two
successive MACS magnetic separation columns, according to the
manufacturer's instructions (Miltenyi Biotec, Auburn, Calif.).
Whole thymocytes were collected from the same mice.
[0326] Cells were cultured at 3.times.10.sup.5 cells/well
(thymocytes) or 10.sup.5 cells/well (mature T cells) with
increasing concentrations of purified murine zalpha11 Ligand (0-30
ng/ml) (commonly owned U.S. patent application Ser. No. 09/522,217)
in 96-well flat bottomed plates pre-coated overnight at 4.degree.
C. with various concentrations of anti-CD3 mAb 2C11 (PharMingen)
for 3 days at 37.degree. C. The anti-CD3 antibody served to
activate the murine T-cells through the T-cell receptor. Each well
was pulsed with 1 .mu.Ci .sup.3H-thymidine on day 2 and plates were
harvested and counted 16 hours later to assess proliferation.
[0327] When we tested zalpha11 Ligand in T cell proliferation
assays, we found that it co-stimulated anti-CD3-activated murine
thymocytes, leading to an accelerated outgrowth of
CD8.sup.+CD4.sup.- cells (the majority of the thymocytes cultured
with anti-CD3+ zalpha11 Ligand were CD8.sup.+CD4.sup.- by day 3 of
culture, while cells cultured with anti-CD3 alone did not
significantly skew to this phenotype until day 5). We did not
observe significant levels of proliferation of thymocytes to
zalpha11 Ligand in the absence of anti-CD3.
[0328] Interestingly, when we assayed mature peripheral murine T
cells for their ability to respond to zalpha11 Ligand+anti-CD3, we
found that only the CD8.sup.+, but not the CD4.sup.+ subset,
responded in a dose-dependent manner to zalpha11 Ligand. We also
observed weak but reproducible proliferation of CD8+ cells (but not
CD4.sup.+ cells) in response to zalpha11 Ligand alone.
Interestingly, this was not observed for human T cells (see Example
22B, below).
B. Human Zalpha11 Ligand Proliferation of Human T-Cells
[0329] Human CD4+ and CD8+ T cells were isolated from PBMC as
described in Example 14. Cells were cultured at about 10.sup.5
cells/well with increasing concentrations of purified human
zalpha11 Ligand (0-50 ng/ml) (commonly owned U.S. patent
application Ser. No. 09/522,217) in 96-well flat bottomed plates
pre-coated overnight at 4.degree. C. with various concentrations of
anti-human CD3 mAb UCHT1 (PharMingen) for 3 days at 37.degree. C.
Each well was pulsed with 1 uCi .sup.3H-thymidine on day 2 and
plates were harvested and counted 16 hours later. Unlike our
results with mouse T cells, our preliminary data suggests that
human zalpha11 Ligand co-stimulates CD4+, but not CD8+, human T
cells in a dose-dependent fashion.
[0330] The soluble zalpha11 receptor or soluble zalpha11
heterodimeric polypeptide, such as soluble zalpha11/IL-2R.gamma.
can be used in this assay to measure binding, antagonist or
inhibitory effects of the soluble zalpha11 receptor or soluble
zalpha11 heterodimeric polypeptide, such as soluble
zalpha11/IL-2R.gamma. on the zalpha11 Ligand
Example 24
Human zalpha11 Receptor Monoclonal Antibodies
[0331] Zalpha11 receptor Monoclonal antibodies were prepared by
immunizing 5 male BalbC mice (Harlan Sprague Dawley, Indianapolis,
Ind.) with the purified recombinant protein, huzalpha11-CEE-BHK
(Example 6). The mice were each given an initial intraperitoneal
(IP) injection of 20 mg of purified protein in Complete Freund's
Adjuvant (Pierce, Rockford, Ill.) followed by booster IP injections
of 10 mg purified protein in Incomplete Freund's Adjuvant every two
weeks. Seven to ten days after the administration of the third
booster injection, the animals were bled and the serum was
collected.
[0332] The mouse sera samples raised to the huzalpha11-CEE-BHK were
characterized by an ELISA titer check using purified recombinant
CHO huzalpha11-Fc protein (Example 10C) as an antibody target. One
mouse serum sample had titer to the specific antibody target at a
dilution of 1:1,000,000 (1:1 E6). Four mouse serum samples had
titer to the specific antibody target at a dilution of 1:100,000
(1:1 E5).
[0333] Splenocytes were harvested from the 4 high-titer mice and
fused to murine SP2/0 myeloma cells using PEG 1500 (Boerhinger
Mannheim, UK) in two separate fusion procedures using a 4:1 fusion
ratio of splenocytes to myeloma cells (Antibodies: A Laboratory
Manual, E. Harlow and D. Lane, Cold Spring Harbor Press). Following
10 days growth post-fusion, specific antibody-producing hybridomas
were identified by ELISA using purified recombinant BHK human
zalpha11-Fc4 protein (Example 6C) as an antibody target and by FACS
using Baf3 cells expressing the huzalpha11 sequence (Example 2) as
an antibody target. The resulting 4 hybridomas positive by both
methods were cloned three times by limiting dilution. The
antibodies were designated: 249.28.2.1.2.2; 247.10.2.15.4.6;
249.19.2.2.3.5; and 249.15.2.4.2.7.
Example 25
Zalpha11 Receptor Purified Recombinant Human Protein Dose-Response
Study in Normal Mice
A. Summary
[0334] Normal nine week old female C57Bl/6 (Harlan Sprague Dawley,
Indianapolis, Ind.) mice were treated by intraperitoneal injection
once daily for seven days with one of three dose levels of purified
recombinant human zalpha11-Fc4 soluble receptor (Example 6C) (5, 50
or 250 .mu.g/mouse/day) or PBS vehicle plus 250 .mu.g per dose of
BSA. Body weights were monitored every other day. On day seven the
five mice from the highest dose group and five of the vehicle
control group were sacrificed. Blood, bone marrow and tissues were
harvested and analyzed. The remaining mice were sacrificed and
harvests done the following day. Potential perturbations in
lymphoid tissues were examined, as well as general physiologic and
toxicologic parameters.
[0335] There was no clinical evidence of toxicity. Liver, kidney,
spleen, thymus and brain were weighed, and there were no
differences between the treatment groups in organ weights. No
histologic changes were found in the examined tissues.
B. Dosing Solution Preparation
[0336] Purified recombinant human zalpha11 receptor-FC4 fusion
protein (zalpha11-FC4) (Example 6C) was diluted into sterile
phosphate buffered saline (PBS) (GibcoBRL, Grand Island, N.Y.) at
concentrations to deliver 5, 50 or 250 micrograms of protein in 0.1
ml of PBS vehicle. Bovine serum albumin (BSA) (Sigma, St. Louis,
Mo.) was dissolved in PBS to make a 250 .mu.g dose per 0.1 ml then
filtered through an 0.2 .mu.m syringe-tip filter for the vehicle
control treatment. The solutions for daily dosing were made on Day
0, aliquotted and frozen in a frosty -20.degree. C. freezer for
use. On the day of administration the appropriate aliquots were
thawed and 0.1 ml of solution was injected intraperitoneally at
approximately mid-morning each day for seven days.
C. Study Design
[0337] The mice were nine weeks old at the start of the study. Each
zalpha11-FC4 treatment group consisted of five mice; the control
group had 10 mice. The mice in the highest dose and half of the
control mice were sacrificed the day after the last of seven
treatments (Day 7). The two lower dose and remaining control groups
were sacrificed the following day (Day 8).
[0338] The body weights of the mice were recorded every other day
during treatment. There was no difference in weight gain between
the treatment groups over the week of treatment.
[0339] At sacrifice, tissues harvested to assess lymphocyte
populations by FACS analysis included bone marrow, thymus and
spleen. Flow Cytometry analysis of the lymphoid organs and bone
marrow was performed with the FACSCalibur, (Becton Dickinson,
Mansfield, Mass.). The tissues harvested for histologic examination
for signs of toxicity of the protein included: spleen, thymus,
liver, kidney, adrenal gland, mesenteric lymph node, duodenum,
pancreas, jejunum, sternum, uterus, ovaries, urinary and gall
bladders, salivary gland, heart and lungs. All tissues fixed for
histology were kept at 4.quadrature.C overnight in 10% Normal
Buffered Saline (Surgipath, Richmond, Ill.). The following day the
NBF was replaced with 70% ethanol and the tissues returned to
4.quadrature.C until processing for histology.
[0340] The tissues were processed and stained for H&E analysis
in house, then sent to the contract pathologist, David Fairchild.
Blood was collected for complete blood cell counts and serum
chemistry profiles. The CBC's were done in-house with the Cell Dyn
3500 Hematology Analyzer (Abbott Diagnostics Division, Abbott Park,
Ill.). The serum was kept frozen in a frosty -20.quadrature.C
freezer until submission to Phoenix Central Laboratory (Everett,
Wash.) for complete serum chemistry panels. To compare
myeloid:erythroid ratios between the 250 .mu.g dose groups of
zalpha11R and BSA, an aliquot of the bone marrow from one femur was
applied to CytoSpin slides (CYTOSPIN 3 CYTOCENTRIFUGE and CYTO
SLIDES, Shandon, Pittsburgh, Pa.). The bone marrow slides were
analyzed at Phoenix Central Laboratories.
D. Study Results
[0341] There were no apparent clinical indications of physiologic
effects or of toxicity of rh-zalpha11R-FC4 fusion protein at doses
tested (250 .mu.g/day or lower). Body weights remained normal for
the duration of the treatments. Red blood cell and platelet counts
were normal. There were two mice in the 250 .mu.g dose zalpha11-FC4
group whose differential WBC count revealed a possible elevation in
the percentage of monocytes, however the other three mice in the
group had monocyte percentages equivalent to the average of the
control mice. The differential white blood cell monocyte count
difference is not considered a significant finding. There were no
other differences in complete blood counts. The bone marrow
cytology did not reveal a shift in the myeloid and erythroid
progenitor populations, and all cell types present appeared normal.
All the standard serum chemistry parameters were in normal ranges.
There were no differences between the treatment groups in the
weights of the thymus, spleen, kidney, liver or brain. Histologic
evaluation of the following tissues showed no evidence of
abnormalities: thymus, spleen, liver, kidney, adrenal gland,
duodenum, pancreas, jejunum, caecum, colon, mesenteric lymph nodes,
uterus, ovaries, salivary gland, heart, trachea, lung and brain.
The absence of physiologic effects in normal mice indicates that
the zalpha11 soluble receptor has low toxicity in vivo, which is
desirable for a therapeutic agent.
Example 26
Zalpha11 Ligand-dependent Proliferation of B-Cell Cells Stimulated
Anti-CD40 or Anti-11M
A. Purification of Human B Cells
[0342] A vial containing 1.times.10.sup.8 frozen, apheresed human
peripheral blood mononuclear cells (PBMCs) was quickly thawed in a
37.degree. C. water bath and resuspended in 25 ml B cell medium
(RPMI Medium 1640 (JRH Biosciences. Lenexa, Kans.), 10% Heat
inactivated fetal bovine serum, 5% L-glutamine, 5% Pen/Strep)
(Gibco BRL)) in a 50 ml tube (Falcon VWR, Seattle, Wash.). Cells
were tested for viability using Trypan Blue (Gibco BRL). Ten
milliliters of Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology
Inc., Piscataway, N.J.) was layered under the cell suspension and
spun for 30 minutes at 1800 rpm and allowed to stop with the brake
off. The interface was then removed and transferred to a fresh 50
ml Falcon tube, brought up to a final volume of 40 ml with PBS and
spun for 10 minutes at 1200 rpm with the brake on. The viability of
the isolated cells was tested using Trypan Blue. Alternately fresh
drawn human blood was diluted 1:1 with PBS (Gibco BRL) and layered
over Ficoll/Hypaque Plus (Pharmacia), spun and washed as above.
Cells isolated from either fresh or frozen sources gave equivalent
results.
[0343] B cells were purified from the Ficoll floated peripheral
blood cells of normal human donors (above) with anti-CD 19 magnetic
beads (Miltenyi Biotec, Auburn, Calif.) following the
manufacturer's instructions. The purity of the resulting
preparations was monitored by flow cytometric analysis with
anti-CD22 FITC Ab (Pharmingen, SanDiego, Calif.). B cell
preparations were typically >90% pure.
B. Purification of Murine B Cells
[0344] A suspension of murine splenocytes was prepared by teasing
adult C57Bl/6 mouse (Charles River Laboratories, Wilmington, Mass.)
spleens apart with bent needles in B cell medium. RBCs were removed
by hypotonic lysis. CD43 positive cells were removed with CD43
magnetic beads (Miltenyi Biotec) following the manufacturer's
instructions. The purity of the resulting preparations was
monitored by flow cytometric analysis with anti-CD45R FITC Ab
(Pharmingen). B cell preparations were typically >90% pure.
C. Proliferation of anti-CD40-stimulated B-Cells in the presence of
human or murine zalpha11 Ligand
[0345] The B cells from either the human or mouse source were
resuspended at a final concentration of 1.times.10.sup.6 cells/ml
in B cell medium and plated at 100 .mu.l/well in a 96 well U bottom
plate (Falcon, VWR) containing various stimulation conditions to
bring the final volume to 200 .mu.l/well. For anti-CD40 stimulation
human cultures were supplemented with 1 ug/ml anti-human CD40
(Genzyme, Cambridge, Mass.) and mouse cultures were supplemented
with 1 .mu.g/ml anti-murine CD40 (Serotec, UK). Human or murine
zalpha11 Ligand (commonly owned U.S. patent application Ser. No.
09/522,217) was added at dilutions ranging from 1 pg/ml-100 ng/ml
as appropriate. The specificity of the effect of zalpha11 Ligand
was confirmed by inhibition of zalpha11 Ligand with 25 mg/ml
soluble human zalpha11CEE (Example 6A). All treatments were
performed in triplicate. The cells were then incubated at
37.degree. C. in a humidified incubator for 120 hours (human) or 72
hours (mouse). Sixteen hours prior to harvesting, 1 .mu.Ci
.sup.3H-thymidine (Amersham, Piscataway, N.J.) was added to all
wells to assess whether the B-cells had proliferated. The cells
were harvested into a 96 well filter plate (UniFilter GF/C,
Packard, Meriden, Conn.) using a cell harvester (Packard) and
collected according to manufacturer's instructions. The plates were
dried at 55.degree. C. for 20-30 minutes and the bottom of the
wells were sealed with an opaque plate sealer. To each well was
added 0.25 ml of scintillation fluid (Microscint-O, Packard) and
the plate was read using a TopCount Microplate Scintillation
Counter (Packard).
[0346] Incubation with Zalpha11 Ligand at concentrations of 3 ng/ml
or more enhanced the proliferation induced by soluble anti-CD40 in
a dose dependent manner in both murine and human B cells by as much
as 30 fold. The murine and human B cells responded equally as well
to their respective zalpha11 Ligand. In both species, the
stimulation was specific to zalpha11 Ligand, as it was reversed by
the presence of soluble zalpha11 receptor in the culture.
D. Proliferation of Anti-11M-Stimulated B-Cells in the Presence of
Human or Murine zalpha11 Ligand
[0347] The B cells from either human or mouse source as described
above (parts A and B) were plated as described above (part C). For
anti-IgM stimulation of human cells the plates were pre-coated
overnight with 10 mg/ml F(ab').sub.2 anti-human IgM Abs (Southern
Biotech Associates, Birmingham, Ala.) and washed with sterile media
just prior to use. The cultures were supplemented with 0-10 ng/ml
hu rIL-4 (R&D Systems, Minneapolis, Minn.). For anti-IgM
stimulation of murine cells soluble anti-IgM (Biosource, Camarillo,
Calif.) was added to the cultures at 10 mg/ml. To each of the
preceding anti-IgM/IL-4 conditions, human or murine Zalpha11 ligand
was added at dilutions ranging from 1 pg/ml-100 ng/ml as described
above.
[0348] The specificity of the effect of zalpha11 Ligand was
confirmed by inhibition with soluble human zalpha11 receptor as
described above (Part C). All treatments were performed in
triplicate. The cells were incubated, labeled with
.sup.3H-thymidine, harvested, and analyzed as described in part C
above.
[0349] Incubation with Zalpha11 ligand at concentrations of 0.3
ng/ml or more inhibited the proliferation induced by insoluble
anti-IgM (mouse) or anti-IgM and IL-4 (human) in a dose-dependent
manner. This inhibition was specific to zalpha11 Ligand, as it was
reversed by the presence of soluble zalpha11 receptor in the
culture.
E. Anti-CD40 B Cell Proliferation requires IL-2 Receptor Gamma
[0350] Murine B-cells were purified and stimulated with anti-CD40
monoclonal antibody as described in Example 26B and C above. The
co-stimulation induced by murine zalpha11 Ligand was completely
blocked by the addition of anti-IL-2 receptor gamma (IL-2R.gamma.)
monoclonal antibodies that block IL-2.gamma. utilization. The
antibodies 3E12 and TUG/m2 (PharMingen, San Diego, Calif.) were
included in the proliferation assay at 50 .mu.g/ml. These results
demonstrate that the IL-2R.gamma. in B cells is physiologically
involved with the zalpha11 Ligand stimulation of B cells. Moreover,
these results provide indirect functional support in vivo for the
finding that the IL-2R.gamma. heterodimerizes with the zalpha11
receptor in vitro (Example 27, below).
F. The Effects of zalpha11 Ligand on B Cells are Inhibited by
Soluble zalpha11 Receptor Constructs
[0351] Murine B-cells were purified and stimulated with anti-CD40
monoclonal antibody or anti-IgM antibodies as described in Example
26C and D above. The effect induced by murine zalpha11 Ligand was
completely blocked by the addition of either purified huzalpha11R:
IL-2R.gamma. a heterodimeric soluble receptor (Example 28), or a
mu-zalpha11-Fc, a homodimeric soluble receptor (Example 6C). Again,
these results provide further functional support for the finding
that the IL-2R.gamma. heterodimerizes with the zalpha11 receptor
(Example 27, below), and acts as an antagonist to the zalpha11
Ligand's effect on B-cells.
Example 27
Human Zalpha11 Receptor Heterodimerizes With IL-2 Receptor
Gamma
[0352] A. Assay Using Conditioned Media from Transfected BHK-570
Cells Expressing Human zalpha11 Ligand
[0353] Soluble human zalpha11 receptor zalpha11CFLAG (Example 6B),
or gp130 (Hibi, M. et al., Cell 63:1149-1157, 1990) were
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 zalpha11 receptor and soluble IL-2
receptor-.gamma. (sIL-2R.gamma.) (R&D Systems, Minneapolis,
Minn.) were 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 zalpha11 receptor were respectively designated Bio-zalpha11
receptor and Ru-zalpha11; the biotinylated and Ru-BPY-NHS-labeled
forms of the soluble IL-2R.gamma. were respectively designated
Bio-IL2R.gamma. and Ru-IL2R.gamma..
[0354] For initial receptor binding characterization of human
zalpha11 Ligand, conditioned media from transfected BHK-570 cells
expressing human zalpha11 Ligand or control media from
untransfected BHK-570 cells was used to determine if zalpha11
Ligand could mediate homodimerization of zalpha11 receptor and if
it could mediate the heterodimerization of zalpha11 receptor with
IL-2R.gamma. or gp130. To do this, 50 .quadrature. l of conditioned
media, from control cells or conditioned media from cells
expressing zalpha11 Ligand, was combined with 50 .quadrature.l of
TBS-B (20 mM Tris, 150 mM NaCl, 1 mg/ml BSA, pH 7.2) containing 400
ng/ml of Ru-zalpha11 receptor and Bio-zalpha11, or 400 ng/ml of
Ru-zalpha11 receptor and Bio-gp130, or 400 ng/ml of Ru-IL2R.gamma.
and Bio-zAlph11. Following incubation for one hour at room
temperature, 30 .mu.g of streptavidin coated, 2.8 mm magnetic beads
(Dynal, Inc., Oslo, Norway) were added and the reaction incubated
an additional hour at room temperature. 200 .mu.l ORIGEN assay
buffer (Igen, Inc., Gaithersburg, Md.) was then added and the
extent of receptor association measured using an M8 ORIGEN analyzer
(Igen, Inc.).
[0355] Conditioned media containing zalpha11 Ligand caused the
heterodimerization of Bio-zalpha11 receptor with Ru-IL2R.gamma.. No
receptor dimerization was observed in the presence of control
media. Conditioned media containing zalpha11 Ligand did not cause
the homodimerization of RU-zalpha11 receptor with Bio-zalpha11
receptor, nor the heterodimerization of Ru-zalpha11 receptor with
Bio-gp130.
B. Assay Using Purified Human zalpha11 Ligand
[0356] To assess the ligand specificity of the heterodimerization
of zalpha11 receptor and IL2R.gamma., 50 .quadrature.l of TBS-B
containing 400 ng/ml of Ru-zalpha11 receptor and Bio-zAlph11, or
400 ng/ml Ru-IL2R.gamma. and Bio-zAlph11 was combined 50
.quadrature.l of TBS-B containing IL-2, IL-4, IL-15 or purified
human zalpha11 Ligand (commonly owned U.S. patent application Ser.
No. 09/522,217) at concentrations from 133 pg/ml to 300 ng/ml.
Following incubation for one hour a room temperature, 3 .mu.g of
streptavidin coated, 2.8 mm magnetic beads (Dynal, Inc.) were added
and the reaction incubated an additional hour at room temperature.
200 .quadrature.l Origlo assay buffer (Igen, Inc.) was then added
and the extent of receptor association measured using an M8 Origen
analyzer (Igen, Inc.). The human zalpha11 Ligand caused the
heterodimerization of Bio-zalpha11 receptor with Ru-IL-2R.gamma. in
a dose dependent manner with a half maximal concentration of 10
ng/ml. No homodimerization of Ru-zalpha11 receptor with
Bio-zalpha11 receptor was observed at any concentration of zalpha11
Ligand tested. No homodimerization of Ru-zalpha11 receptor with
Bio-zalpha11 receptor or heterodimerization of Bio-zalpha11
receptor with Ru-IL2R.gamma. was observed with IL-2, IL-4 or IL-15,
at any of the concentrations tested. Thus, the results show that
the human zalpha11 receptor heterodimerizes specifically with the
IL-2 receptor-.gamma. in the presence of human zalpha11 Ligand, and
that the zalpha11 receptor does not homodimerize or heterodimerize
in the presence of other cytokines tested.
Example 28
Construct for Generating Human Zalpha11 Receptor/IL-2R.gamma.
Heterodimer
[0357] A vector expressing a secreted human hzalpha11/hIL2Rgamma
heterodimer was constructed. In this construct, the extracellular
domain of hzalpha11 was fused to the heavy chain of IgG gamma 1
(IgG.gamma.1) (SEQ ID NO:16), while the extracellular portion of
hIL-2R.gamma. was fused to a human kappa light chain (human K light
chain) (SEQ ID NO:18).
A. Construction of IgG Gamma 1 and Human .kappa. Light Chain Fusion
Vectors
[0358] The heavy chain of IgG.gamma.1 was cloned into the Zem229R
mammalian expression vector (ATCC deposit No. 69447) such that any
extracellular portion of a receptor 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 was made by using PCR to isolate the IgG.gamma.1
sequence from a Clontech hFetal Liver cDNA library as a template. A
PCR reaction using oligos ZC11,450 (SEQ ID NO:50) and ZC11,443 (SEQ
ID NO:51) was run as follows: 40 cycles of 94.degree. C. for 60
sec., 53.degree. C. for 60 sec., and 72.degree. C. for 120 sec.;
and 72.degree. C. for 7 min. PCR products were separated by agarose
gel electrophoresis and purified using a QiaQuick.TM. (Qiagen) gel
extraction kit. The isolated, 990 bp, DNA fragment was digested
with Mlu I and EcoRI (Boerhinger-Mannheim), ethanol precipitated
and ligated with oligos ZC11,440 (SEQ ID NO:52) and ZC11,441 (SEQ
ID NO:53), which comprise an MluI/EcoRI linker, into Zem229R
previously digested with and EcoRI using standard molecular biology
techniques disclosed herein. This generic cloning vector was called
Vector#76 hIgGgamma1 w/ Ch1 #786 Zem229R (Vector #76). The
polynucleotide sequence of the extracellular domain of hzalpha11
fused to the heavy chain of IgG gamma 1 is shown in SEQ ID NO:15
and the corresponding polypeptide sequence shown in SEQ ID
NO:16.
[0359] The human .kappa. light chain was cloned in the Zem228R
mammalian expression vector (ATCC deposit No. 69446) such that any
extracellular portion of a receptor having a 5' EcoRI site and a 3'
KpnI site can be cloned in resulting in a N-terminal extracellular
domain-C-terminal human .kappa. light chain fusion. The human K
light chain fragment used in this construct was made by using PCR
to isolate the human .kappa. light chain sequence from the same
Clontech hFetal Liver cDNA library used above. A PCR reaction using
oligos ZC11,501 (SEQ ID NO:54) and ZC11,451 (SEQ ID NO:55) was run
under conditions described above. PCR products were separated by
agarose gel electrophoresis and purified using a QiaQuick.TM.
(Qiagen) gel extraction kit. The isolated, 315 bp, DNA fragment was
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. This
generic cloning vector was called Vector #77 hKlight #774 Zem228R
(Vector #77). The polynucleotide sequence of the extracellular
portion of hIL-2R.gamma. was fused to a human kappa light chain is
shown in SEQ ID NO:17 and the corresponding polypeptide sequence
shown in SEQ ID NO:18.
B. Insertion of zalpha11 Receptor or IL-2R.gamma. Extracellular
Domains into Fusion Vector Constructs
[0360] Using the construction vectors above, a construct having
human zalpha11 fused to IgG.gamma.1 was made. This construction was
done by PCRing human zalpha11 receptor from a CD4+ bone marrow
library (selected, and made in house) with oligos ZC24,052 (SEQ ID
NO:56) and ZC24,053 (SEQ ID NO:57), under conditions described 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. The resulting PCR product was digested with EcoRI and NheI,
gel purified, as described herein, and ligated into a previously
EcoRI and NheI digested and band-purified Vector#76 (above). The
resulting vector was sequenced to confirm that the human
zalpha11/IgG gamma 1 fusion (hzalpha11/Ch1 IgG) was correct. The
hzalpha11/Ch1 IgG gamma1 vector was called Vector #190.
[0361] A separate construct having IL-2R.gamma. fused to .kappa.
light was also constructed. The IL-2R.gamma./human .kappa. light
chain construction was performed as above by PCRing from the same
CD4+ library mentioned above with oligos ZC12,834 (SEQ ID NO:58)
and ZC12,831 (SEQ ID NO:59), digesting the resulting band with
EcoRI and KpnI and then ligating this product into a previously
EcoRI and KpnI digested and band-purified Vec#77 (above). The
resulting vector was sequenced to confirm that the human
IL-2R.gamma./ human .kappa. light chain fusion
(hIL-2R.gamma./Klight) was correct. This hIL-2gamma/Klight #1052
Zem228R vector was called Vector #101.
D. Co-Expression of the Human zalpha11 and human IL-2R.gamma.
Receptors
[0362] Approximately 16 .mu.g of each of Vectors #190 and #101,
above, were co-transfected into BHK-570 cells (ATCC No. CRL-10314)
using LipofectaminePlus.TM. reagent (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 of
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 of MTX and 0.5 mg/ml G418 for 10
days.
[0363] The resulting pool of doubly-selected cells was used to
generate protein. Three Factories (Nunc, Denmark) of this pool were
used to generate 10 L of serum free conditioned medium. This
conditioned media was passed over a 1 ml protein-A column and
eluted in (10) 750 microliter fractions. 4 of these fractions found
to have the highest concentration were pooled and dialyzed (10 kD
MW cutoff) against PBS. Finally the dialyzed material was submitted
for amino acid analysis (AAA) and found to have a concentration of
227.17 .mu.g/ml AAA. A total of 681.5 .mu.g was obtained from this
10 L purification. The purified soluble human zalpha11
receptor/IL-2R.gamma. receptor was used to assess its ability to
compete with the human zalpha11 Ligand a BaF3 proliferation assay
(Example 29, below).
Example 29
Soluble Human zalpha11 Receptor/Human IL2 Gamma Receptor-Fc as a
zalpha11 Ligand Antagonist
[0364] BaF3 cells stably expressing the human zalpha11 receptor
(Example 2) were plated at 5500 cells per well in standard 96-well
tissue culture plates in base medium plus 3 ng/ml human zalpha11
Ligand. Base medium is 500 ml RPMI 1640 (JRH Biosciences), 5 ml
100.times. Sodium Pyruvate (Gibco BRL), 5 ml 100.times.L-glutamine
(Gibco BRL), and 50 ml heat-inactivated Fetal Bovine Serum (FBS)
(Hyclone Laboratories). To the cells, a decreasing dose of either
purified soluble human zalpha11 receptor-Fc homodimer (Example 6C)
or purified soluble human zalpha11 receptor/human IL2 gamma
receptor-Fc heterodimer (Example 27) were added. An Alamar Blue
proliferation assay was run and fluorimetry performed as per
Example 2B.
[0365] The zalpha11 receptor/IL2 gamma receptor-Fc heterodimer
inhibited human zalpha11 Ligand activity in a dose dependent
manner, with 0.312 .mu.g/ml able to completely inhibit the activity
of 3 ng/ml human zalpha11 Ligand. The soluble zalpha11 receptor-Fc
homodimer also was able to inhibit zalpha11 Ligand activity in a
dose dependent manner, however it required about 10 .mu.g/ml of
soluble homodimer to completely inhibit the activity of 3 ng/ml
zalpha11 Ligand. These data suggested the zalpha11 receptor/IL2
gamma receptor-Fc heterodimer soluble receptor is approximately 30
to 100 fold more potent than the homodimeric soluble zalpha11
receptor in inhibiting human zalpha11 Ligand.
Example 30
Zalpha11 Receptor Distribution
[0366] To assess zalpha11 receptor distribution on various cells
types, we generated both rabbit polyclonal and mouse monoclonal
antibodies (mAbs) directed against the human receptor (Example 24
and Example 10) and conjugated these antibodies to biotin for use
in flow cytometry. We initially used the polyclonal antibodies,
which were of relatively low affinity, to stain a panel of cell
lines: IL-3 dependent murine pre-B cell line wild-type BaF3 cells
(Palacios and Steinmetz, ibid.; Mathey-Prevot et al., ibid.); BaF3
cells transfected with human zalpha11 (Example 2); human Burkitt's
lymphoma cell lines Raji (ATCC No. CCL-86), Ramos (ATCC No.
CRL-1596), RPMI 8226 (ATCC No. CCL-155), and Daudi (ATCC No.
CCL-213); human T cell leukemia cell line Jurkat (ATCC No.
TIB-152); human myelomonocytic leukemia cell lines Thp-1 (ATCC No.
TIB-202) and U937 (ATCC No.CRL-1593.2); human pro-myelomonocytic
cells HL-60 (ATCC No. CCL-240); murine B cell lymphoma cell line
A20 (ATCC No TIB-208); and murine thymoma cell line EL4 (ATCC No.
TIB-39).
[0367] The cells were harvested, washed once with FACS wash buffer
with serum (WBS). WBS consisted of Hank's balanced salt solution
(Gibco/BRL)+10 mM HEPES (Gibco/BRL)+1% BSA (Sigma)+10% normal goat
serum (Gemini Bioproducts, Woodland, Calif.)+10% normal rabbit
serum (Sigma); wash buffer (WB) was identical to WBS except that it
is serum free. After washing, the cells were resuspended in 100
.mu.l WB containing 10 .mu.g/ml rabbit anti-zalpha11 polyclonal
antibodies (Example 10). The cells were kept on ice with Ab for 20
min, then washed with WB and resuspended in WB containing goat
anti-rabbit-FITC (BioSource, International), incubated another 20
min on ice, then washed and resuspended in 400 .mu.l WB for
analysis on a FACSCalibur flow cytometer (Becton Dickinson).
Control samples were stained with the secondary goat
anti-rabbit-FITC Ab only. Positive staining was defined as a shift
above the staining with secondary alone. Although the polyclonal
antibodies were of low affinity, we were reasonably confident that
we detected zalpha11 expression on the BaF3/zalpha11 transfectant,
on all four human Burkitt's lymphomas (Raji, Ramos, Daudi, and RPMI
8226), and on Jurkat T cells. Our data with the monocytic cell
lines were more ambiguous. Resting (undifferentiated) HL-60 cells
did not bind the anti-zalpha11 antibodies, but we did detect a
positive signal on HL-60 cells activated for 24 hours with PMA
(Calbiochem, La Jolla, Calif.) which induces HL-60 cell
differentiation into a monocyte-like cell. We also saw a positive
signal on U937 and Thp-1 cells, although this signal may have been
due to non-specific binding. The polyclonal antibodies weakly
cross-reacted on the mouse B cell line A20, but we saw no staining
of the EL4 murine thymoma. The four anti-zalpha11 monoclonal
antibodies (Example 24) were conjugated to biotin, and a subset of
the cells described above were screened for zalpha11 receptor
expression (BaF3, BaF3/zalpha11, Raji, Jurkat, and resting HL-60).
Cells were harvested, washed, then resuspended in 100 .mu.l WB
containing 15 .mu.g/ml of one of each of the 4 biotinylated mAbs.
The cells were incubated with mAb for 20 min on ice, then washed
with 1.5 ml WB and pelleted in a centrifuge. The supernatant was
removed by aspiration and the pellets were resuspended in 100 .mu.l
of CyChrome-conjugated streptavidin (CyC-SA; PharMingen), then
incubated on ice for another 20 min and washed and pelleted as
before.
[0368] Control tubes contained cells stained only with CyC-SA.
Pellets were resuspended in 400 .mu.l WB and flow cytometry
performed as above. Positive staining was defined as a signal
exceeding the background level of staining with CyC-SA alone. Using
the BaF3/zalpha11 transfectant as a control, we were able to rank
the 4 mAbs in terms of their respective mean fluorescence
intensities (MFI), which can reflect antibody affinity and/or the
extent of biotinylation of the mAbs. The mAbs were ranked as
follows, from highest to lowest MFI: 249.28.2.1.2.2,
247.10.2.15.4.6, 249.19.2.2.3.5, and 249.15.2.4.2.7. This pattern
was essentially the same on both Raji and Jurkat cells, indicating
that zalpha11 is expressed on these B and T cell lines. The
staining patterns on non-activated HL60 cells were identical for
all the mAbs, and the signal was very weak. We speculate that this
does not reflect actual expression of zalpha11 by this cell line,
but rather is a function of non-specific binding of the mouse mAbs
to the human cells, probably via Fc-receptors.
Example 31
Reconstitution of Human zalpha11 Receptor In Vitro
[0369] To identify components involved in the zalpha11-signaling
complex, receptor reconstitution studies were performed as follows.
BHK 570 cells (ATCC No. CRL-10314) transfected, using standard
methods described herein, with the KZ134 luciferase reporter
plasmid (Example 19) served as a bioassay cell line to measure
signal transduction response from a transfected zalpha11 receptor
complex to the luciferase reporter in the presence of zalpha11
Ligand. BHK cells do not endogenously express the zalpha11
receptor. The bioassay cell line was transfected with zalpha11
receptor alone, or co-transfected with zalpha11 receptor along with
one of a variety of other known receptor subunits. Each receptor
subunit was cloned using PCR followed by ligation into appropriate
expression vectors; correct sequence of each construct was
confirmed before transfection. Cell lines were tested for receptor
expression by RT/PCR prior to assays. Receptor complexes tested
included: zalpha11 receptor alone; zalpha11 receptor with
IL-2R.gamma.; zalpha11 receptor with IL-2R.gamma. and IL-2R.beta.;
zalpha11 receptor with IL-2R.gamma. and IL-13R.alpha.; zalpha11
receptor with IL-2R.gamma. and IL-2R.beta.; and zalpha11 receptor
with IL-2R.gamma. and IL-4R.alpha.. Each independent receptor
complex cell line was assayed in the presence of human zalpha11
Ligand and luciferase activity measured as described in Example 19.
The untransfected bioassay cell line served as a control for the
background luciferase activity, and was used as a baseline to
compare signaling by the various receptor complex combinations. In
each cell line containing both zalpha11 receptor and IL-2R.gamma.,
maximal luciferase activity was about two-fold over background in
the presence of zalpha11 Ligand. No increase in signal was observed
in the presence of any other receptor subunit tested (IL-2R.beta.,
IL-2R.alpha., IL-4R.alpha., or IL-13R.alpha.).
[0370] Other zalpha11 receptor complexes that can be assessed by
this method include combinations of zalpha11 receptor with one or
more of the IL-4/IL-13 receptor family receptor components
(IL-13R.alpha.), as well as other Interleukin receptors (e.g.,
IL-15 R.alpha., IL-7R.alpha., IL-9R.alpha.).
Example 32
.sup.125I-labeled Human zalpha11 Ligand Binding Study in Cell
Lines
[0371] 25 micrograms of purified human zalpha11 Ligand (commonly
owned U.S. patent application Ser. No. 09/522,217) was labeled with
2 mCI 1251 using iodobeads (Pierce, Rockford Ill.), according to
manufacturer's instructions. This labeled protein was used to
assess human zalpha11 Ligand binding to human Raji cells (ATCC No.
CCL-86), using binding to wild-type murine BaF3 cells, and BaF3
cells transfected with zalpha11 receptor (BaF3/hzalpha11 cells) as
controls. Zalpha11 Ligand binding to BaF3/hzalpha11 cells was
expected (positive control), while no binding to wild-type BaF3
cells was expected (negative control), based on proliferation assay
results (Example 2). About 5.times.10.sup.5 Raji cells/well,
1.times.10.sup.6 BaF3/hzalpha11 and 1.times.10.sup.6 BaF3 cells
cells/well, were each plated in 96-well plates. Ten ng/ml of
labeled human zalpha11 Ligand was added in duplicate to wells, with
a dilution series of unlabeled human zalpha11 Ligand competitor
added from 250 fold molar excess in 1:4 dilutions down to 0.061
fold molar excess. Each point was run in duplicate. After the
labeled human zalpha11 Ligand was added to wells, it was allowed to
incubate at 4.degree. C. for 2 h to allow for binding of Ligand to
the cells. The cells were then washed 3.times. in binding buffer
(RPMI-1710 (JRH Biosciences) with 1% BSA (Sigma)), and counted on
the COBRA II AUTO-GAMMA gamma counter (Packard Instrument Company,
Meriden, Conn.).
[0372] Binding of the labeled zalpha11 Ligand to cells was evident
in the Raji and the BaF3/hzalpha11 cells. In addition, for Raji
cells, an average 250 fold molar excess of unlabeled zalpha11
Ligand decreased binding 3 fold in the presence of a non-specific
unlabeled competitor (Interferon Gamma from R&D Systems,
Minneapolis, Minn.), and 3.7 fold relative to no competitor.
Competition was observed in a dose dependent fashion for the
specific unlabeled competitor, human zalpha11 Ligand. Thus, the
zalpha11 Ligand binding to Raji cells was specific. Similarly, for
positive control BaF3/zalpha11 cells, the 250 fold molar excess of
unlabeled zalpha11 Ligand decreased binding 2 fold relative to the
non-specific competitor and 3.06 fold relative to no competitor.
Thus, the zalpha11 Ligand binding to BaF3/zalpha11 cells also was
specific. No competable binding was observed with the wild-type
BaF3 cells. Thus, the zalpha11 Ligand was shown to bind
specifically to Raji cells, and to BaF3/hzalpha11 cells, but not to
the negative control BaB3 cells.
[0373] The bound radiolabeled zalpha11 Ligand is then cross-linked
to the molecule to which it binds on the cell surface of Raji cells
using standard cross-linking methods, to identify the receptor
complex to which it binds on these cells. Moreover, anti-zalpha11
receptor antibodies (Example 24 and Example 10), and other
anti-cytokine receptor subunit antibodies are employed to assess
which subunit components comprise a functional hzalpha11 receptor
complex, for example, on the Raji cells and other cell lines to
which zalpha11 Ligand binds. Such antibodies can be used to compete
for zalpha11 Ligand in a binding assay as described above, and
hence show which receptor subunits are present of the Raji cell
surface, and on other cell lines to which zalpha11 Ligand binds.
Moreover, such antibodies can be used to immunoprecipitate
radiolabeled zalpha11 Ligand cross-linked material using methods
known in the art and described herein. In addition anti-zalpha11
Ligand antibodies (commonly owned U.S. patent application Ser. No.
09/522,217) can be used to immunoprecipitate radiolabeled zalpha11
Ligand cross-linked material.
Example 33
Zalpha11 Receptor Expression On Human Blood Cells
A. Preparation and Culture of Human Peripheral Blood Cells
[0374] Fresh drawn human blood was diluted 1:1 with PBS (GIBCO BRL)
and layered over Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology
Inc., Piscataway, N.J.) and spun for 30 minutes at 1800 rpm and
allowed to stop with the brake off. The interface layer was removed
and transferred to a fresh 50 ml Falcon tube (Falcon, VWR, Seattle,
Wash.), brought up to a final volume of 40 ml with PBS and spun for
10 minutes at 1200 rpm with the brake on. The viability of the
isolated cells was tested using Trypan Blue (GIBCO BRL) and the
cells were resuspended at a final concentration of 1.times.10.sup.6
cells/ml cell medium (RPMI Medium 1640, 10% Heat inactivated fetal
bovine serum, 5% L-glutamine, 5% Pen/Strep) (GIBCO BRL).
[0375] Cells were cultured in 6 well plates (Falcon, VWR) for 0, 4
or 24 hours with a variety of different stimuli described below.
Anti-IgM, anti-CD40 and anti-CD3 stimulation were done as in
Example 26. Phorbol myristate acetate (PMA) and ionomycin (Sigma,
St. Louis, Mo.) were added to appropriate wells at 10 ng/ml and 0.5
mg/ml respectively. The cells were incubated at 37.degree. C. in a
humidified incubator for various times.
B. Antibody Staining and Analysis
[0376] Cells were collected out of the plates, washed and
resuspended in ice cold staining media (HBSS, 1% fetal bovine
serum, 0.1% sodium azide) at a concentration of about ten million
cells per milliliter. Blocking of Fc receptor and non-specific
binding of antibodies to the cells was achieved by adding 10%
normal goat serum (Gemini Bioproducts, Woodland, Calif.) and 10%
normal human serum (Ultraserum, Gemini) to the cell suspension.
Aliquots of the cell suspensions were mixed with a FITC labeled
monoclonal antibody against one of the lineage markers CD3, CD19 or
CD14 (PharMingen, La Jolla, Calif.) and a biotinylated monoclonal
antibody against the human zalpha11 receptor (hu-zalpha11) (Example
24). After incubation on ice for 60 minutes the cells were washed
twice with ice cold staining media and resuspended in 50 ml
staining media containing streptavidin-PE (Caltag, Burlingame,
Calif.).
[0377] After a 30 minute incubation on ice, the cells were washed
twice with ice cold wash buffer (PBS, 1% fetal bovine serum, 0.1%
sodium azide) and resuspended in wash buffer containing 1 mg/ml
7-AAD (Molecular Probes, Eugene, Oreg.) as a viability marker. Flow
data was acquired on living cells using a FACSCalibur flow
cytometer (BD Immunocytometry Systems, San Jose, Calif.). Both
acquisition and analysis were performed using CellQuest software
(BD Immunocytometry Systems).
[0378] Results showed that the human zalpha11 receptor is expressed
on human peripheral blood cells expressing either CD3, CD19 or
CD14. Activation of either T cells with anti-CD3 or B cells with
anti-CD40 resulted in an increased level of cell surface zalpha11
at 24 hours. No increase in the level of expression of zalpha11 was
seen at 4 hours with any stimulus on either cell population.
Treatment of the cells with zalpha11ligand resulted in a decrease
of zalpha11 staining on CD3 positive and CD19 positive cells but
not CD14 positive cells at both 4 and 24 hours.
Example 34
Human zalpha11 Ligand Activity is Blocked With Anti-IL-2R.gamma.
Antibodies in a BaF3/zalpha11 Proliferation Assay
[0379] The role of the IL-2.quadrature. receptor was investigated
using anti-IL-2.quadrature. receptor monoclonal antibodies to
assess whether they would block zalpha11 Ligand activity in a
BaF3/zalpha11 proliferation assay (Example 2). Conditioned-media
from BHK570 cells transfected with the human zalpha11 Ligand was
added to the assay at 5%, 2.5%, 1.25% and 0.625% concentrations,
with or without IL-2 receptor antibodies.
[0380] The following mouse anti-IL-2 receptor monoclonal antibodies
from PharMingen International, San Diego, Calif. were added at 50
.quadrature.g/ml each: (a) 4G3+TUGm2 or (b) TM-.quadrature.1. 4G3
and TUGm2 are purified rat anti-mouse .gamma..sub.C chain
antibodies, TM-.quadrature.1 is a purified rat anti-mouse CD 122
(IL-2 receptor F chain) antibody. Assay results demonstrated almost
complete inhibition of the zalpha11 Ligand response with the
4G3+TUGm2 antibody combination in comparison to the no-antibody
control. The TM-.quadrature.1 antibody had no effect. These results
suggest a role for the IL-2 .quadrature. receptor in the zalpha11
Ligand proliferative response, and further supports that the
IL-2R.gamma. heterodimerizes with the zalpha11 receptor to elicit
that response.
Example 35
Post-Translational Mannosylation of zalpha11 Receptor Polypeptide
on a Highly Conserved Trp Residue
[0381] Mannosylation of the human zalpha11 receptor was assessed
using the method for C-2 mannosylation of Tryptophan as described
in Hofsteenge, J et al., Biochemistry 33:13524-13530, 1994, and
Loeffler, A et al., Biochemistry 35:12005-14, 1996. Moreover, these
investigators showed that in a motif of amino acids, WXXW (SEQ ID
NO:67), that Trp can be mannosylated.
[0382] A soluble zalpha11 receptor bearing a C-terminal Glu-Glu
(CEE) (SEQ ID NO: 14) or FLAG (SEQ ID NO:23) tag was expressed in
BHK cells and purified by anti-Flag or anti-EE affinity
chromatography (Example 4A). A soluble zalpha11 receptor
C-terminally tagged with an Fc4 tag (SEQ ID NO:25) and expressed in
CHO cells was affinity purified by anti-Fc4 affinity chromatography
(Example 4B). These polypeptides were enzymatically cleaved to
generate peptide fragments for the study.
[0383] All enzymatic digestions were performed overnight at a
protein concentration of 1.0 mg/ml. PNGaseF (Oxford GlycoSciences,
Abingdon, Oxford UK) digestion was performed by diluting each
soluble zalpha11 receptor polypeptide into a 50 mM EDTA, 20 mM
Na-Phosphate pH 7.5 buffer and incubating it with 0.4 U of enzyme
per .mu.g of protein. Glu-C (Roche Molecular Biochemicals,
Indianapolis, Ind.) digestion was performed at a 1:50 ratio of
enzyme to protein by buffer exchanging the sample into 25 mM
NH.sub.4HCO.sub.3 pH 7.8 and incubating it at 25.degree. C., except
for the Fc4 tagged material, which was digested in 50 mM
Na-Phosphate pH 7.8+5% Acetonitrile (EM Science, Darmstadt,
Germany) at 37.degree. C. The Glu-C digestion generated a zalpha11
WSXWS-containing peptide as shown from amino acid 178 (Leu) to
amino acid 199 (Ser) of SEQ ID NO:6 (197 (Leu) to amino acid 218
(Ser) of SEQ ID NO:2). Asp-N (Roche Molecular Biochemicals,
Indianapolis, Ind.) digestion was performed by buffer exchanging
the protein into 50 mM Na-Phosphate pH 7.7 and incubating it at
37.degree. C. with enzyme at a 1:50 ratio to zalpha11 receptor
polypeptide. The Asp-N digestion generated a zalpha11
WSXWS-containing peptide as shown from amino acid 179 (Glu) to
amino acid 210 (Ser) of SEQ ID NO:6 (198 (Leu) to amino acid 229
(Glu) of SEQ ID NO:2).
[0384] LCMS and LCMS-MS analyses were performed on a Magic HPLC
(Michrom Bioresources, Auburn, Calif.) connected in-line to a
Finnigan LCQ mass spectrometer (Finnigan MAT, San Jose, Calif.). LC
separation was done on a Vydac C4 5.mu. 300 .ANG. column (Michrom
Bioresources) with an elution gradient of 20%-80% solvent B over 80
minutes where solvent A was 2% Acetonitrile+0.1% TFA and solvent B
was 90% Acetonitrile+0.095% TFA (EM Science; Sigma, St. Louis,
Mo.). The LCQ mass spectrometer was set to collect MS spectra for
the duration of the run. LCMS-MS analysis of polypeptide digests
was performed on the same instrument system using a Vydac C18 5p
300 .ANG. column (Michrom Bioresources) with an elution gradient of
5-65% solvent B over 80 minutes with the same solvent system
described for LCMS analysis above. The LCQ mass spectrometer was
configured to collect MS, zoom-scan and MS-MS spectra for each ion
over a minimum threshold.
[0385] The extent of tryptophan mannosylation was estimated by
comparing ion intensities for the 2+ and 3+ ions of the peptides
containing the WSXWS motif (SEQ ID NO:13) from both Glu-C and Asp-N
digestion described above. Peak composition was first determined
utilizing the MS data and a peptide map was generated. Next, an
average spectrum was created starting approximately 1 minute before
the early eluting mannosylated WSXWS (SEQ ID NO:13) containing
peptide and ending approximately 1 minute after its later eluting
non-mannosylated companion peptide. The normalized intensities of
the ions corresponding to mannosylated and non-mannosylated peptide
were compared and used to generate a percentage occupancy number.
Values generated for both 2+ and 3+ charge states were averaged to
generate a percent occupancy value for each digest. This value was
then averaged with the value from the companion digest for each lot
of protein to generate a final value.
[0386] Table 7 below summarizes the data that were calculated for
each Peptide-tag and host cell used for zalpha11 soluble receptor
expression.
TABLE-US-00007 TABLE 7 C-terminal-Tag Expression Host % WSXWS
Mannosylated Glu-Glu BHK ~46% FLAG BHK ~35% Fc4 CHO ~11%
[0387] One of skill in the art would appreciate that mannosylation
or non-mannosylation of the zalpha11 receptor WSXWS motif (SEQ ID
NO:13) can affect the ability of the zalpha11 receptor or zalpha11
soluble receptor to homodimerize, heterodimerize, and/or it's
ability to bind the zalpha11 Ligand. As the mannosylation on
zalpha11 receptor appears to differ depending on the cell type in
which the receptor so expressed, optimization of the expression and
production of zalpha11 receptor and soluble receptor polypeptides
may take into consideration whether the zalpha11 receptor produced
by the cell is mannosylated or non-mannosylated. As such, one of
skill in the art would appreciate that the polypeptides of the
present invention can be either mannosylated or
non-mannosylated.
[0388] As the mannosylation event is within the WSXWS motif (SEQ ID
NO:13) of the zalpha11 class I cytokine receptor, the mannosylation
of the Trp or the lack thereof can affect the polypeptide
functionally. For example, insertions or deletions in the WSXWS
motif (SEQ ID NO:13) of the EPOR can abrogate cell surface
expression, destroy or reduce proliferative response, decrease
receptor internalization, and affect EPO binding (Yoshimura, A et
al., J. Biol. Chem. 267:11619-11625, 1992; Quelle, D E et al., Mol.
Cell. Biol. 12:4553-4561, 1992; Hilton, D J et al., Proc. Natl.
Acad. Sci. USA 92:190-194, 1995). However, mutation in the WSXWS
motif (SEQ ID NO:13) can also result in more efficient export from
the ER and greater expression of the receptor on the cell surface
(Hilton, D J et al., supra.). Effects on cell surface expression,
ligand binding and stimulatory response have also been seen with
studies on WSXWS motif (SEQ ID NO: 13) and related motifs in
mutational analysis on IL-2R.beta., GM-CSFR, and GHR (Miyazaki, et
al., EMBO J. 10:3191-3197, 1991; Ronco, L. V. et al., J. Biol.
Chem. 269:277-283, 1994; Baumgartner, J W et al., J. Biol. Chem.
269:29094-29101, 1994).
[0389] Similarly, mannosylation of the first Trp residue in the
WSXWS motif (SEQ ID NO:13) of zalpha11 receptor polypeptides,
including full-length and soluble receptors described herein, can
have important structural and functional implications such as
having affects on the overall stability of the receptor, rate of
proteolysis, intracellular processing, antigenicity, cell surface
expression, dimerization or multimerization, co-receptor binding,
signaling or internalization, affects on zalpha11 Ligand binding
and stability of receptor-ligand interaction. Comparison of
mannosylated and non-mannosylated zalpha11 receptors can be made
using X-ray crystallography or NMR on purified zalpha11
polypeptides (e.g., soluble receptors), or functional studies
comparing zalpha11 expressed in cell lines that either mannosylated
(e.g., BHK or other cell line) or are defective or reduced in
mannosylation (e.g., CHO or other cell line) and comparing the
receptors in the various assays described herein.
Example 36
BHK Tranfectant Binding Studies
[0390] Purified human zalpha11 Ligand (25 .mu.g) protein (commonly
owned U.S. patent application Ser. No. 09/522,217) was iodinated
with .sup.125I (Amersham) using iodo-beads (Pierce) and purified on
a Sephadex G25 PD-10 column (Pharmacia). BHK tranfectants (Example
31) expressing either human zalpha11 alone or human zalpha11+human
IL-2R.quadrature. receptor were plated at 30K/well in a 24-well
dish 24 hours prior to the binding study. BHK tranfectants were
incubated for 2 hours at 4.degree. C. with 2.5 ng (0.147 pMoles)
.sup.125I zalpha11 Ligand (specific activity 6.4.times.10.sup.7
cpm/ug) in the presence of various concentrations of cold zalpha11
Ligand (in a range from about a 10,884 fold excess to no
competition in 15, 4-fold dilutions). Cells were washed three times
with binding buffer before lysis in 0.8M NaOH, followed by gamma
emission counting. Analysis of these data yielded an affinity of
approximately 1 nM for the zalpha11 transfectants and approximately
0.1 nM for the human zalpha11+human IL-2R.quadrature. receptor
transfectants. This result suggested that zalpha11 Ligand has high
affinity on both the homodimeric human zalpha11 or heterodimeric
human zalpha11+ human IL-2R.quadrature. receptor, and that the
affinity is higher for the heterodimer.
Example 37
Murine Homodimeric zalpha11Receptor-mG2a Fusion Protein
[0391] The expression vector pEZE2 was used to express the murine
zalpha11receptor-murine IgGamma2a Fc fusion protein
(zalpha11m-mG2a). The mouse zalpha11 extracellular domain murine
immunoglobulin gamma 2a heavy chain Fc region fusion protein
(zalpha11m-mG2a) DNA sequence is shown in SEQ ID NO:72, and the
corresponding polypeptide sequence is shown in SEQ ID NO:73.
[0392] The pEZE2 vector is a plasmid derived from pDC312 (Immunex
Corp., Seattle, Wash.), and contains an EASE segment as described
in WIPO Publication WO 97/25420. The presence of the EASE segment
in an expression vector can improve expression of recombinant
proteins about two to eight fold in stable cell pools. The pEZE2
plasmid is a dicistronic expression vector that can be used to
express two different proteins in mammalian cells, such as Chinese
Hamster Ovary (CHO) cells. The pEZE2 expression unit contains a CMV
enhancer/promoter; an adenovirus tripartite leader sequence; a
multiple cloning site (MCS) for insertion of the coding region for
the recombinant protein of interest; an encephalomyocarditis virus
internal ribosome entry site; a coding segment for mouse
dihydrofolate reductase; and the SV40 transcription terminator. In
addition, pEZE2 contains an E. coli origin of replication and the
bacterial beta lactamase gene.
[0393] The zalpha11m-mG2a fusion protein is a disulfide-linked
homodimer consisting of two chains of the mouse zalpha11
extracellular domain fused to a wild type murine immunoglobulin
gamma 2a Fc region. The murine immunoglobulin gamma 2a Fc confers
effector functions, Fc.gamma.RI binding and C1q complement
fixation.
[0394] The mouse zalpha11 extracellular domain murine
immunoglobulin gamma 2a Fc constant region fusion construct was
generated by overlap PCR of three separate DNA fragments, each
generated by separate PCR amplification reactions. The first
fragment contained an optimized tPA (tissue plasminogen activator)
signal sequence (SEQ ID NO:80). The optimized tPA (otPA) signal
sequence was amplified using oligonucleotide primers ZC26,644 (SEQ
ID NO:74) and ZC26,641 (SEQ ID NO:75) using an in-house previously
generated expression vector as the template. PCR reaction mix
contained 20 pmoles of each primer, 10 ng template cDNA, 20 .mu.M
each dNTP, 1.times. Taq buffer (Life Technologies, Gaithersburg,
Md.), 0.5 .mu.l Taq polymerase in a 100 .mu.l reaction. PCR
conditions: 1 cycle, 94.degree. C., 2 minutes, 25 cycles,
94.degree. C., 30 seconds, 60.degree. C., 30 seconds, 72.degree.
C., 30 seconds, 1 cycle, 72.degree. C., 5 minutes. The second
fragment contained the mouse zalpha11 extracellular domain coding
region of amino acids 20 to 257 of SEQ ID NO:12. Oligonucleotide
primers ZC26,642 (SEQ ID NO:76) and ZC26,662 (SEQ ID NO:77) were
used to amplify this mouse zalpha11 segment using a previously
generated clone of mouse zalpha11 (SEQ ID NO: 11) as the template.
This PCR fragment was made using the same PCR reaction mix
specified above. PCR reaction conditions were as follows: 1 cycle,
94.degree. C., 2 minutes, 25 cycles, 94.degree. C., 30 seconds,
50.degree. C., 30 seconds, 72.degree. C., 45 seconds, 1 cycle,
72.degree. C., 5 minutes.
[0395] The murine gamma 2a heavy chain Fc region was generated from
a previously generated clone of murine 1 .mu.g gamma 2a heavy chain
cDNA. The segment containing the hinge, C.sub.H2, and C.sub.H3
domains of the murine immunoglobulin gamma 2a heavy chain constant
region was generated by PCR amplification using oligonucleotide
primers ZC26,643 (SEQ ID NO:78) and ZC26,645 (SEQ ID NO:79). This
PCR fragment was made using the same reaction mix specified above.
PCR conditions were as follows: 1 cycle, 94.degree. C., 2 minutes,
25 cycles, 94.degree. C., 30 seconds, 60.degree. C., 30 seconds,
72.degree. C., 30 seconds, 1 cycle, 72.degree. C., 5 minutes.
[0396] To prepare the fusion protein-coding segment, three protein
coding domains were linked by overlap PCR using oligonucleotides
ZC26,644 (SEQ ID NO:74) and ZC26,662 (SEQ ID NO:77) to link the
first two PCR products and ZC26,644 (SEQ ID NO:74) and ZC26,645
(SEQ ID NO:79) to link in the Fc region. Two reactions were set up:
The first ran 25 cycles of 94.degree. C. for 2 min., 55.degree. C.
for 30 sec. and 72.degree. C. for 1 min. 30 sec. The other reaction
ran 25 cycles of 94 degrees C. for 2 min., 60.degree. C. for 30 sec
and 72.degree. C. for 1 min. and 30 sec. The PCR products in the
two reactions were pooled and purified by using the QIAquick PCR
purification kit (Qiagen) as per manufacturer's protocol. The
product was eluted in 60 .mu.l of buffer. 30 .mu.l of this eluate
was digested with Fse1 and Asc1 restriction enzymes in diluted NEB
10.times. buffer No. 4 (New England Biolabs, Beverly, Mass.) as per
manufacturer's directions. The material was then run on a 1% TAE
agarose gel and the approximately 1500 bp band was excised and the
DNA purified using a Qiagen Agarose gel extraction kit (Qiagen) as
per manufacturers instructions. The fragment was eluted in 30 .mu.l
H.sub.2O.
[0397] To prepare the recipient vector for the insert, about 3
.mu.g of pEZE2 vector was digested w/ Asc1 and Fse1 in the same
manner as above, with the exception of 1 .mu.l of Calf Intestinal
Phosphatase (CIP) (New England Biolabs) added after restriction
enzyme digest (the reaction was allowed to proceed an additional 2
hrs). The vector was then run on an agarose gel and purified as per
above. The material was eluted in 30 .mu.l of H2O.
[0398] The fusion protein-coding segment was cloned into the MCS of
pEZE2 from the FseI site to the AscI site in the polylinker, and
ligated in 20 .mu.l using standard molecular biological reagents
and procedures. The ligation reaction was incubated O/N at
16.degree. C. About 4 .mu.l of this ligation mix was electroporated
into 50 .mu.l of DH12s E. Coli electrocompetent cells (Life
Technologies, Rockville, Md.) and the cells rescued in 1 ml of LB
media and allowed to shake/incubate for 1 hr. and 100 .mu.l spread
on Amp 100 agar plates. The plates were allowed to incubate o/n at
37.degree. C. A single colony was sequence analyzed. A mutation was
found that would result in a change from Glu to Lys at position 25
in SEQ ID NO:73. This amino acid substitution is within the otPA
leader, and may have resulted in improper processing of the signal
peptide, as N-terminal showed that the leader sequence was
incompletely cleaved and started at a pyroglutamine residue
upstream of the predicted start. However, this homodimeric
construct was still active in inhibiting the zalpha11 Ligand
(Example 40).
[0399] A large prep was created using the Qiagen Maxi prep kit
(Qiagen) as per manufacturers instructions. The plasmid was used to
transfect CHO cells. The cells were selected in medium without
hypoxanthine or thymidine and the transgene was amplified using
methotrexate (Example 38). The presence of protein was assayed by
Western blotting using anti human gamma 1 heavy chain constant
region and anti human kappa light chain antibodies (Rockland
Immunochemicals, Gilbertsville Pa.).
Example 38
Production of zAlpha11m-mG2A in DG-44 CHO Cells
[0400] 20 .mu.g of a zAlpha11m-mG2A/pEZE2 construct (Example 37)
was digested with 40 units of Pvu I at 37.degree. C. for three
hours and was then precipitated with isopropanol and pelleted in a
1.5 mL microfuge tube. The supernatant was aspirated away from the
pellet and the pellet was resuspended in 100 .mu.l of water. About
200 .mu.g (20 .mu.l) of sheared salmon sperm DNA was added to the
digested zAlpha11m-mG2A/pEZE2 construct. The DNA mixture was
co-precipitated using 0.1 volumes of sodium acetate (pH 5.2) and
2.2 volumes of ethanol. The tube was placed on dry ice for 15
minutes then was spun down in a microfuge at 14,000 RPM for 15
minutes forming a DNA pellet. The supernatant was aspirated off the
pellet, and the pellet washed with 1 mL of 70% ethanol and allowed
to incubate for 5 minutes at room temperature. The tube was spun in
a microfuge for 10 minutes at 14,000 RPM and the supernatant was
aspirated off the pellet. The pellet was allowed to air dry for 30
minutes. The pellet was then resuspended in 100 .mu.l of water and
allowed to incubate at room temperature for 10 minutes. 500 .mu.l
containing about 5.times.10.sup.6 DG-44 CHO cells was added to the
DNA in the microfuge tube, then the DNA/cell mixture was placed in
a 0.4 cm gap cuvette and electroporated using the following
parameters: 1,070 .mu.F, high capacitance and 376 V. The contents
of the cuvette were then removed and diluted to 25 mLs with
EX-CELL.TM. 325 PF-CHO Protein Free Media (JRH Biosciences, Lenexa,
Kans.) with 3 mM L-Glutamine and placed in a 125 mL shake flask.
The flask was placed in an incubator on a shaker at 37.degree. C.,
6% CO.sub.2 and shaking at 120 RPM.
[0401] The DG-44 CHO zAlpha11m-mG2A culture was amplified with
methotrexate (MTX) using standard methods to a final MTX level of
50 nM MTX. The culture was dilution-cloned and screened using a
series of western blots. A final clone was chosen and further
selected in MTX to a level of 200 nM MTX and was then scaled up for
production. Production of each lot of the clone was accomplished by
seeding 8.times. 4 L spinner flasks with 2 L of culture at
approximately 5.times.10.sup.5 cells/mL. Cultures were spun at 70
RPM, maintained at 37.degree. C., 6% CO.sub.2 and allowed to
incubate for either 72 or 96 hours. The cells were spun down and
the supernatants were 0.2 .mu.m filtered. A sufficient number of
cells were recovered to seed the next series of flasks. Four total
lots were produced in this manner for protein purification (Example
39).
Example 39
Purification of the Homodimeric zalpha11m-mG2a Soluble Receptor
Protein
[0402] All procedures performed at 4.degree. C., unless otherwise
noted. Conditioned media (Example 38) was directly captured on an
appropriately sized POROS 50 A (coupled protein A; PerSeptive
BioSystems, Framingham, Mass.) column at an optimal capture flow
rate. The column washed with 20 column volumes (CV) of loading
buffer, then rapidly eluted with 3 CV of 0.1 M Glycine pH 2.5. 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.
[0403] Brilliant Blue (Sigma) stained NuPAGE gels were ran to
analyze the elution. Fractions of interested were pooled and
concentrated against 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 (Pharmacia) to
remove aggregates and to buffer exchange the protein into PBS pH
7.2.
[0404] Brilliant Blue (Sigma) stained NuPAGE gels (NOVEX) were
again used to analyze the elution. Fractions were pooled and
concentrated as before to .about.1-2 mgs/ml. Western and Brilliant
Blue (Sigma) stained NuPAGE gels (NOVEX) were ran to confirm purity
and content. In addition, the protein was submitted for amino acid
analysis (AAA), and N-terminal sequencing for further analysis.
Example 40
Soluble Homodimeric zalpha11m-mg2a fusion protein as a zalpha11
Ligand Antagonist
[0405] BaF3 cells stably expressing the mouse zalpha11 receptor
(constructed as per Example 2 using primers to SEQ ID NO: 11) were
plated at 5500 cells per well in standard 96-well tissue culture
plates in base medium plus 3 ng/ml human zalpha11 Ligand. Base
medium is 500 ml RPMI 1640 (JRH Biosciences), 5 ml 100.times.
Sodium Pyruvate (Gibco BRL), 5 ml 100.times.L-glutamine (Gibco
BRL), and 50 ml heat-inactivated Fetal Bovine Serum (FBS) (Hyclone
Laboratories). To the cells, a decreasing dose of either purified
homodimeric zalpha11m-mg2a (Example 39) was added. An Alamar Blue
proliferation assay was run and fluorimetry performed as per
Example 2B.
[0406] The homodimeric zalpha11m-mG2a fusion protein inhibited
human zalpha11 Ligand activity in a dose dependent manner, with 1-5
.mu.g/ml able to inhibit the activity of 1.25 ng/ml human zalpha11
Ligand.
[0407] 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
8611614DNAHomo sapiensCDS(1)...(1614) 1atg ccg cgt ggc tgg gcc gcc
ccc ttg ctc ctg ctg ctg ctc cag gga 48Met Pro Arg Gly Trp Ala Ala
Pro Leu Leu Leu Leu Leu Leu Gln Gly1 5 10 15ggc tgg ggc tgc ccc gac
ctc gtc tgc tac acc gat tac ctc cag acg 96Gly Trp Gly Cys Pro Asp
Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr20 25 30gtc atc tgc atc ctg
gaa atg tgg aac ctc cac ccc agc acg ctc acc 144Val Ile Cys Ile Leu
Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr35 40 45ctt acc tgg caa
gac cag tat gaa gag ctg aag gac gag gcc acc tcc 192Leu Thr Trp Gln
Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser50 55 60tgc agc ctc
cac agg tcg gcc cac aat gcc acg cat gcc acc tac acc 240Cys Ser Leu
His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr65 70 75 80tgc
cac atg gat gta ttc cac ttc atg gcc gac gac att ttc agt gtc 288Cys
His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val85 90
95aac atc aca gac cag tct ggc aac tac tcc cag gag tgt ggc agc ttt
336Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser
Phe100 105 110ctc ctg gct gag agc atc aag ccg gct ccc cct ttc aac
gtg act gtg 384Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn
Val Thr Val115 120 125acc ttc tca gga cag tat aat atc tcc tgg cgc
tca gat tac gaa gac 432Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg
Ser Asp Tyr Glu Asp130 135 140cct gcc ttc tac atg ctg aag ggc aag
ctt cag tat gag ctg cag tac 480Pro Ala Phe Tyr Met Leu Lys Gly Lys
Leu Gln Tyr Glu Leu Gln Tyr145 150 155 160agg aac cgg gga gac ccc
tgg gct gtg agt ccg agg aga aag ctg atc 528Arg Asn Arg Gly Asp Pro
Trp Ala Val Ser Pro Arg Arg Lys Leu Ile165 170 175tca gtg gac tca
aga agt gtc tcc ctc ctc ccc ctg gag ttc cgc aaa 576Ser Val Asp Ser
Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys180 185 190gac tcg
agc tat gag ctg cag gtg cgg gca ggg ccc atg cct ggc tcc 624Asp Ser
Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser195 200
205tcc tac cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc ttt cag
672Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe
Gln210 215 220acc cag tca gag gag tta aag gaa ggc tgg aac cct cac
ctg ctg ctt 720Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His
Leu Leu Leu225 230 235 240ctc ctc ctg ctt gtc ata gtc ttc att cct
gcc ttc tgg agc ctg aag 768Leu Leu Leu Leu Val Ile Val Phe Ile Pro
Ala Phe Trp Ser Leu Lys245 250 255acc cat cca ttg tgg agg cta tgg
aag aag ata tgg gcc gtc ccc agc 816Thr His Pro Leu Trp Arg Leu Trp
Lys Lys Ile Trp Ala Val Pro Ser260 265 270cct gag cgg ttc ttc atg
ccc ctg tac aag ggc tgc agc gga gac ttc 864Pro Glu Arg Phe Phe Met
Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe275 280 285aag aaa tgg gtg
ggt gca ccc ttc act ggc tcc agc ctg gag ctg gga 912Lys Lys Trp Val
Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly290 295 300ccc tgg
agc cca gag gtg ccc tcc acc ctg gag gtg tac agc tgc cac 960Pro Trp
Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His305 310 315
320cca cca cgg agc ccg gcc aag agg ctg cag ctc acg gag cta caa gaa
1008Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln
Glu325 330 335cca gca gag ctg gtg gag tct gac ggt gtg ccc aag ccc
agc ttc tgg 1056Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro
Ser Phe Trp340 345 350ccg aca gcc cag aac tcg ggg ggc tca gct tac
agt gag gag agg gat 1104Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr
Ser Glu Glu Arg Asp355 360 365cgg cca tac ggc ctg gtg tcc att gac
aca gtg act gtg cta gat gca 1152Arg Pro Tyr Gly Leu Val Ser Ile Asp
Thr Val Thr Val Leu Asp Ala370 375 380gag ggg cca tgc acc tgg ccc
tgc agc tgt gag gat gac ggc tac cca 1200Glu Gly Pro Cys Thr Trp Pro
Cys Ser Cys Glu Asp Asp Gly Tyr Pro385 390 395 400gcc ctg gac ctg
gat gct ggc ctg gag ccc agc cca ggc cta gag gac 1248Ala Leu Asp Leu
Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp405 410 415cca ctc
ttg gat gca ggg acc aca gtc ctg tcc tgt ggc tgt gtc tca 1296Pro Leu
Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser420 425
430gct ggc agc cct ggg cta gga ggg ccc ctg gga agc ctc ctg gac aga
1344Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp
Arg435 440 445cta aag cca ccc ctt gca gat ggg gag gac tgg gct ggg
gga ctg ccc 1392Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly
Gly Leu Pro450 455 460tgg ggt ggc cgg tca cct gga ggg gtc tca gag
agt gag gcg ggc tca 1440Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu
Ser Glu Ala Gly Ser465 470 475 480ccc ctg gcc ggc ctg gat atg gac
acg ttt gac agt ggc ttt gtg ggc 1488Pro Leu Ala Gly Leu Asp Met Asp
Thr Phe Asp Ser Gly Phe Val Gly485 490 495tct gac tgc agc agc cct
gtg gag tgt gac ttc acc agc ccc ggg gac 1536Ser Asp Cys Ser Ser Pro
Val Glu Cys Asp Phe Thr Ser Pro Gly Asp500 505 510gaa gga ccc ccc
cgg agc tac ctc cgc cag tgg gtg gtc att cct ccg 1584Glu Gly Pro Pro
Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro515 520 525cca ctt
tcg agc cct gga ccc cag gcc agc 1614Pro Leu Ser Ser Pro Gly Pro Gln
Ala Ser530 5352538PRTHomo sapiens 2Met Pro Arg Gly Trp Ala Ala Pro
Leu Leu Leu Leu Leu Leu Gln Gly1 5 10 15Gly Trp Gly Cys Pro Asp Leu
Val Cys Tyr Thr Asp Tyr Leu Gln Thr20 25 30Val Ile Cys Ile Leu Glu
Met Trp Asn Leu His Pro Ser Thr Leu Thr35 40 45Leu Thr Trp Gln Asp
Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser50 55 60Cys Ser Leu His
Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr65 70 75 80Cys His
Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val85 90 95Asn
Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe100 105
110Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr
Val115 120 125Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp
Tyr Glu Asp130 135 140Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln
Tyr Glu Leu Gln Tyr145 150 155 160Arg Asn Arg Gly Asp Pro Trp Ala
Val Ser Pro Arg Arg Lys Leu Ile165 170 175Ser Val Asp Ser Arg Ser
Val Ser Leu Leu Pro Leu Glu Phe Arg Lys180 185 190Asp Ser Ser Tyr
Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser195 200 205Ser Tyr
Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln210 215
220Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu
Leu225 230 235 240Leu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe
Trp Ser Leu Lys245 250 255Thr His Pro Leu Trp Arg Leu Trp Lys Lys
Ile Trp Ala Val Pro Ser260 265 270Pro Glu Arg Phe Phe Met Pro Leu
Tyr Lys Gly Cys Ser Gly Asp Phe275 280 285Lys Lys Trp Val Gly Ala
Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly290 295 300Pro Trp Ser Pro
Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His305 310 315 320Pro
Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu325 330
335Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe
Trp340 345 350Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu
Glu Arg Asp355 360 365Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val
Thr Val Leu Asp Ala370 375 380Glu Gly Pro Cys Thr Trp Pro Cys Ser
Cys Glu Asp Asp Gly Tyr Pro385 390 395 400Ala Leu Asp Leu Asp Ala
Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp405 410 415Pro Leu Leu Asp
Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser420 425 430Ala Gly
Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg435 440
445Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu
Pro450 455 460Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu
Ala Gly Ser465 470 475 480Pro Leu Ala Gly Leu Asp Met Asp Thr Phe
Asp Ser Gly Phe Val Gly485 490 495Ser Asp Cys Ser Ser Pro Val Glu
Cys Asp Phe Thr Ser Pro Gly Asp500 505 510Glu Gly Pro Pro Arg Ser
Tyr Leu Arg Gln Trp Val Val Ile Pro Pro515 520 525Pro Leu Ser Ser
Pro Gly Pro Gln Ala Ser530 5353696DNAHomo sapiensCDS(1)...(696)
3ctg aac acg aca att ctg acg ccc aat ggg aat gaa gac acc aca gct
48Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala1
5 10 15gat ttc ttc ctg acc act atg ccc act gac tcc ctc agt gtt tcc
act 96Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser
Thr20 25 30ctg ccc ctc cca gag gtt cag tgt ttt gtg ttc aat gtc gag
tac atg 144Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu
Tyr Met35 40 45aat tgc act tgg aac agc agc tct gag ccc cag cct acc
aac ctc act 192Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr
Asn Leu Thr50 55 60ctg cat tat tgg tac aag aac tcg gat aat gat aaa
gtc cag aag tgc 240Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys
Val Gln Lys Cys65 70 75 80agc cac tat cta ttc tct gaa gaa atc act
tct ggc tgt cag ttg caa 288Ser His Tyr Leu Phe Ser Glu Glu Ile Thr
Ser Gly Cys Gln Leu Gln85 90 95aaa aag gag atc cac ctc tac caa aca
ttt gtt gtt cag ctc cag gac 336Lys Lys Glu Ile His Leu Tyr Gln Thr
Phe Val Val Gln Leu Gln Asp100 105 110cca cgg gaa ccc agg aga cag
gcc aca cag atg cta aaa ctg cag aat 384Pro Arg Glu Pro Arg Arg Gln
Ala Thr Gln Met Leu Lys Leu Gln Asn115 120 125ctg gtg atc ccc tgg
gct cca gag aac cta aca ctt cac aaa ctg agt 432Leu Val Ile Pro Trp
Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser130 135 140gaa tcc cag
cta gaa ctg aac tgg aac aac aga ttc ttg aac cac tgt 480Glu Ser Gln
Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys145 150 155
160ttg gag cac ttg gtg cag tac cgg act gac tgg gac cac agc tgg act
528Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp
Thr165 170 175gaa caa tca gtg gat tat aga cat aag ttc tcc ttg cct
agt gtg gat 576Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro
Ser Val Asp180 185 190ggg cag aaa cgc tac acg ttt cgt gtt cgg agc
cgc ttt aac cca ctc 624Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser
Arg Phe Asn Pro Leu195 200 205tgt gga agt gct cag cat tgg agt gaa
tgg agc cac cca atc cac tgg 672Cys Gly Ser Ala Gln His Trp Ser Glu
Trp Ser His Pro Ile His Trp210 215 220ggg agc aat act tca aaa gag
aat 696Gly Ser Asn Thr Ser Lys Glu Asn225 2304232PRTHomo sapiens
4Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala1 5
10 15Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser
Thr20 25 30Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu
Tyr Met35 40 45Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr
Asn Leu Thr50 55 60Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys
Val Gln Lys Cys65 70 75 80Ser His Tyr Leu Phe Ser Glu Glu Ile Thr
Ser Gly Cys Gln Leu Gln85 90 95Lys Lys Glu Ile His Leu Tyr Gln Thr
Phe Val Val Gln Leu Gln Asp100 105 110Pro Arg Glu Pro Arg Arg Gln
Ala Thr Gln Met Leu Lys Leu Gln Asn115 120 125Leu Val Ile Pro Trp
Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser130 135 140Glu Ser Gln
Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys145 150 155
160Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp
Thr165 170 175Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro
Ser Val Asp180 185 190Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser
Arg Phe Asn Pro Leu195 200 205Cys Gly Ser Ala Gln His Trp Ser Glu
Trp Ser His Pro Ile His Trp210 215 220Gly Ser Asn Thr Ser Lys Glu
Asn225 2305654DNAHomo sapiensCDS(1)...(654) 5tgc ccc gac ctc gtc
tgc tac acc gat tac ctc cag acg gtc atc tgc 48Cys Pro Asp Leu Val
Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys1 5 10 15atc ctg gaa atg
tgg aac ctc cac ccc agc acg ctc acc ctt acc tgg 96Ile Leu Glu Met
Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp20 25 30caa gac cag
tat gaa gag ctg aag gac gag gcc acc tcc tgc agc ctc 144Gln Asp Gln
Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu35 40 45cac agg
tcg gcc cac aat gcc acg cat gcc acc tac acc tgc cac atg 192His Arg
Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met50 55 60gat
gta ttc cac ttc atg gcc gac gac att ttc agt gtc aac atc aca 240Asp
Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr65 70 75
80gac cag tct ggc aac tac tcc cag gag tgt ggc agc ttt ctc ctg gct
288Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu
Ala85 90 95gag agc atc aag ccg gct ccc cct ttc aac gtg act gtg acc
ttc tca 336Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr
Phe Ser100 105 110gga cag tat aat atc tcc tgg cgc tca gat tac gaa
gac cct gcc ttc 384Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu
Asp Pro Ala Phe115 120 125tac atg ctg aag ggc aag ctt cag tat gag
ctg cag tac agg aac cgg 432Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu
Leu Gln Tyr Arg Asn Arg130 135 140gga gac ccc tgg gct gtg agt ccg
agg aga aag ctg atc tca gtg gac 480Gly Asp Pro Trp Ala Val Ser Pro
Arg Arg Lys Leu Ile Ser Val Asp145 150 155 160tca aga agt gtc tcc
ctc ctc ccc ctg gag ttc cgc aaa gac tcg agc 528Ser Arg Ser Val Ser
Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser165 170 175tat gag ctg
cag gtg cgg gca ggg ccc atg cct ggc tcc tcc tac cag 576Tyr Glu Leu
Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln180 185 190ggg
acc tgg agt gaa tgg agt gac ccg gtc atc ttt cag acc cag tca 624Gly
Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser195 200
205gag gag tta aag gaa ggc tgg aac cct cac 654Glu Glu Leu Lys Glu
Gly Trp Asn Pro His210 2156218PRTHomo sapiens 6Cys Pro Asp Leu Val
Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys1 5 10 15Ile Leu Glu Met
Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp20 25 30Gln Asp Gln
Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu35 40 45His Arg
Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met50 55 60Asp
Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr65 70 75
80Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala85
90 95Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe
Ser100 105 110Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp
Pro Ala Phe115 120 125Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu
Gln Tyr Arg Asn Arg130 135 140Gly Asp Pro Trp Ala Val Ser Pro Arg
Arg Lys Leu Ile Ser Val Asp145 150 155 160Ser Arg Ser Val Ser Leu
Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser165 170 175Tyr Glu Leu Gln
Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln180 185 190Gly Thr
Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser195 200
205Glu Glu Leu Lys Glu Gly Trp Asn Pro His210
2157654DNAArtificial
SequenceDegenerate polynucleotide sequence of soluble zalpha11
Receptor polypeptide as shown in SEQ ID NO6 7tgyccngayy tngtntgyta
yacngaytay ytncaracng tnathtgyat hytngaratg 60tggaayytnc ayccnwsnac
nytnacnytn acntggcarg aycartayga rgarytnaar 120gaygargcna
cnwsntgyws nytncaymgn wsngcncaya aygcnacnca ygcnacntay
180acntgycaya tggaygtntt ycayttyatg gcngaygaya thttywsngt
naayathacn 240gaycarwsng gnaaytayws ncargartgy ggnwsnttyy
tnytngcnga rwsnathaar 300ccngcnccnc cnttyaaygt nacngtnacn
ttywsnggnc artayaayat hwsntggmgn 360wsngaytayg argayccngc
nttytayatg ytnaarggna arytncarta ygarytncar 420taymgnaaym
gnggngaycc ntgggcngtn wsnccnmgnm gnaarytnat hwsngtngay
480wsnmgnwsng tnwsnytnyt nccnytngar ttymgnaarg aywsnwsnta
ygarytncar 540gtnmgngcng gnccnatgcc nggnwsnwsn taycarggna
cntggwsnga rtggwsngay 600ccngtnatht tycaracnca rwsngargar
ytnaargarg gntggaaycc ncay 6548696DNAArtificial SequenceDegenerate
polynucleotide sequence of IL-2Rgamma polypeptide as shown in SEQ
ID NO4 8ytnaayacna cnathytnac nccnaayggn aaygargaya cnacngcnga
yttyttyytn 60acnacnatgc cnacngayws nytnwsngtn wsnacnytnc cnytnccnga
rgtncartgy 120ttygtnttya aygtngarta yatgaaytgy acntggaayw
snwsnwsnga rccncarccn 180acnaayytna cnytncayta ytggtayaar
aaywsngaya aygayaargt ncaraartgy 240wsncaytayy tnttywsnga
rgarathacn wsnggntgyc arytncaraa raargarath 300cayytntayc
aracnttygt ngtncarytn cargayccnm gngarccnmg nmgncargcn
360acncaratgy tnaarytnca raayytngtn athccntggg cnccngaraa
yytnacnytn 420cayaarytnw sngarwsnca rytngarytn aaytggaaya
aymgnttyyt naaycaytgy 480ytngarcayy tngtncarta ymgnacngay
tgggaycayw sntggacnga rcarwsngtn 540gaytaymgnc ayaarttyws
nytnccnwsn gtngayggnc araarmgnta yacnttymgn 600gtnmgnwsnm
gnttyaaycc nytntgyggn wsngcncarc aytggwsnga rtggwsncay
660ccnathcayt ggggnwsnaa yacnwsnaar garaay 6969486DNAHomo
sapiensCDS(1)...(486) 9atg aga tcc agt cct ggc aac atg gag agg att
gtc atc tgt ctg atg 48Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile
Val Ile Cys Leu Met1 5 10 15gtc atc ttc ttg ggg aca ctg gtc cac aaa
tca agc tcc caa ggt caa 96Val Ile Phe Leu Gly Thr Leu Val His Lys
Ser Ser Ser Gln Gly Gln20 25 30gat cgc cac atg att aga atg cgt caa
ctt ata gat att gtt gat cag 144Asp Arg His Met Ile Arg Met Arg Gln
Leu Ile Asp Ile Val Asp Gln35 40 45ctg aaa aat tat gtg aat gac ttg
gtc cct gaa ttt ctg cca gct cca 192Leu Lys Asn Tyr Val Asn Asp Leu
Val Pro Glu Phe Leu Pro Ala Pro50 55 60gaa gat gta gag aca aac tgt
gag tgg tca gct ttt tcc tgt ttt cag 240Glu Asp Val Glu Thr Asn Cys
Glu Trp Ser Ala Phe Ser Cys Phe Gln65 70 75 80aag gcc caa cta aag
tca gca aat aca gga aac aat gaa agg ata atc 288Lys Ala Gln Leu Lys
Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile85 90 95aat gta tca att
aaa aag ctg aag agg aaa cca cct tcc aca aat gca 336Asn Val Ser Ile
Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala100 105 110ggg aga
aga cag aaa cac aga cta aca tgc cct tca tgt gat tct tat 384Gly Arg
Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr115 120
125gag aaa aaa cca ccc aaa gaa ttc cta gaa aga ttc aaa tca ctt ctc
432Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu
Leu130 135 140caa aag atg att cat cag cat ctg tcc tct aga aca cac
gga agt gaa 480Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His
Gly Ser Glu145 150 155 160gat tcc 486Asp Ser10162PRTHomo sapiens
10Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met1
5 10 15Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly
Gln20 25 30Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val
Asp Gln35 40 45Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu
Pro Ala Pro50 55 60Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe
Ser Cys Phe Gln65 70 75 80Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly
Asn Asn Glu Arg Ile Ile85 90 95Asn Val Ser Ile Lys Lys Leu Lys Arg
Lys Pro Pro Ser Thr Asn Ala100 105 110Gly Arg Arg Gln Lys His Arg
Leu Thr Cys Pro Ser Cys Asp Ser Tyr115 120 125Glu Lys Lys Pro Pro
Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu130 135 140Gln Lys Met
Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu145 150 155
160Asp Ser111735DNAmus musculusCDS(143)...(1729) 11ctgcccacct
caaaccttca cctcccacca ccaccactcc gagtcccgct gtgactccca 60cgcccaggag
accacccaag tgccccagcc taaagaatgg ctttctgaga aagaccctga
120aggagtaggt ctgggacaca gc atg ccc cgg ggc cca gtg gct gcc tta ctc
172Met Pro Arg Gly Pro Val Ala Ala Leu Leu1 5 10ctg ctg att ctc cat
gga gct tgg agc tgc ctg gac ctc act tgc tac 220Leu Leu Ile Leu His
Gly Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr15 20 25act gac tac ctc
tgg acc atc acc tgt gtc ctg gag aca cgg agc ccc 268Thr Asp Tyr Leu
Trp Thr Ile Thr Cys Val Leu Glu Thr Arg Ser Pro30 35 40aac ccc agc
ata ctc agt ctc acc tgg caa gat gaa tat gag gaa ctt 316Asn Pro Ser
Ile Leu Ser Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu45 50 55cag gac
caa gag acc ttc tgc agc cta cac agg tct ggc cac aac acc 364Gln Asp
Gln Glu Thr Phe Cys Ser Leu His Arg Ser Gly His Asn Thr60 65 70aca
cat ata tgg tac acg tgc cat atg cgc ttg tct caa ttc ctg tcc 412Thr
His Ile Trp Tyr Thr Cys His Met Arg Leu Ser Gln Phe Leu Ser75 80 85
90gat gaa gtt ttc att gtc aat gtg acg gac cag tct ggc aac aac tcc
460Asp Glu Val Phe Ile Val Asn Val Thr Asp Gln Ser Gly Asn Asn
Ser95 100 105caa gag tgt ggc agc ttt gtc ctg gct gag agc atc aaa
cca gct ccc 508Gln Glu Cys Gly Ser Phe Val Leu Ala Glu Ser Ile Lys
Pro Ala Pro110 115 120ccc ttg aac gtg act gtg gcc ttc tca gga cgc
tat gat atc tcc tgg 556Pro Leu Asn Val Thr Val Ala Phe Ser Gly Arg
Tyr Asp Ile Ser Trp125 130 135gac tca gct tat gac gaa ccc tcc aac
tac gtg ctg agg ggc aag cta 604Asp Ser Ala Tyr Asp Glu Pro Ser Asn
Tyr Val Leu Arg Gly Lys Leu140 145 150caa tat gag ctg cag tat cgg
aac ctc aga gac ccc tat gct gtg agg 652Gln Tyr Glu Leu Gln Tyr Arg
Asn Leu Arg Asp Pro Tyr Ala Val Arg155 160 165 170ccg gtg acc aag
ctg atc tca gtg gac tca aga aac gtc tct ctt ctc 700Pro Val Thr Lys
Leu Ile Ser Val Asp Ser Arg Asn Val Ser Leu Leu175 180 185cct gaa
gag ttc cac aaa gat tct agc tac cag ctg cag gtg cgg gca 748Pro Glu
Glu Phe His Lys Asp Ser Ser Tyr Gln Leu Gln Val Arg Ala190 195
200gcg cct cag cca ggc act tca ttc agg ggg acc tgg agt gag tgg agt
796Ala Pro Gln Pro Gly Thr Ser Phe Arg Gly Thr Trp Ser Glu Trp
Ser205 210 215gac ccc gtc atc ttt cag acc cag gct ggg gag ccc gag
gca ggc tgg 844Asp Pro Val Ile Phe Gln Thr Gln Ala Gly Glu Pro Glu
Ala Gly Trp220 225 230gac cct cac atg ctg ctg ctc ctg gct gtc ttg
atc att gtc ctg gtt 892Asp Pro His Met Leu Leu Leu Leu Ala Val Leu
Ile Ile Val Leu Val235 240 245 250ttc atg ggt ctg aag atc cac ctg
cct tgg agg cta tgg aaa aag ata 940Phe Met Gly Leu Lys Ile His Leu
Pro Trp Arg Leu Trp Lys Lys Ile255 260 265tgg gca cca gtg ccc acc
cct gag agt ttc ttc cag ccc ctg tac agg 988Trp Ala Pro Val Pro Thr
Pro Glu Ser Phe Phe Gln Pro Leu Tyr Arg270 275 280gag cac agc ggg
aac ttc aag aaa tgg gtt aat acc cct ttc acg gcc 1036Glu His Ser Gly
Asn Phe Lys Lys Trp Val Asn Thr Pro Phe Thr Ala285 290 295tcc agc
ata gag ttg gtg cca cag agt tcc aca aca aca tca gcc tta 1084Ser Ser
Ile Glu Leu Val Pro Gln Ser Ser Thr Thr Thr Ser Ala Leu300 305
310cat ctg tca ttg tat cca gcc aag gag aag aag ttc ccg ggg ctg ccg
1132His Leu Ser Leu Tyr Pro Ala Lys Glu Lys Lys Phe Pro Gly Leu
Pro315 320 325 330ggt ctg gaa gag caa ctg gag tgt gat gga atg tct
gag cct ggt cac 1180Gly Leu Glu Glu Gln Leu Glu Cys Asp Gly Met Ser
Glu Pro Gly His335 340 345tgg tgc ata atc ccc ttg gca gct ggc caa
gcg gtc tca gcc tac agt 1228Trp Cys Ile Ile Pro Leu Ala Ala Gly Gln
Ala Val Ser Ala Tyr Ser350 355 360gag gag aga gac cgg cca tat ggt
ctg gtg tcc att gac aca gtg act 1276Glu Glu Arg Asp Arg Pro Tyr Gly
Leu Val Ser Ile Asp Thr Val Thr365 370 375gtg gga gat gca gag ggc
ctg tgt gtc tgg ccc tgt agc tgt gag gat 1324Val Gly Asp Ala Glu Gly
Leu Cys Val Trp Pro Cys Ser Cys Glu Asp380 385 390gat ggc tat cca
gcc atg aac ctg gat gct ggc cga gag tct ggc cct 1372Asp Gly Tyr Pro
Ala Met Asn Leu Asp Ala Gly Arg Glu Ser Gly Pro395 400 405 410aat
tca gag gat ctg ctc ttg gtc aca gac cct gct ttt ctg tct tgc 1420Asn
Ser Glu Asp Leu Leu Leu Val Thr Asp Pro Ala Phe Leu Ser Cys415 420
425ggc tgt gtc tca ggt agt ggt ctc agg ctt gga ggc tcc cca ggc agc
1468Gly Cys Val Ser Gly Ser Gly Leu Arg Leu Gly Gly Ser Pro Gly
Ser430 435 440cta ctg gac agg ttg agg ctg tca ttt gca aag gaa ggg
gac tgg aca 1516Leu Leu Asp Arg Leu Arg Leu Ser Phe Ala Lys Glu Gly
Asp Trp Thr445 450 455gca gac cca acc tgg aga act ggg tcc cca gga
ggg ggc tct gag agt 1564Ala Asp Pro Thr Trp Arg Thr Gly Ser Pro Gly
Gly Gly Ser Glu Ser460 465 470gaa gca ggt tcc ccc cct ggt ctg gac
atg gac aca ttt gac agt ggc 1612Glu Ala Gly Ser Pro Pro Gly Leu Asp
Met Asp Thr Phe Asp Ser Gly475 480 485 490ttt gca ggt tca gac tgt
ggc agc ccc gtg gag act gat gaa gga ccc 1660Phe Ala Gly Ser Asp Cys
Gly Ser Pro Val Glu Thr Asp Glu Gly Pro495 500 505cct cga agc tat
ctc cgc cag tgg gtg gtc agg acc cct cca cct gtg 1708Pro Arg Ser Tyr
Leu Arg Gln Trp Val Val Arg Thr Pro Pro Pro Val510 515 520gac agt
gga gcc cag agc agc tagcat 1735Asp Ser Gly Ala Gln Ser
Ser52512529PRTmus musculus 12Met Pro Arg Gly Pro Val Ala Ala Leu
Leu Leu Leu Ile Leu His Gly1 5 10 15Ala Trp Ser Cys Leu Asp Leu Thr
Cys Tyr Thr Asp Tyr Leu Trp Thr20 25 30Ile Thr Cys Val Leu Glu Thr
Arg Ser Pro Asn Pro Ser Ile Leu Ser35 40 45Leu Thr Trp Gln Asp Glu
Tyr Glu Glu Leu Gln Asp Gln Glu Thr Phe50 55 60Cys Ser Leu His Arg
Ser Gly His Asn Thr Thr His Ile Trp Tyr Thr65 70 75 80Cys His Met
Arg Leu Ser Gln Phe Leu Ser Asp Glu Val Phe Ile Val85 90 95Asn Val
Thr Asp Gln Ser Gly Asn Asn Ser Gln Glu Cys Gly Ser Phe100 105
110Val Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Leu Asn Val Thr
Val115 120 125Ala Phe Ser Gly Arg Tyr Asp Ile Ser Trp Asp Ser Ala
Tyr Asp Glu130 135 140Pro Ser Asn Tyr Val Leu Arg Gly Lys Leu Gln
Tyr Glu Leu Gln Tyr145 150 155 160Arg Asn Leu Arg Asp Pro Tyr Ala
Val Arg Pro Val Thr Lys Leu Ile165 170 175Ser Val Asp Ser Arg Asn
Val Ser Leu Leu Pro Glu Glu Phe His Lys180 185 190Asp Ser Ser Tyr
Gln Leu Gln Val Arg Ala Ala Pro Gln Pro Gly Thr195 200 205Ser Phe
Arg Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln210 215
220Thr Gln Ala Gly Glu Pro Glu Ala Gly Trp Asp Pro His Met Leu
Leu225 230 235 240Leu Leu Ala Val Leu Ile Ile Val Leu Val Phe Met
Gly Leu Lys Ile245 250 255His Leu Pro Trp Arg Leu Trp Lys Lys Ile
Trp Ala Pro Val Pro Thr260 265 270Pro Glu Ser Phe Phe Gln Pro Leu
Tyr Arg Glu His Ser Gly Asn Phe275 280 285Lys Lys Trp Val Asn Thr
Pro Phe Thr Ala Ser Ser Ile Glu Leu Val290 295 300Pro Gln Ser Ser
Thr Thr Thr Ser Ala Leu His Leu Ser Leu Tyr Pro305 310 315 320Ala
Lys Glu Lys Lys Phe Pro Gly Leu Pro Gly Leu Glu Glu Gln Leu325 330
335Glu Cys Asp Gly Met Ser Glu Pro Gly His Trp Cys Ile Ile Pro
Leu340 345 350Ala Ala Gly Gln Ala Val Ser Ala Tyr Ser Glu Glu Arg
Asp Arg Pro355 360 365Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val
Gly Asp Ala Glu Gly370 375 380Leu Cys Val Trp Pro Cys Ser Cys Glu
Asp Asp Gly Tyr Pro Ala Met385 390 395 400Asn Leu Asp Ala Gly Arg
Glu Ser Gly Pro Asn Ser Glu Asp Leu Leu405 410 415Leu Val Thr Asp
Pro Ala Phe Leu Ser Cys Gly Cys Val Ser Gly Ser420 425 430Gly Leu
Arg Leu Gly Gly Ser Pro Gly Ser Leu Leu Asp Arg Leu Arg435 440
445Leu Ser Phe Ala Lys Glu Gly Asp Trp Thr Ala Asp Pro Thr Trp
Arg450 455 460Thr Gly Ser Pro Gly Gly Gly Ser Glu Ser Glu Ala Gly
Ser Pro Pro465 470 475 480Gly Leu Asp Met Asp Thr Phe Asp Ser Gly
Phe Ala Gly Ser Asp Cys485 490 495Gly Ser Pro Val Glu Thr Asp Glu
Gly Pro Pro Arg Ser Tyr Leu Arg500 505 510Gln Trp Val Val Arg Thr
Pro Pro Pro Val Asp Ser Gly Ala Gln Ser515 520
525Ser135PRTArtificial SequenceWSXWS polypeptide consensus motif
13Trp Ser Xaa Trp Ser1 5146PRTArtificial SequenceGlu-Glu (CEE) Tag
amino acid sequence 14Glu Tyr Met Pro Met Glu1 5151701DNAArtificial
Sequencesoluble zalpha11R/IgGgamma1 construct 15atg ccg cgt ggc tgg
gcc gcc ccc ttg ctc ctg ctg ctg ctc cag gga 48Met Pro Arg Gly Trp
Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly1 5 10 15ggc tgg ggc tgc
ccc gac ctc gtc tgc tac acc gat tac ctc cag acg 96Gly Trp Gly Cys
Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr20 25 30gtc atc tgc
atc ctg gaa atg tgg aac ctc cac ccc agc acg ctc acc 144Val Ile Cys
Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr35 40 45ctt acc
tgg caa gac cag tat gaa gag ctg aag gac gag gcc acc tcc 192Leu Thr
Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser50 55 60tgc
agc ctc cac agg tcg gcc cac aat gcc acg cat gcc acc tac acc 240Cys
Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr65 70 75
80tgc cac atg gat gta ttc cac ttc atg gcc gac gac att ttc agt gtc
288Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser
Val85 90 95aac atc aca gac cag tct ggc aac tac tcc cag gag tgt ggc
agc ttt 336Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly
Ser Phe100 105 110ctc ctg gct gag agc atc aag ccg gct ccc cct ttc
aac gtg act gtg 384Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe
Asn Val Thr Val115 120 125acc ttc tca gga cag tat aat atc tcc tgg
cgc tca gat tac gaa gac 432Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp
Arg Ser Asp Tyr Glu Asp130 135 140cct gcc ttc tac atg ctg aag ggc
aag ctt cag tat gag ctg cag tac 480Pro Ala Phe Tyr Met Leu Lys Gly
Lys Leu Gln Tyr Glu Leu Gln Tyr145 150 155 160agg aac cgg gga gac
ccc tgg gct gtg agt ccg agg aga aag ctg atc 528Arg Asn Arg Gly Asp
Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile165 170 175tca gtg gac
tca aga agt gtc tcc ctc ctc ccc ctg gag ttc cgc aaa 576Ser Val Asp
Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys180 185 190gac
tcg agc tat gag ctg cag gtg cgg gca ggg ccc atg cct ggc tcc 624Asp
Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser195 200
205tcc tac cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc ttt cag
672Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe
Gln210 215 220acc cag tca gag gag tta aag gaa ggc tgg aac cct cac
gct agc acc 720Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His
Ala Ser Thr225 230 235 240aag ggc cca tcg gtc ttc ccc ctg gca ccc
tcc tcc aag agc acc tct 768Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser245 250 255ggg ggc aca gcg gcc ctg ggc tgc
ctg gtc aag gac tac ttc ccc gaa 816Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu260
265 270ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg
cac 864Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His275 280 285acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc
ctc agc agc 912Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser290 295 300gtg gtg acc gtg ccc tcc agc agc ttg ggc acc
cag acc tac atc tgc 960Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys305 310 315 320aac gtg aat cac aag ccc agc aac
acc aag gtg gac aag aaa gtt gag 1008Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu325 330 335ccc aaa tct tgt gac aaa
act cac aca tgc cca ccg tgc cca gca cct 1056Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro340 345 350gaa ctc ctg ggg
gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag 1104Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys355 360 365gac acc
ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg 1152Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val370 375
380gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac
1200Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp385 390 395 400ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg
gag gag cag tac 1248Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr405 410 415aac agc acg tac cgt gtg gtc agc gtc ctc
acc gtc ctg cac cag gac 1296Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp420 425 430tgg ctg aat ggc aag gag tac aag
tgc aag gtc tcc aac aaa gcc ctc 1344Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu435 440 445cca gcc ccc atc gag aaa
acc atc tcc aaa gcc aaa ggg cag ccc cga 1392Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg450 455 460gaa cca cag gtg
tac acc ctg ccc cca tcc cgg gat gag ctg acc aag 1440Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys465 470 475 480aac
cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac 1488Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp485 490
495atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag
1536Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys500 505 510acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc
ctc tac agc 1584Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser515 520 525aag ctc acc gtg gac aag agc agg tgg cag cag
ggg aac gtc ttc tca 1632Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser530 535 540tgc tcc gtg atg cat gag gct ctg cac
aac cac tac acg cag aag agc 1680Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser545 550 555 560ctc tcc ctg tct ccg ggt
aaa 1701Leu Ser Leu Ser Pro Gly Lys56516567PRTArtificial
Sequencesoluble zalpha11R/IgGgamma1 construct 16Met Pro Arg Gly Trp
Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly1 5 10 15Gly Trp Gly Cys
Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr20 25 30Val Ile Cys
Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr35 40 45Leu Thr
Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser50 55 60Cys
Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr65 70 75
80Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val85
90 95Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser
Phe100 105 110Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn
Val Thr Val115 120 125Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg
Ser Asp Tyr Glu Asp130 135 140Pro Ala Phe Tyr Met Leu Lys Gly Lys
Leu Gln Tyr Glu Leu Gln Tyr145 150 155 160Arg Asn Arg Gly Asp Pro
Trp Ala Val Ser Pro Arg Arg Lys Leu Ile165 170 175Ser Val Asp Ser
Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys180 185 190Asp Ser
Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser195 200
205Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe
Gln210 215 220Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His
Ala Ser Thr225 230 235 240Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser245 250 255Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu260 265 270Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His275 280 285Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser290 295 300Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys305 310 315
320Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu325 330 335Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro340 345 350Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys355 360 365Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val370 375 380Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp385 390 395 400Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr405 410 415Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp420 425
430Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu435 440 445Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg450 455 460Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys465 470 475 480Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp485 490 495Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys500 505 510Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser515 520 525Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser530 535
540Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser545 550 555 560Leu Ser Leu Ser Pro Gly Lys565171083DNAArtificial
Sequencesoluble human IL-2Rgamma/human kappa light chainconstruct
17atg ttg aag cca tca tta cca ttc aca tcc ctc tta ttc ctg cag ctg
48Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu1
5 10 15ccc ctg ctg gga gtg ggg ctg aac acg aca att ctg acg ccc aat
ggg 96Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn
Gly20 25 30aat gaa gac acc aca gct gat ttc ttc ctg acc act atg ccc
act gac 144Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro
Thr Asp35 40 45tcc ctc agt gtt tcc act ctg ccc ctc cca gag gtt cag
tgt ttt gtg 192Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln
Cys Phe Val50 55 60ttc aat gtc gag tac atg aat tgc act tgg aac agc
agc tct gag ccc 240Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser
Ser Ser Glu Pro65 70 75 80cag cct acc aac ctc act ctg cat tat tgg
tac aag aac tcg gat aat 288Gln Pro Thr Asn Leu Thr Leu His Tyr Trp
Tyr Lys Asn Ser Asp Asn85 90 95gat aaa gtc cag aag tgc agc cac tat
cta ttc tct gaa gaa atc act 336Asp Lys Val Gln Lys Cys Ser His Tyr
Leu Phe Ser Glu Glu Ile Thr100 105 110tct ggc tgt cag ttg caa aaa
aag gag atc cac ctc tac caa aca ttt 384Ser Gly Cys Gln Leu Gln Lys
Lys Glu Ile His Leu Tyr Gln Thr Phe115 120 125gtt gtt cag ctc cag
gac cca cgg gaa ccc agg aga cag gcc aca cag 432Val Val Gln Leu Gln
Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln130 135 140atg cta aaa
ctg cag aat ctg gtg atc ccc tgg gct cca gag aac cta 480Met Leu Lys
Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu145 150 155
160aca ctt cac aaa ctg agt gaa tcc cag cta gaa ctg aac tgg aac aac
528Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn
Asn165 170 175aga ttc ttg aac cac tgt ttg gag cac ttg gtg cag tac
cgg act gac 576Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr
Arg Thr Asp180 185 190tgg gac cac agc tgg act gaa caa tca gtg gat
tat aga cat aag ttc 624Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp
Tyr Arg His Lys Phe195 200 205tcc ttg cct agt gtg gat ggg cag aaa
cgc tac acg ttt cgt gtt cgg 672Ser Leu Pro Ser Val Asp Gly Gln Lys
Arg Tyr Thr Phe Arg Val Arg210 215 220agc cgc ttt aac cca ctc tgt
gga agt gct cag cat tgg agt gaa tgg 720Ser Arg Phe Asn Pro Leu Cys
Gly Ser Ala Gln His Trp Ser Glu Trp225 230 235 240agc cac cca atc
cac tgg ggg agc aat act tca aaa gag aat act gtg 768Ser His Pro Ile
His Trp Gly Ser Asn Thr Ser Lys Glu Asn Thr Val245 250 255gct gca
cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa 816Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys260 265
270tct ggt acc gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga
864Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg275 280 285gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa
tcg ggt aac 912Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn290 295 300tcc cag gag agt gtc aca gag cag gac agc aag
gac agc acc tac agc 960Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser305 310 315 320ctc agc agc acc ctg acg ctg agc
aaa gca gac tac gag aaa cac aaa 1008Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys325 330 335gtc tac gcc tgc gaa gtc
acc cat cag ggc ctg agc tcg ccc gtc aca 1056Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr340 345 350aag agc ttc aac
agg gga gag tgt tag 1083Lys Ser Phe Asn Arg Gly Glu Cys *355
36018360PRTArtificial Sequencesoluble human IL-2Rgamma/human kappa
light 18Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln
Leu1 5 10 15Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro
Asn Gly20 25 30Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met
Pro Thr Asp35 40 45Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val
Gln Cys Phe Val50 55 60Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn
Ser Ser Ser Glu Pro65 70 75 80Gln Pro Thr Asn Leu Thr Leu His Tyr
Trp Tyr Lys Asn Ser Asp Asn85 90 95Asp Lys Val Gln Lys Cys Ser His
Tyr Leu Phe Ser Glu Glu Ile Thr100 105 110Ser Gly Cys Gln Leu Gln
Lys Lys Glu Ile His Leu Tyr Gln Thr Phe115 120 125Val Val Gln Leu
Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln130 135 140Met Leu
Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu145 150 155
160Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn
Asn165 170 175Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr
Arg Thr Asp180 185 190Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp
Tyr Arg His Lys Phe195 200 205Ser Leu Pro Ser Val Asp Gly Gln Lys
Arg Tyr Thr Phe Arg Val Arg210 215 220Ser Arg Phe Asn Pro Leu Cys
Gly Ser Ala Gln His Trp Ser Glu Trp225 230 235 240Ser His Pro Ile
His Trp Gly Ser Asn Thr Ser Lys Glu Asn Thr Val245 250 255Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys260 265
270Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg275 280 285Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn290 295 300Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser305 310 315 320Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys325 330 335Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr340 345 350Lys Ser Phe Asn
Arg Gly Glu Cys355 3601936DNAArtificial SequenceOligonucleotide
primer ZC19905 19acaggatccg tcagcatgcc gcgtggctgg gccgcc
362033DNAArtificial SequenceOligonucleotide primer ZC19906
20acagaattct tagctggcct ggggtccagg cgt 332136DNAArtificial
SequenceOligonucleotide primer ZC19931 21ggttggtacc gcaagatgcc
gcgtggctgg gccgcc 362229DNAArtificial SequenceOligonucleotide
primer ZC19932 22cggaggatcc gtgagggttc cagccttcc 29238PRTArtificial
SequenceFLAG tag amino acid sequence 23Asp Tyr Lys Asp Asp Asp Asp
Lys1 52466DNAArtificial SequenceOligonucleotide primer spanning
vector flanking region and the 5' end of the zalpha11 24tccactttgc
ctttctctcc acaggtgtcc agggaattca tcgataatgc cgcgtggctg 60ggccgc
6625699DNAHomo sapiens 25gagcccagat cttcagacaa aactcacaca
tgcccaccgt gcccagcacc tgaagccgag 60ggggcaccgt cagtcttcct cttcccccca
aaacccaagg acaccctcat gatctcccgg 120acccctgagg tcacatgcgt
ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180aactggtacg
tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
240tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga
ctggctgaat 300ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc
catcctccat cgagaaaacc 360atctccaaag ccaaagggca gccccgagaa
ccacaggtgt acaccctgcc cccatcccgg 420gatgagctga ccaagaacca
ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480gacatcgccg
tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
540cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt
ggacaagagc 600aggtggcagc aggggaacgt cttctcatgc tccgtgatgc
atgaggctct gcacaaccac 660tacacgcaga agagcctctc cctgtctccg ggtaaataa
6992662DNAArtificial SequenceFirst Oligonucleotide primer spanning
3' end of the zalpha11 extracellular domain and the 5' end of Fc4
26gcacggtggg catgtgtgag ttttgtctga agatctgggc tcgtgagggt tccagccttc
60ct 622761DNAArtificial SequenceSecond Oligonucleotide primer
spanning 3' end of the zalpha11 extracellular domain and the 5' end
of Fc4 27agacccagtc agaggagtta aaggaaggct ggaaccctca cgagcccaga
tcttcagaca 60a 612867DNAArtificial SequenceOligonucleotide primer
spanning the 3' end of Fc4 and the vector flanking region
28gtgggcctct ggggtgggta caaccccaga gctgttttaa tctagattat ttacccggag
60acaggga 67291821DNAArtificial SequencePolynucleotide encoding
MBP-human zalpha11 soluble receptor fusion 29atg aaa atc gaa gaa
ggt aaa ctg gta atc tgg att aac ggc gat aaa 48Met Lys Ile Glu Glu
Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys1 5 10 15ggc tat aac ggt
ctc gct gaa gtc ggt aag aaa ttc gag aaa gat acc 96Gly Tyr Asn Gly
Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr20 25 30gga att aaa
gtc acc gtt gag cat ccg gat aaa ctg gaa gag aaa ttc 144Gly Ile Lys
Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe35 40 45cca cag
gtt gcg gca act ggc gat ggc cct gac att atc ttc tgg gca 192Pro Gln
Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala50 55 60cac
gac cgc ttt ggt ggc tac gct caa tct ggc ctg ttg gct gaa atc 240His
Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile65 70 75
80acc ccg gac aaa gcg ttc cag gac aag ctg tat ccg ttt acc tgg gat
288Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp
Asp85 90 95gcc gta cgt tac aac ggc aag ctg att gct tac ccg atc gct
gtt gaa 336Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala
Val Glu100 105 110gcg
tta tcg ctg att tat aac aaa gat ctg ctg ccg aac ccg cca aaa 384Ala
Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys115 120
125acc tgg gaa gag atc ccg gcg ctg gat aaa gaa ctg aaa gcg aaa ggt
432Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys
Gly130 135 140aag agc gcg ctg atg ttc aac ctg caa gaa ccg tac ttc
acc tgg ccg 480Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe
Thr Trp Pro145 150 155 160ctg att gct gct gac ggg ggt tat gcg ttc
aag tat gaa aac ggc aag 528Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe
Lys Tyr Glu Asn Gly Lys165 170 175tac gac att aaa gac gtg ggc gtg
gat aac gct ggc gcg aaa gcg ggt 576Tyr Asp Ile Lys Asp Val Gly Val
Asp Asn Ala Gly Ala Lys Ala Gly180 185 190ctg acc ttc ctg gtt gac
ctg att aaa aac aaa cac atg aat gca gac 624Leu Thr Phe Leu Val Asp
Leu Ile Lys Asn Lys His Met Asn Ala Asp195 200 205acc gat tac tcc
atc gca gaa gct gcc ttt aat aaa ggc gaa aca gcg 672Thr Asp Tyr Ser
Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala210 215 220atg acc
atc aac ggc ccg tgg gca tgg tcc aac atc gac acc agc aaa 720Met Thr
Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys225 230 235
240gtg aat tat ggt gta acg gta ctg ccg acc ttc aag ggt caa cca tcc
768Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro
Ser245 250 255aaa ccg ttc gtt ggc gtg ctg agc gca ggt att aac gcc
gcc agt ccg 816Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala
Ala Ser Pro260 265 270aac aaa gag ctg gca aaa gag ttc ctc gaa aac
tat ctg ctg act gat 864Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn
Tyr Leu Leu Thr Asp275 280 285gaa ggt ctg gaa gcg gtt aat aaa gac
aaa ccg ctg ggt gcc gta gcg 912Glu Gly Leu Glu Ala Val Asn Lys Asp
Lys Pro Leu Gly Ala Val Ala290 295 300ctg aag tct tac gag gaa gag
ttg gcg aaa gat cca cgt att gcc gcc 960Leu Lys Ser Tyr Glu Glu Glu
Leu Ala Lys Asp Pro Arg Ile Ala Ala305 310 315 320acc atg gaa aac
gcc cag aaa ggt gaa atc atg ccg aac atc ccg cag 1008Thr Met Glu Asn
Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln325 330 335atg tcc
gct ttc tgg tat gcc gtg cgt act gcg gtg atc aac gcc gcc 1056Met Ser
Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala340 345
350agc ggt cgt cag act gtc gat gaa gcc ctg aaa gac gcg cag act aat
1104Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr
Asn355 360 365tcg agc tcc cac cat cac cat cac cac gcg aat tcg gta
ccg ctg gtt 1152Ser Ser Ser His His His His His His Ala Asn Ser Val
Pro Leu Val370 375 380ccg cgt gga tcc tgc ccc gac ctc gtc tgc tac
acc gat tac ctc cag 1200Pro Arg Gly Ser Cys Pro Asp Leu Val Cys Tyr
Thr Asp Tyr Leu Gln385 390 395 400acg gtc atc tgc atc ctg gaa atg
tgg aac ctc cac ccc agc acg ctc 1248Thr Val Ile Cys Ile Leu Glu Met
Trp Asn Leu His Pro Ser Thr Leu405 410 415acc ctt acc tgg caa gac
cag tat gaa gag ctg aag gac gag gcc acc 1296Thr Leu Thr Trp Gln Asp
Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr420 425 430tcc tgc agc ctc
cac agg tcg gcc cac aat gcc acg cat gcc acc tac 1344Ser Cys Ser Leu
His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr435 440 445acc tgc
cac atg gat gta ttc cac ttc atg gcc gac gac att ttc agt 1392Thr Cys
His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser450 455
460gtc aac atc aca gac cag tct ggc aac tac tcc cag gag tgt ggc agc
1440Val Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly
Ser465 470 475 480ttt ctc ctg gct gag agc atc aag ccg gct ccc cct
ttc aac gtg act 1488Phe Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro
Phe Asn Val Thr485 490 495gtg acc ttc tca gga cag tat aat atc tcc
tgg cgc tca gat tac gaa 1536Val Thr Phe Ser Gly Gln Tyr Asn Ile Ser
Trp Arg Ser Asp Tyr Glu500 505 510gac cct gcc ttc tac atg ctg aag
ggc aag ctt cag tat gag ctg cag 1584Asp Pro Ala Phe Tyr Met Leu Lys
Gly Lys Leu Gln Tyr Glu Leu Gln515 520 525tac agg aac cgg gga gac
ccc tgg gct gtg agt ccg agg aga aag ctg 1632Tyr Arg Asn Arg Gly Asp
Pro Trp Ala Val Ser Pro Arg Arg Lys Leu530 535 540atc tca gtg gac
tca aga agt gtc tcc ctc ctc ccc ctg gag ttc cgc 1680Ile Ser Val Asp
Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg545 550 555 560aaa
gac tcg agc tat gag ctg cag gtg cgg gca ggg ccc atg cct ggc 1728Lys
Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly565 570
575tcc tcc tac cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc ttt
1776Ser Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile
Phe580 585 590cag acc cag tca gag gag tta aag gaa ggc tgg aac cct
cac tag 1821Gln Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His
*595 600 60530606PRTArtificial SequencePolynucleotide encoding
MBP-human zalpha11 soluble receptor fusion 30Met Lys Ile Glu Glu
Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys1 5 10 15Gly Tyr Asn Gly
Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr20 25 30Gly Ile Lys
Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe35 40 45Pro Gln
Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala50 55 60His
Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile65 70 75
80Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp85
90 95Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val
Glu100 105 110Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn
Pro Pro Lys115 120 125Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu
Leu Lys Ala Lys Gly130 135 140Lys Ser Ala Leu Met Phe Asn Leu Gln
Glu Pro Tyr Phe Thr Trp Pro145 150 155 160Leu Ile Ala Ala Asp Gly
Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys165 170 175Tyr Asp Ile Lys
Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly180 185 190Leu Thr
Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp195 200
205Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr
Ala210 215 220Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp
Thr Ser Lys225 230 235 240Val Asn Tyr Gly Val Thr Val Leu Pro Thr
Phe Lys Gly Gln Pro Ser245 250 255Lys Pro Phe Val Gly Val Leu Ser
Ala Gly Ile Asn Ala Ala Ser Pro260 265 270Asn Lys Glu Leu Ala Lys
Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp275 280 285Glu Gly Leu Glu
Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala290 295 300Leu Lys
Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala305 310 315
320Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro
Gln325 330 335Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile
Asn Ala Ala340 345 350Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys
Asp Ala Gln Thr Asn355 360 365Ser Ser Ser His His His His His His
Ala Asn Ser Val Pro Leu Val370 375 380Pro Arg Gly Ser Cys Pro Asp
Leu Val Cys Tyr Thr Asp Tyr Leu Gln385 390 395 400Thr Val Ile Cys
Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu405 410 415Thr Leu
Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr420 425
430Ser Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr
Tyr435 440 445Thr Cys His Met Asp Val Phe His Phe Met Ala Asp Asp
Ile Phe Ser450 455 460Val Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser
Gln Glu Cys Gly Ser465 470 475 480Phe Leu Leu Ala Glu Ser Ile Lys
Pro Ala Pro Pro Phe Asn Val Thr485 490 495Val Thr Phe Ser Gly Gln
Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu500 505 510Asp Pro Ala Phe
Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln515 520 525Tyr Arg
Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu530 535
540Ile Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe
Arg545 550 555 560Lys Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly
Pro Met Pro Gly565 570 575Ser Ser Tyr Gln Gly Thr Trp Ser Glu Trp
Ser Asp Pro Val Ile Phe580 585 590Gln Thr Gln Ser Glu Glu Leu Lys
Glu Gly Trp Asn Pro His595 600 60531657DNAArtificial SequenceA
fragment of human zalpha11 cDNA 31tgccccgacc tcgtctgcta caccgattac
ctccagacgg tcatctgcat cctggaaatg 60tggaacctcc accccagcac gctcaccctt
acctggcaag accagtatga agagctgaag 120gacgaggcca cctcctgcag
cctccacagg tcggcccaca atgccacgca tgccacctac 180acctgccaca
tggatgtatt ccacttcatg gccgacgaca ttttcagtgt caacatcaca
240gaccagtctg gcaactactc ccaggagtgt ggcagctttc tcctggctga
gagcatcaag 300ccggctcccc ctttcaacgt gactgtgacc ttctcaggac
agtataatat ctcctggcgc 360tcagattacg aagaccctgc cttctacatg
ctgaagggca agcttcagta tgagctgcag 420tacaggaacc ggggagaccc
ctgggctgtg agtccgagga gaaagctgat ctcagtggac 480tcaagaagtg
tctccctcct ccccctggag ttccgcaaag actcgagcta tgagctgcag
540gtgcgggcag ggcccatgcc tggctcctcc taccagggga cctggagtga
atggagtgac 600ccggtcatct ttcagaccca gtcagaggag ttaaaggaag
gctggaaccc tcactag 6573265DNAArtificial SequenceOligonucleotide
primerZC20187 32tcaccacgcg aattcggtac cgctggttcc gcgtggatcc
tgccccgacc tcgtctgcta 60caccg 653368DNAArtificial
SequenceOligonucleotide primerZC20185 33tctgtatcag gctgaaaatc
ttatctcatc cgccaaaaca ctagtgaggg ttccagcctt 60cctttaac
683440DNAArtificial SequenceOligonucleotide primerZC19372
34tgtcgatgaa gccctgaaag acgcgcagac taattcgagc 403560DNAArtificial
SequenceOligonucleotide primerZC19351 35acgcgcagac taattcgagc
tcccaccatc accatcacca cgcgaattcg gtaccgctgg 603660DNAArtificial
SequenceOligonucleotide primerZC19352 36actcactata gggcgaattg
cccgggggat ccacgcggaa ccagcggtac cgaattcgcg 603742DNAArtificial
SequenceOligonucleotide primerZC19371 37acggccagtg aattgtaata
cgactcacta tagggcgaat tg 423824DNAArtificial
SequenceOligonucleotide primerZC19907 38atggatgtat tccacttcat ggcc
243924DNAArtificial SequenceOligonucleotide primerZC19908
39actgtcaaac gtgtccatat ccag 244020DNAArtificial
SequenceOligonucleotide primerZC22277 40ccaggagtgt ggcagctttc
204121DNAArtificial SequenceOligonucleotide primerZC22276
41gcttgccctt cagcatgtag a 214223DNAArtificial SequenceZalpha11
TaqMan Probe, ZG31 42cggctccccc tttcaacgtg act 234320DNAArtificial
SequenceOligonucleotide primer, rRNA forward primer 43cggctaccac
atccaaggaa 204418DNAArtificial SequenceOligonucleotide primer, rRNA
reverse primer 44gctggaatta ccgcggct 184522DNAArtificial
SequencerRNA TaqMan probe 45tgctggcacc agacttgccc tc 22463072DNAMus
musculusCDS(54)...(491) 46gagaaccaga ccaaggccct gtcatcagct
cctggagact cagttctggt ggc atg 56Met1gag agg acc ctt gtc tgt ctg gta
gtc atc ttc ttg ggg aca gtg gcc 104Glu Arg Thr Leu Val Cys Leu Val
Val Ile Phe Leu Gly Thr Val Ala5 10 15cat aaa tca agc ccc caa ggg
cca gat cgc ctc ctg att aga ctt cgt 152His Lys Ser Ser Pro Gln Gly
Pro Asp Arg Leu Leu Ile Arg Leu Arg20 25 30cac ctt att gac att gtt
gaa cag ctg aaa atc tat gaa aat gac ttg 200His Leu Ile Asp Ile Val
Glu Gln Leu Lys Ile Tyr Glu Asn Asp Leu35 40 45gat cct gaa ctt cta
tca gct cca caa gat gta aag ggg cac tgt gag 248Asp Pro Glu Leu Leu
Ser Ala Pro Gln Asp Val Lys Gly His Cys Glu50 55 60 65cat gca gct
ttt gcc tgt ttt cag aag gcc aaa ctc aag cca tca aac 296His Ala Ala
Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser Asn70 75 80cct gga
aac aat aag aca ttc atc att gac ctc gtg gcc cag ctc agg 344Pro Gly
Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu Arg85 90 95agg
agg ctg cct gcc agg agg gga gga aag aaa cag aag cac ata gct 392Arg
Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His Ile Ala100 105
110aaa tgc cct tcc tgt gat tcg tat gag aaa agg aca ccc aaa gaa ttc
440Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr Pro Lys Glu
Phe115 120 125cta gaa aga cta aaa tgg ctc ctt caa aag atg att cat
cag cat ctc 488Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys Met Ile His
Gln His Leu130 135 140 145tcc tagaacacat aggacccgaa gattcctgag
gatccgagaa gattcccgag 541Sergactgaggag acgccggaca ctatagacgc
tcacgaatgc aggagtacat cttgcctctt 601gggattgcaa gtggagaagt
acgatacgtt atgataagaa caactcagaa aagctatagg 661ttaagatcct
ttcgcccatt aactaagcag acattgtggt tccctgcaca gactccatgc
721tgtcaacatg gaaaatctca actcaacaag agcccagctt cccgtgtcag
ggatttctgg 781tgcttctcaa gctgtggctt catcttattg cccaactgtg
acattctttg attggaaggg 841gaaaactaaa gcttttagca aaaatacagc
tagggaattt gtcgatctgc gagagtaaga 901cctcttatga tcctaacgga
atgatgtaag ctggaaataa taagcataag atgaaattga 961aaattgaagt
ctttattctt taagaaaaac tttgtacttg aaagcatgtc tgaagagttt
1021actcattacc acaaacatct agcatattga taactaacat ctttatactc
tacaagagag 1081gctttccaga taggtacagt ttttcttctc tattaggtct
atcaaaattt aacctattat 1141gagggtcacc cctggctttc actgtttttc
taaagaggca agggtgtagt aagaagcagg 1201cttaagttgc cttcctccca
atgtcaagtt cctttataag ctaatagttt aatcttgtga 1261agatggcaat
gaaagcctgt ggaagtgcaa acctcactat cttctggagc caagtagaat
1321tttcaagttt gtagctctca cctcaagtgg ttatgggtgt cctgtgatga
atctgctagc 1381tccagcctca gtctcctctc ccacatcctt tcctttcttt
cctctttgaa acttctaaga 1441aaaagcaatc caaacaagtt cagcacttaa
gacacattgc atgcacactt ttgataagtt 1501aaatccaacc atctatttaa
aatcaaaatc aggagatgag ccaagagacc agaggttctg 1561ttccagtttt
aaacagactt ttactgaaca tcccaatctt ttaaccacag aggctaaatt
1621gagcaaatag ttttgccatt tgatataatt tccaacagta tgtttcaatg
tcaagttaaa 1681aagtctacaa agctattttc cctggagtgg tatcatcgct
ttgagaattt cttatggtta 1741aaatggatct gagatccaag catggcctgg
gggatggttt tgatctaagg aaaaaggtgt 1801ctgtacctca cagtgccttt
aaaacaagca gagatcccgt gtaccgccct aagatagcac 1861agactagtgt
taactgattc ccagaaaagt gtcacaatca gaaccaacgc attctcttaa
1921actttaaaaa tatgtattgc aaagaacttg tgtaactgta aatgtgtgac
tgttgatgac 1981attatacaca catagcccac gtaagtgtcc aatggtgcta
gcattggttg ctgagtttgc 2041tgctcgaaag ctgaagcaga gatgcagtcc
ttcacaaagc aatgatggac agagagggga 2101gtctccatgt tttattcttt
tgttgtttct ggctgtgtaa ctgttgactt cttgacattg 2161tgatttttat
atttaagaca atgtatttat tttggtgtgt ttattgttct agccttttaa
2221atcactgaca atttctaatc aagaagtaca aataattcaa tgcagcacag
gctaagagct 2281tgtatcgttt ggaaaagcca gtgaaggctt ctccactagc
catgggaaag ctacgcttta 2341gagtaaacta gacaaaattg cacagcagtc
ttgaacctct ctgtgctcaa gactcagcca 2401gtcctttgac attattgttc
actgtgggtg ggaacacatt ggacctgaca cactgttgtg 2461tgtccatgaa
ggttgccact ggtgtaagct ttttttggtt ttcattctct tatctgtaga
2521acaagaatgt ggggctttcc taagtctatt ctgtatttta ttctgaactt
cgtatgtctg 2581agttttaatg ttttgagtac tcttacagga acacctgacc
acacttttga gttaaatttt 2641atcccaagtg tgatatttag ttgttcaaaa
agggaaggga tatacataca tacatacata 2701catacataca tatatatata
tatatataca tatatatata tatatatatg tatatatata 2761tatatataga
gagagagaga gagagagaga gagaaagaga gagaggttgt tgtaggtcat
2821aggagttcag aggaaatcag ttatggccgt taatactgta gctgaaagtg
ttttctttgt 2881gaataaattc atagcattat tgatctatgt tattgctctg
ttttatttac agtcacacct 2941gagaatttag ttttaatatg aatgatgtac
tttataactt aatgattatt tattatgtat 3001ttggttttga atgtttgtgt
tcatggcttc ttatttaaga cctgatcata ttaaatgcta 3061cccagtccgg a
307247146PRTMus musculus 47Met Glu Arg Thr Leu Val Cys Leu Val Val
Ile Phe Leu Gly Thr Val1 5 10 15Ala His Lys Ser Ser Pro Gln Gly Pro
Asp Arg Leu Leu Ile Arg Leu20 25 30Arg His Leu Ile Asp Ile Val Glu
Gln Leu Lys Ile Tyr Glu Asn Asp35 40 45Leu Asp
Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys50 55 60Glu
His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser65 70 75
80Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu85
90 95Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His
Ile100 105 110Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr
Pro Lys Glu115 120 125Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys
Met Ile His Gln His130 135 140Leu Ser14548100DNAArtificial
SequenceOligonucleotide primer ZC12749 48gtaccttccc gtaaatccct
ccccttcccg gaattacacc cgcgtatttc ccagaaaagg 60aactgtagat ttctaggaat
tcaatccttg gccacgcgtc 10049100DNAArtificial SequenceOligonucleotide
primer ZC12748 49tcgagacgcg tggccaagga ttgaattcct agaaatctac
agttcctttt ctgggaaata 60cgcgggtgta attccgggaa ggggagggat ttacgggaag
1005036DNAArtificial SequenceOligonucleotide primer ZC11450
50acttgtggaa ttcgctagca ccaagggccc atcggt 365132DNAArtificial
SequenceOligonucleotide primer ZC11443 51gcctagaacg cgttcattta
cccggagaca gg 32528DNAArtificial SequenceOligonucleotide primer
ZC11440 52aattgaga 8538DNAArtificial SequenceOligonucleotide primer
ZC11441 53cgcgtctc 85437DNAArtificial SequenceOligonucleotide
primer ZC11501 54gtcacttgaa ttcggtaccg cctctgttgt gtgcctg
375532DNAArtificial SequenceOligonucleotide primer ZC11451
55gacctgaacg cgtctaacac tctcccctgt tg 325641DNAArtificial
SequenceOligonucleotide primer ZC24052 56tcagtcggaa ttcgcagaag
ccatgccgcg tggctgggcc g 415734DNAArtificial SequenceOligonucleotide
primer ZC24053 57ctgtgacgct agcgtgaggg ttccagcctt cctt
345841DNAArtificial SequenceOligonucleotide primer ZC12834
58tcagtcggaa ttcgcagaag ccatgttgaa gccatcatta c 415990DNAArtificial
SequenceOligonucleotide primer ZC12831 59aagacggtac cagatttcaa
ctgctcatca gatggcggga agatgaagac agatggtgca 60gccacagtag gattctcttt
tgaagtattg 906020DNAArtificial SequenceOligonucleotide primer
ZC23684 60tcacccttac ctggcaagac 206143DNAArtificial
SequenceOligonucleotide primer ZC23656 61taatacgact cactataggg
agggggagac acttcttgag tcc 436220DNAArtificial
SequenceOligonucleotide primer ZC23685 62aggtctgaat cccgactctg
206343DNAArtificial SequenceOligonucleotide primer ZC23657
63taatacgact cactataggg aggacgtaat tggtgtttaa taa 43641707DNAhomo
sapiensCDS(1)...(1707) 64atg ccg cgt ggc tgg gcc gcc ccc ttg ctc
ctg ctg ctg ctc cag gga 48Met Pro Arg Gly Trp Ala Ala Pro Leu Leu
Leu Leu Leu Leu Gln Gly1 5 10 15gcc ctc gag ggg atg gag agg aag ctc
tgc agt ccc aag cca ccc ccc 96Ala Leu Glu Gly Met Glu Arg Lys Leu
Cys Ser Pro Lys Pro Pro Pro20 25 30acc aag gcc tct ctc ccc act gac
cct cca ggc tgg ggc tgc ccc gac 144Thr Lys Ala Ser Leu Pro Thr Asp
Pro Pro Gly Trp Gly Cys Pro Asp35 40 45ctc gtc tgc tac acc gat tac
ctc cag acg gtc atc tgc atc ctg gaa 192Leu Val Cys Tyr Thr Asp Tyr
Leu Gln Thr Val Ile Cys Ile Leu Glu50 55 60atg tgg aac ctc cac ccc
agc acg ctc acc ctt acc tgg ata ctt tct 240Met Trp Asn Leu His Pro
Ser Thr Leu Thr Leu Thr Trp Ile Leu Ser65 70 75 80aat aat act ggg
tgc tat atc aag gac aga aca ctg gac ctc agg caa 288Asn Asn Thr Gly
Cys Tyr Ile Lys Asp Arg Thr Leu Asp Leu Arg Gln85 90 95gac cag tat
gaa gag ctg aag gac gag gcc acc tcc tgc agc ctc cac 336Asp Gln Tyr
Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His100 105 110agg
tcg gcc cac aat gcc acg cat gcc acc tac acc tgc cac atg gat 384Arg
Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp115 120
125gta ttc cac ttc atg gcc gac gac att ttc agt gtc aac atc aca gac
432Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr
Asp130 135 140cag tct ggc aac tac tcc cag gag tgt ggc agc ttt ctc
ctg gct gag 480Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu
Leu Ala Glu145 150 155 160agc aga cag tat aat atc tcc tgg cgc tca
gat tac gaa gac cct gcc 528Ser Arg Gln Tyr Asn Ile Ser Trp Arg Ser
Asp Tyr Glu Asp Pro Ala165 170 175ttc tac atg ctg aag ggc aag ctt
cag tat gag ctg cag tac agg aac 576Phe Tyr Met Leu Lys Gly Lys Leu
Gln Tyr Glu Leu Gln Tyr Arg Asn180 185 190cgg gga gac ccc tgg gct
gtg agt ccg agg aga aag ctg atc tca gtg 624Arg Gly Asp Pro Trp Ala
Val Ser Pro Arg Arg Lys Leu Ile Ser Val195 200 205gac tca aga agt
gtc tcc ctc ctc ccc ctg gag ttc cgc aaa gac tcg 672Asp Ser Arg Ser
Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser210 215 220agc tat
gag ctg cag gtg cgg gca ggg ccc atg cct ggc tcc tcc tac 720Ser Tyr
Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr225 230 235
240cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc ttt cag acc cag
768Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr
Gln245 250 255tca gag gag tta aag gaa ggc tgg aac cct cac ctg ctg
ctt ctc ctc 816Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu
Leu Leu Leu260 265 270ctg ctt gtc ata gtc ttc att cct gcc ttc tgg
agc ctg aag acc cat 864Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp
Ser Leu Lys Thr His275 280 285cca ttg tgg agg cta tgg aag aag ata
tgg gcc gtc ccc agc cct gag 912Pro Leu Trp Arg Leu Trp Lys Lys Ile
Trp Ala Val Pro Ser Pro Glu290 295 300cgg ttc ttc atg ccc ctg tac
aag ggc tgc agc gga gac ttc aag aaa 960Arg Phe Phe Met Pro Leu Tyr
Lys Gly Cys Ser Gly Asp Phe Lys Lys305 310 315 320tgg gtg ggt gca
ccc ttc act ggc tcc agc ctg gag ctg gga ccc tgg 1008Trp Val Gly Ala
Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly Pro Trp325 330 335agc cca
gag gtg ccc tcc acc ctg gag gtg tac agc tgc cac cca cca 1056Ser Pro
Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His Pro Pro340 345
350cgg agc ccg gcc aag agg ctg cag ctc acg gag cta caa gaa cca gca
1104Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu Pro
Ala355 360 365gag ctg gtg gag tct gac ggt gtg ccc aag ccc agc ttc
tgg ccg aca 1152Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe
Trp Pro Thr370 375 380gcc cag aac tcg ggg ggc tca gct tac agt gag
gag agg gat cgg cca 1200Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu
Glu Arg Asp Arg Pro385 390 395 400tac ggc ctg gtg tcc att gac aca
gtg act gtg cta gat gca gag ggg 1248Tyr Gly Leu Val Ser Ile Asp Thr
Val Thr Val Leu Asp Ala Glu Gly405 410 415cca tgc acc tgg ccc tgc
agc tgt gag gat gac ggc tac cca gcc ctg 1296Pro Cys Thr Trp Pro Cys
Ser Cys Glu Asp Asp Gly Tyr Pro Ala Leu420 425 430gac ctg gat gct
ggc ctg gag ccc agc cca ggc cta gag gac cca ctc 1344Asp Leu Asp Ala
Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp Pro Leu435 440 445ttg gat
gca ggg acc aca gtc ctg tcc tgt ggc tgt gtc tca gct ggc 1392Leu Asp
Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser Ala Gly450 455
460agc cct ggg cta gga ggg ccc ctg gga agc ctc ctg gac aga cta aag
1440Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg Leu
Lys465 470 475 480cca ccc ctt gca gat ggg gag gac tgg gct ggg gga
ctg ccc tgg ggt 1488Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly
Leu Pro Trp Gly485 490 495ggc cgg tca cct gga ggg gtc tca gag agt
gag gcg ggc tca ccc ctg 1536Gly Arg Ser Pro Gly Gly Val Ser Glu Ser
Glu Ala Gly Ser Pro Leu500 505 510gcc ggc ctg gat atg gac acg ttt
gac agt ggc ttt gtg ggc tct gac 1584Ala Gly Leu Asp Met Asp Thr Phe
Asp Ser Gly Phe Val Gly Ser Asp515 520 525tgc agc agc cct gtg gag
tgt gac ttc acc agc ccc ggg gac gaa gga 1632Cys Ser Ser Pro Val Glu
Cys Asp Phe Thr Ser Pro Gly Asp Glu Gly530 535 540ccc ccc cgg agc
tac ctc cgc cag tgg gtg gtc att cct ccg cca ctt 1680Pro Pro Arg Ser
Tyr Leu Arg Gln Trp Val Val Ile Pro Pro Pro Leu545 550 555 560tcg
agc cct gga ccc cag gcc agc taa 1707Ser Ser Pro Gly Pro Gln Ala Ser
*56565568PRThomo sapiens 65Met Pro Arg Gly Trp Ala Ala Pro Leu Leu
Leu Leu Leu Leu Gln Gly1 5 10 15Ala Leu Glu Gly Met Glu Arg Lys Leu
Cys Ser Pro Lys Pro Pro Pro20 25 30Thr Lys Ala Ser Leu Pro Thr Asp
Pro Pro Gly Trp Gly Cys Pro Asp35 40 45Leu Val Cys Tyr Thr Asp Tyr
Leu Gln Thr Val Ile Cys Ile Leu Glu50 55 60Met Trp Asn Leu His Pro
Ser Thr Leu Thr Leu Thr Trp Ile Leu Ser65 70 75 80Asn Asn Thr Gly
Cys Tyr Ile Lys Asp Arg Thr Leu Asp Leu Arg Gln85 90 95Asp Gln Tyr
Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His100 105 110Arg
Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp115 120
125Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr
Asp130 135 140Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu
Leu Ala Glu145 150 155 160Ser Arg Gln Tyr Asn Ile Ser Trp Arg Ser
Asp Tyr Glu Asp Pro Ala165 170 175Phe Tyr Met Leu Lys Gly Lys Leu
Gln Tyr Glu Leu Gln Tyr Arg Asn180 185 190Arg Gly Asp Pro Trp Ala
Val Ser Pro Arg Arg Lys Leu Ile Ser Val195 200 205Asp Ser Arg Ser
Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser210 215 220Ser Tyr
Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr225 230 235
240Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr
Gln245 250 255Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu
Leu Leu Leu260 265 270Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp
Ser Leu Lys Thr His275 280 285Pro Leu Trp Arg Leu Trp Lys Lys Ile
Trp Ala Val Pro Ser Pro Glu290 295 300Arg Phe Phe Met Pro Leu Tyr
Lys Gly Cys Ser Gly Asp Phe Lys Lys305 310 315 320Trp Val Gly Ala
Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly Pro Trp325 330 335Ser Pro
Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His Pro Pro340 345
350Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu Pro
Ala355 360 365Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe
Trp Pro Thr370 375 380Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu
Glu Arg Asp Arg Pro385 390 395 400Tyr Gly Leu Val Ser Ile Asp Thr
Val Thr Val Leu Asp Ala Glu Gly405 410 415Pro Cys Thr Trp Pro Cys
Ser Cys Glu Asp Asp Gly Tyr Pro Ala Leu420 425 430Asp Leu Asp Ala
Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp Pro Leu435 440 445Leu Asp
Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val Ser Ala Gly450 455
460Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu Asp Arg Leu
Lys465 470 475 480Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly
Leu Pro Trp Gly485 490 495Gly Arg Ser Pro Gly Gly Val Ser Glu Ser
Glu Ala Gly Ser Pro Leu500 505 510Ala Gly Leu Asp Met Asp Thr Phe
Asp Ser Gly Phe Val Gly Ser Asp515 520 525Cys Ser Ser Pro Val Glu
Cys Asp Phe Thr Ser Pro Gly Asp Glu Gly530 535 540Pro Pro Arg Ser
Tyr Leu Arg Gln Trp Val Val Ile Pro Pro Pro Leu545 550 555 560Ser
Ser Pro Gly Pro Gln Ala Ser56566741DNAArtificial SequenceDegenerate
polynucleotide sequence of SEQ ID NO69 66atggarmgna arytntgyws
nccnaarccn ccnccnacna argcnwsnyt nccnacngay 60ccnccnggnt ggggntgycc
ngayytngtn tgytayacng aytayytnca racngtnath 120tgyathytng
aratgtggaa yytncayccn wsnacnytna cnytnacntg gathytnwsn
180aayaayacng gntgytayat haargaymgn acnytngayy tnmgncarga
ycartaygar 240garytnaarg aygargcnac nwsntgywsn ytncaymgnw
sngcncayaa ygcnacncay 300gcnacntaya cntgycayat ggaygtntty
cayttyatgg cngaygayat httywsngtn 360aayathacng aycarwsngg
naaytaywsn cargartgyg gnwsnttyyt nytngcngar 420wsnmgncart
ayaayathws ntggmgnwsn gaytaygarg ayccngcntt ytayatgytn
480aarggnaary tncartayga rytncartay mgnaaymgng gngayccntg
ggcngtnwsn 540ccnmgnmgna arytnathws ngtngaywsn mgnwsngtnw
snytnytncc nytngartty 600mgnaargayw snwsntayga rytncargtn
mgngcnggnc cnatgccngg nwsnwsntay 660carggnacnt ggwsngartg
gwsngayccn gtnathttyc aracncarws ngargarytn 720aargarggnt
ggaayccnca y 741674PRTArtificial SequenceWXXW motif 67Trp Xaa Xaa
Trp168741DNAHomo sapiensCDS(1)...(741) 68atg gag agg aag ctc tgc
agt ccc aag cca ccc ccc acc aag gcc tct 48Met Glu Arg Lys Leu Cys
Ser Pro Lys Pro Pro Pro Thr Lys Ala Ser1 5 10 15ctc ccc act gac cct
cca ggc tgg ggc tgc ccc gac ctc gtc tgc tac 96Leu Pro Thr Asp Pro
Pro Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr20 25 30acc gat tac ctc
cag acg gtc atc tgc atc ctg gaa atg tgg aac ctc 144Thr Asp Tyr Leu
Gln Thr Val Ile Cys Ile Leu Glu Met Trp Asn Leu35 40 45cac ccc agc
acg ctc acc ctt acc tgg ata ctt tct aat aat act ggg 192His Pro Ser
Thr Leu Thr Leu Thr Trp Ile Leu Ser Asn Asn Thr Gly50 55 60tgc tat
atc aag gac aga aca ctg gac ctc agg caa gac cag tat gaa 240Cys Tyr
Ile Lys Asp Arg Thr Leu Asp Leu Arg Gln Asp Gln Tyr Glu65 70 75
80gag ctg aag gac gag gcc acc tcc tgc agc ctc cac agg tcg gcc cac
288Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His Arg Ser Ala
His85 90 95aat gcc acg cat gcc acc tac acc tgc cac atg gat gta ttc
cac ttc 336Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp Val Phe
His Phe100 105 110atg gcc gac gac att ttc agt gtc aac atc aca gac
cag tct ggc aac 384Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr Asp
Gln Ser Gly Asn115 120 125tac tcc cag gag tgt ggc agc ttt ctc ctg
gct gag agc aga cag tat 432Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu
Ala Glu Ser Arg Gln Tyr130 135 140aat atc tcc tgg cgc tca gat tac
gaa gac cct gcc ttc tac atg ctg 480Asn Ile Ser Trp Arg Ser Asp Tyr
Glu Asp Pro Ala Phe Tyr Met Leu145 150 155 160aag ggc aag ctt cag
tat gag ctg cag tac agg aac cgg gga gac ccc 528Lys Gly Lys Leu Gln
Tyr Glu Leu Gln Tyr Arg Asn Arg Gly Asp Pro165 170 175tgg gct gtg
agt ccg agg aga aag ctg atc tca gtg gac tca aga agt 576Trp Ala Val
Ser Pro Arg Arg Lys Leu Ile Ser Val Asp Ser Arg Ser180 185 190gtc
tcc ctc ctc ccc ctg gag ttc cgc aaa gac tcg agc tat gag ctg 624Val
Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser Tyr Glu Leu195 200
205cag gtg cgg gca ggg ccc atg cct ggc tcc tcc tac cag ggg acc tgg
672Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln Gly Thr
Trp210 215 220agt gaa tgg agt gac ccg gtc atc ttt cag acc cag tca
gag gag tta 720Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser
Glu Glu Leu225 230 235 240aag gaa ggc tgg aac cct cac 741Lys Glu
Gly Trp Asn Pro His24569247PRTHomo sapiens 69Met Glu Arg Lys Leu
Cys Ser Pro Lys Pro Pro Pro Thr Lys Ala Ser1 5 10 15Leu Pro Thr Asp
Pro Pro Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr20 25 30Thr Asp Tyr
Leu Gln Thr Val Ile Cys Ile Leu Glu Met Trp Asn Leu35 40 45His Pro
Ser Thr Leu Thr Leu Thr Trp Ile Leu Ser Asn Asn Thr Gly50 55 60Cys
Tyr Ile Lys Asp Arg Thr Leu Asp Leu Arg Gln Asp Gln Tyr Glu65 70 75
80Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His Arg Ser Ala
His85
90 95Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp Val Phe His
Phe100 105 110Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr Asp Gln
Ser Gly Asn115 120 125Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala
Glu Ser Arg Gln Tyr130 135 140Asn Ile Ser Trp Arg Ser Asp Tyr Glu
Asp Pro Ala Phe Tyr Met Leu145 150 155 160Lys Gly Lys Leu Gln Tyr
Glu Leu Gln Tyr Arg Asn Arg Gly Asp Pro165 170 175Trp Ala Val Ser
Pro Arg Arg Lys Leu Ile Ser Val Asp Ser Arg Ser180 185 190Val Ser
Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser Tyr Glu Leu195 200
205Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln Gly Thr
Trp210 215 220Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser
Glu Glu Leu225 230 235 240Lys Glu Gly Trp Asn Pro
His245704PRTArtificial SequenceDomain linker motif; PAPP motif
70Pro Ala Pro Pro171261PRTArtificial SequenceRepresentative variant
soluble receptor with domain linker 71Met Glu Arg Lys Leu Cys Ser
Pro Lys Pro Pro Pro Thr Lys Ala Ser1 5 10 15Leu Pro Thr Asp Pro Pro
Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr20 25 30Thr Asp Tyr Leu Gln
Thr Val Ile Cys Ile Leu Glu Met Trp Asn Leu35 40 45His Pro Ser Thr
Leu Thr Leu Thr Trp Ile Leu Ser Asn Asn Thr Gly50 55 60Cys Tyr Ile
Lys Asp Arg Thr Leu Asp Leu Arg Gln Asp Gln Tyr Glu65 70 75 80Glu
Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His Arg Ser Ala His85 90
95Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp Val Phe His
Phe100 105 110Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr Asp Gln
Ser Gly Asn115 120 125Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala
Glu Ser Xaa Xaa Pro130 135 140Ala Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Arg Gln Tyr Asn Ile145 150 155 160Ser Trp Arg Ser Asp Tyr
Glu Asp Pro Ala Phe Tyr Met Leu Lys Gly165 170 175Lys Leu Gln Tyr
Glu Leu Gln Tyr Arg Asn Arg Gly Asp Pro Trp Ala180 185 190Val Ser
Pro Arg Arg Lys Leu Ile Ser Val Asp Ser Arg Ser Val Ser195 200
205Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser Tyr Glu Leu Gln
Val210 215 220Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln Gly Thr
Trp Ser Glu225 230 235 240Trp Ser Asp Pro Val Ile Phe Gln Thr Gln
Ser Glu Glu Leu Lys Glu245 250 255Gly Trp Asn Pro
His260721461DNAArtificial SequenceMouse zalpha11 extracellular
domain murine immunoglobulin gamma 2a heavy chain Fc region fusion
protein (zalpha11m-mG2a) Polynucleotide 72atg gat gca atg aag aga
ggg ctc tgc tgt gtg ctg ctg ctg tgt ggc 48Met Asp Ala Met Lys Arg
Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15gcc gtc ttc gtt tcg
ctc agc cag aaa atc cat gcc gag ttg aga cgc 96Ala Val Phe Val Ser
Leu Ser Gln Lys Ile His Ala Glu Leu Arg Arg20 25 30ttc cgg aga tgc
ctg gac ctc act tgc tac act gac tac ctc tgg acc 144Phe Arg Arg Cys
Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp Thr35 40 45atc acc tgt
gtc ctg gag aca cgg agc ccc aac ccc agc ata ctc agt 192Ile Thr Cys
Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile Leu Ser50 55 60ctc acc
tgg caa gat gaa tat gag gaa ctt cag gac caa gag acc ttc 240Leu Thr
Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln Glu Thr Phe65 70 75
80tgc agc cta cac agg tct ggc cac aac acc aca cat ata tgg tac acg
288Cys Ser Leu His Arg Ser Gly His Asn Thr Thr His Ile Trp Tyr
Thr85 90 95tgc cat atg cgc ttg tct caa ttc ctg tcc gat gaa gtt ttc
att gtc 336Cys His Met Arg Leu Ser Gln Phe Leu Ser Asp Glu Val Phe
Ile Val100 105 110aat gtg acg gac cag tct ggc aac aac tcc caa gag
tgt ggc agc ttt 384Asn Val Thr Asp Gln Ser Gly Asn Asn Ser Gln Glu
Cys Gly Ser Phe115 120 125gtc ctg gct gag agc atc aaa cca gct ccc
ccc ttg aac gtg act gtg 432Val Leu Ala Glu Ser Ile Lys Pro Ala Pro
Pro Leu Asn Val Thr Val130 135 140gcc ttc tca gga cgc tat gat atc
tcc tgg gac tca gct tat gac gaa 480Ala Phe Ser Gly Arg Tyr Asp Ile
Ser Trp Asp Ser Ala Tyr Asp Glu145 150 155 160ccc tcc aac tac gtg
ctg agg ggc aag cta caa tat gag ctg cag tat 528Pro Ser Asn Tyr Val
Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr165 170 175cgg aac ctc
aga gac ccc tat gct gtg agg ccg gtg acc aag ctg atc 576Arg Asn Leu
Arg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu Ile180 185 190tca
gtg gac tca aga aac gtc tct ctt ctc cct gaa gag ttc cac aaa 624Ser
Val Asp Ser Arg Asn Val Ser Leu Leu Pro Glu Glu Phe His Lys195 200
205gat tct agc tac cag ctg cag gtg cgg gca gcg cct cag cca ggc act
672Asp Ser Ser Tyr Gln Leu Gln Val Arg Ala Ala Pro Gln Pro Gly
Thr210 215 220tca ttc agg ggg acc tgg agt gag tgg agt gac ccc gtc
atc ttt cag 720Ser Phe Arg Gly Thr Trp Ser Glu Trp Ser Asp Pro Val
Ile Phe Gln225 230 235 240acc cag gct ggg gag ccc gag gca ggc tgg
gac cct cac gag ccc aga 768Thr Gln Ala Gly Glu Pro Glu Ala Gly Trp
Asp Pro His Glu Pro Arg245 250 255tct ccc aca atc aag ccc tgt cct
cca tgc aaa tgc cca gca cct aac 816Ser Pro Thr Ile Lys Pro Cys Pro
Pro Cys Lys Cys Pro Ala Pro Asn260 265 270ctc ttg ggt gga cca tcc
gtc ttc atc ttc cct cca aag atc aag gat 864Leu Leu Gly Gly Pro Ser
Val Phe Ile Phe Pro Pro Lys Ile Lys Asp275 280 285gta ctc atg atc
tcc ctg agc ccc ata gtc aca tgt gtg gtg gtg gat 912Val Leu Met Ile
Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp290 295 300gtg agc
gag gat gac cca gat gtc cag atc agc tgg ttt gtg aac aac 960Val Ser
Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn305 310 315
320gtg gaa gta cac aca gct cag aca caa acc cat aga gag gat tac aac
1008Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr
Asn325 330 335agt act ctc cgg gtg gtc agt gcc ctc ccc atc cag cac
cag gac tgg 1056Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His
Gln Asp Trp340 345 350atg agt ggc aag gag ttc aaa tgc aag gtc aac
aac aaa gac ctc cca 1104Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn
Asn Lys Asp Leu Pro355 360 365gcg ccc atc gag aga acc atc tca aaa
ccc aaa ggg tca gta aga gct 1152Ala Pro Ile Glu Arg Thr Ile Ser Lys
Pro Lys Gly Ser Val Arg Ala370 375 380cca cag gta tat gtc ttg cct
cca cca gaa gaa gag atg act aag aaa 1200Pro Gln Val Tyr Val Leu Pro
Pro Pro Glu Glu Glu Met Thr Lys Lys385 390 395 400cag gtc act ctg
acc tgc atg gtc aca gac ttc atg cct gaa gac att 1248Gln Val Thr Leu
Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile405 410 415tac gtg
gag tgg acc aac aac ggg aaa aca gag cta aac tac aag aac 1296Tyr Val
Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn420 425
430act gaa cca gtc ctg gac tct gat ggt tct tac ttc atg tac agc aag
1344Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser
Lys435 440 445ctg aga gtg gaa aag aag aac tgg gtg gaa aga aat agc
tac tcc tgt 1392Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser
Tyr Ser Cys450 455 460tca gtg gtc cac gag ggt ctg cac aat cac cac
acg act aag agc ttc 1440Ser Val Val His Glu Gly Leu His Asn His His
Thr Thr Lys Ser Phe465 470 475 480tcc cgg act ccg ggt aaa taa
1461Ser Arg Thr Pro Gly Lys *48573486PRTArtificial SequenceMouse
zalpha11 extracellular domain murine immunoglobulin gamma 2a heavy
chain Fc region fusion protein (zalpha11m-mG2a) Polynucleotide
73Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1
5 10 15Ala Val Phe Val Ser Leu Ser Gln Lys Ile His Ala Glu Leu Arg
Arg20 25 30Phe Arg Arg Cys Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu
Trp Thr35 40 45Ile Thr Cys Val Leu Glu Thr Arg Ser Pro Asn Pro Ser
Ile Leu Ser50 55 60Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp
Gln Glu Thr Phe65 70 75 80Cys Ser Leu His Arg Ser Gly His Asn Thr
Thr His Ile Trp Tyr Thr85 90 95Cys His Met Arg Leu Ser Gln Phe Leu
Ser Asp Glu Val Phe Ile Val100 105 110Asn Val Thr Asp Gln Ser Gly
Asn Asn Ser Gln Glu Cys Gly Ser Phe115 120 125Val Leu Ala Glu Ser
Ile Lys Pro Ala Pro Pro Leu Asn Val Thr Val130 135 140Ala Phe Ser
Gly Arg Tyr Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu145 150 155
160Pro Ser Asn Tyr Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln
Tyr165 170 175Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro Val Thr
Lys Leu Ile180 185 190Ser Val Asp Ser Arg Asn Val Ser Leu Leu Pro
Glu Glu Phe His Lys195 200 205Asp Ser Ser Tyr Gln Leu Gln Val Arg
Ala Ala Pro Gln Pro Gly Thr210 215 220Ser Phe Arg Gly Thr Trp Ser
Glu Trp Ser Asp Pro Val Ile Phe Gln225 230 235 240Thr Gln Ala Gly
Glu Pro Glu Ala Gly Trp Asp Pro His Glu Pro Arg245 250 255Ser Pro
Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn260 265
270Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys
Asp275 280 285Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val
Val Val Asp290 295 300Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser
Trp Phe Val Asn Asn305 310 315 320Val Glu Val His Thr Ala Gln Thr
Gln Thr His Arg Glu Asp Tyr Asn325 330 335Ser Thr Leu Arg Val Val
Ser Ala Leu Pro Ile Gln His Gln Asp Trp340 345 350Met Ser Gly Lys
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro355 360 365Ala Pro
Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala370 375
380Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys
Lys385 390 395 400Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
Pro Glu Asp Ile405 410 415Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr
Glu Leu Asn Tyr Lys Asn420 425 430Thr Glu Pro Val Leu Asp Ser Asp
Gly Ser Tyr Phe Met Tyr Ser Lys435 440 445Leu Arg Val Glu Lys Lys
Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys450 455 460Ser Val Val His
Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe465 470 475 480Ser
Arg Thr Pro Gly Lys4857423DNAArtificial SequenceOligonucleotide
primer ZC26644 74ggggtcgacg gccggccacc atg 237535DNAArtificial
SequenceOligonucleotide primer ZC26641 75caagtgaggt ccaggcatct
ccggaagcgt ctcaa 357635DNAArtificial SequenceOligonucleotide primer
ZC26642 76ttgagacgct tccggagatg cctggacctc acttg
357736DNAArtificial SequenceOligonucleotide primer ZC26662
77tgtgggagat ctgggctcgt gagggtccca gcctgc 367830DNAArtificial
SequenceOligonucleotide primer ZC26643 78gagcccagat ctcccacaat
caagccctgt 307921DNAArtificial SequenceOligonucleotide primer
ZC26645 79aaacgcggcc gcggatccgg c 218035PRTHomo sapiens 80Met Asp
Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala
Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg20 25
30Phe Arg Arg3581966DNAHomo sapiensCDS(1)...(966) 81ggg ggc ggg ggc
gcc gcg cct acg gaa act cag cca cct gtg aca aat 48Gly Gly Gly Gly
Ala Ala Pro Thr Glu Thr Gln Pro Pro Val Thr Asn1 5 10 15ttg agt gtc
tct gtt gaa aac ctc tgc aca gta ata tgg aca tgg aat 96Leu Ser Val
Ser Val Glu Asn Leu Cys Thr Val Ile Trp Thr Trp Asn20 25 30cca ccc
gag gga gcc agc tca aat tgt agt cta tgg tat ttt agt cat 144Pro Pro
Glu Gly Ala Ser Ser Asn Cys Ser Leu Trp Tyr Phe Ser His35 40 45ttt
ggc gac aaa caa gat aag aaa ata gct ccg gaa act cgt cgt tca 192Phe
Gly Asp Lys Gln Asp Lys Lys Ile Ala Pro Glu Thr Arg Arg Ser50 55
60ata gaa gta ccc ctg aat gag agg att tgt ctg caa gtg ggg tcc cag
240Ile Glu Val Pro Leu Asn Glu Arg Ile Cys Leu Gln Val Gly Ser
Gln65 70 75 80tgt agc acc aat gag agt gag aag cct agc att ttg gtt
gaa aaa tgc 288Cys Ser Thr Asn Glu Ser Glu Lys Pro Ser Ile Leu Val
Glu Lys Cys85 90 95atc tca ccc cca gaa ggt gat cct gag tct gct gtg
act gag ctt caa 336Ile Ser Pro Pro Glu Gly Asp Pro Glu Ser Ala Val
Thr Glu Leu Gln100 105 110tgc att tgg cac aac ctg agc tac atg aag
tgt tct tgg ctc cct gga 384Cys Ile Trp His Asn Leu Ser Tyr Met Lys
Cys Ser Trp Leu Pro Gly115 120 125agg aat acc agt ccc gac act aac
tat act ctc tac tat tgg cac aga 432Arg Asn Thr Ser Pro Asp Thr Asn
Tyr Thr Leu Tyr Tyr Trp His Arg130 135 140agc ctg gaa aaa att cat
caa tgt gaa aac atc ttt aga gaa ggc caa 480Ser Leu Glu Lys Ile His
Gln Cys Glu Asn Ile Phe Arg Glu Gly Gln145 150 155 160tac ttt ggt
tgt tcc ttt gat ctg acc aaa gtg aag gat tcc agt ttt 528Tyr Phe Gly
Cys Ser Phe Asp Leu Thr Lys Val Lys Asp Ser Ser Phe165 170 175gaa
caa cac agt gtc caa ata atg gtc aag gat aat gca gga aaa att 576Glu
Gln His Ser Val Gln Ile Met Val Lys Asp Asn Ala Gly Lys Ile180 185
190aaa cca tcc ttc aat ata gtg cct tta act tcc cgt gtg aaa cct gat
624Lys Pro Ser Phe Asn Ile Val Pro Leu Thr Ser Arg Val Lys Pro
Asp195 200 205cct cca cat att aaa aac ctc tcc ttc cac aat gat gac
cta tat gtg 672Pro Pro His Ile Lys Asn Leu Ser Phe His Asn Asp Asp
Leu Tyr Val210 215 220caa tgg gag aat cca cag aat ttt att agc aga
tgc cta ttt tat gaa 720Gln Trp Glu Asn Pro Gln Asn Phe Ile Ser Arg
Cys Leu Phe Tyr Glu225 230 235 240gta gaa gtc aat aac agc caa act
gag aca cat aat gtt ttc tac gtc 768Val Glu Val Asn Asn Ser Gln Thr
Glu Thr His Asn Val Phe Tyr Val245 250 255caa gag gct aaa tgt gag
aat cca gaa ttt gag aga aat gtg gag aat 816Gln Glu Ala Lys Cys Glu
Asn Pro Glu Phe Glu Arg Asn Val Glu Asn260 265 270aca tct tgt ttc
atg gtc cct ggt gtt ctt cct gat act ttg aac aca 864Thr Ser Cys Phe
Met Val Pro Gly Val Leu Pro Asp Thr Leu Asn Thr275 280 285gtc aga
ata aga gtc aaa aca aat aag tta tgc tat gag gat gac aaa 912Val Arg
Ile Arg Val Lys Thr Asn Lys Leu Cys Tyr Glu Asp Asp Lys290 295
300ctc tgg agt aat tgg agc caa gaa atg agt ata ggt aag aag cgc aat
960Leu Trp Ser Asn Trp Ser Gln Glu Met Ser Ile Gly Lys Lys Arg
Asn305 310 315 320tcc aca 966Ser Thr82322PRTHomo sapiens 82Gly Gly
Gly Gly Ala Ala Pro Thr Glu Thr Gln Pro Pro Val Thr Asn1 5 10 15Leu
Ser Val Ser Val Glu Asn Leu Cys Thr Val Ile Trp Thr Trp Asn20 25
30Pro Pro Glu Gly Ala Ser Ser Asn Cys Ser Leu Trp Tyr Phe Ser His35
40 45Phe Gly Asp Lys Gln Asp Lys Lys Ile Ala Pro Glu Thr Arg Arg
Ser50 55 60Ile Glu Val Pro Leu Asn Glu Arg Ile Cys Leu Gln Val Gly
Ser Gln65 70 75 80Cys Ser Thr Asn Glu Ser Glu Lys Pro Ser Ile Leu
Val Glu Lys Cys85 90 95Ile Ser Pro Pro Glu Gly Asp Pro Glu Ser Ala
Val Thr Glu Leu Gln100 105 110Cys Ile Trp His Asn Leu Ser Tyr Met
Lys Cys Ser Trp Leu Pro Gly115 120 125Arg Asn Thr Ser Pro Asp Thr
Asn Tyr Thr Leu Tyr Tyr Trp His Arg130 135 140Ser Leu Glu Lys Ile
His Gln Cys Glu Asn Ile Phe Arg Glu Gly Gln145 150 155
160Tyr Phe Gly Cys Ser Phe Asp Leu Thr Lys Val Lys Asp Ser Ser
Phe165 170 175Glu Gln His Ser Val Gln Ile Met Val Lys Asp Asn Ala
Gly Lys Ile180 185 190Lys Pro Ser Phe Asn Ile Val Pro Leu Thr Ser
Arg Val Lys Pro Asp195 200 205Pro Pro His Ile Lys Asn Leu Ser Phe
His Asn Asp Asp Leu Tyr Val210 215 220Gln Trp Glu Asn Pro Gln Asn
Phe Ile Ser Arg Cys Leu Phe Tyr Glu225 230 235 240Val Glu Val Asn
Asn Ser Gln Thr Glu Thr His Asn Val Phe Tyr Val245 250 255Gln Glu
Ala Lys Cys Glu Asn Pro Glu Phe Glu Arg Asn Val Glu Asn260 265
270Thr Ser Cys Phe Met Val Pro Gly Val Leu Pro Asp Thr Leu Asn
Thr275 280 285Val Arg Ile Arg Val Lys Thr Asn Lys Leu Cys Tyr Glu
Asp Asp Lys290 295 300Leu Trp Ser Asn Trp Ser Gln Glu Met Ser Ile
Gly Lys Lys Arg Asn305 310 315 320Ser Thr83951DNAHomo
sapiensCDS(1)...(951) 83gac acc gag ata aaa gtt aac cct cct cag gat
ttt gag ata gtg gat 48Asp Thr Glu Ile Lys Val Asn Pro Pro Gln Asp
Phe Glu Ile Val Asp1 5 10 15ccc gga tac tta ggt tat ctc tat ttg caa
tgg caa ccc cca ctg tct 96Pro Gly Tyr Leu Gly Tyr Leu Tyr Leu Gln
Trp Gln Pro Pro Leu Ser20 25 30ctg gat cat ttt aag gaa tgc aca gtg
gaa tat gaa cta aaa tac cga 144Leu Asp His Phe Lys Glu Cys Thr Val
Glu Tyr Glu Leu Lys Tyr Arg35 40 45aac att ggt agt gaa aca tgg aag
acc atc att act aag aat cta cat 192Asn Ile Gly Ser Glu Thr Trp Lys
Thr Ile Ile Thr Lys Asn Leu His50 55 60tac aaa gat ggg ttt gat ctt
aac aag ggc att gaa gcg aag ata cac 240Tyr Lys Asp Gly Phe Asp Leu
Asn Lys Gly Ile Glu Ala Lys Ile His65 70 75 80acg ctt tta cca tgg
caa tgc aca aat gga tca gaa gtt caa agt tcc 288Thr Leu Leu Pro Trp
Gln Cys Thr Asn Gly Ser Glu Val Gln Ser Ser85 90 95tgg gca gaa act
act tat tgg ata tca cca caa gga att cca gaa act 336Trp Ala Glu Thr
Thr Tyr Trp Ile Ser Pro Gln Gly Ile Pro Glu Thr100 105 110aaa gtt
cag gat atg gat tgc gta tat tac aat tgg caa tat tta ctc 384Lys Val
Gln Asp Met Asp Cys Val Tyr Tyr Asn Trp Gln Tyr Leu Leu115 120
125tgt tct tgg aaa cct ggc ata ggt gta ctt ctt gat acc aat tac aac
432Cys Ser Trp Lys Pro Gly Ile Gly Val Leu Leu Asp Thr Asn Tyr
Asn130 135 140ttg ttt tac tgg tat gag ggc ttg gat cat gca tta cag
tgt gtt gat 480Leu Phe Tyr Trp Tyr Glu Gly Leu Asp His Ala Leu Gln
Cys Val Asp145 150 155 160tac atc aag gct gat gga caa aat ata gga
tgc aga ttt ccc tat ttg 528Tyr Ile Lys Ala Asp Gly Gln Asn Ile Gly
Cys Arg Phe Pro Tyr Leu165 170 175gag gca tca gac tat aaa gat ttc
tat att tgt gtt aat gga tca tca 576Glu Ala Ser Asp Tyr Lys Asp Phe
Tyr Ile Cys Val Asn Gly Ser Ser180 185 190gag aac aag cct atc aga
tcc agt tat ttc act ttt cag ctt caa aat 624Glu Asn Lys Pro Ile Arg
Ser Ser Tyr Phe Thr Phe Gln Leu Gln Asn195 200 205ata gtt aaa cct
ttg ccg cca gtc tat ctt act ttt act cgg gag agt 672Ile Val Lys Pro
Leu Pro Pro Val Tyr Leu Thr Phe Thr Arg Glu Ser210 215 220tca tgt
gaa att aag ctg aaa tgg agc ata cct ttg gga cct att cca 720Ser Cys
Glu Ile Lys Leu Lys Trp Ser Ile Pro Leu Gly Pro Ile Pro225 230 235
240gca agg tgt ttt gat tat gaa att gag atc aga gaa gat gat act acc
768Ala Arg Cys Phe Asp Tyr Glu Ile Glu Ile Arg Glu Asp Asp Thr
Thr245 250 255ttg gtg act gct aca gtt gaa aat gaa aca tac acc ttg
aaa aca aca 816Leu Val Thr Ala Thr Val Glu Asn Glu Thr Tyr Thr Leu
Lys Thr Thr260 265 270aat gaa acc cga caa tta tgc ttt gta gta aga
agc aaa gtg aat att 864Asn Glu Thr Arg Gln Leu Cys Phe Val Val Arg
Ser Lys Val Asn Ile275 280 285tat tgc tca gat gac gga att tgg agt
gag tgg agt gat aaa caa tgc 912Tyr Cys Ser Asp Asp Gly Ile Trp Ser
Glu Trp Ser Asp Lys Gln Cys290 295 300tgg gaa ggt gaa gac cta tcg
aag aaa act ttg cta cgt 951Trp Glu Gly Glu Asp Leu Ser Lys Lys Thr
Leu Leu Arg305 310 31584317PRTHomo sapiens 84Asp Thr Glu Ile Lys
Val Asn Pro Pro Gln Asp Phe Glu Ile Val Asp1 5 10 15Pro Gly Tyr Leu
Gly Tyr Leu Tyr Leu Gln Trp Gln Pro Pro Leu Ser20 25 30Leu Asp His
Phe Lys Glu Cys Thr Val Glu Tyr Glu Leu Lys Tyr Arg35 40 45Asn Ile
Gly Ser Glu Thr Trp Lys Thr Ile Ile Thr Lys Asn Leu His50 55 60Tyr
Lys Asp Gly Phe Asp Leu Asn Lys Gly Ile Glu Ala Lys Ile His65 70 75
80Thr Leu Leu Pro Trp Gln Cys Thr Asn Gly Ser Glu Val Gln Ser Ser85
90 95Trp Ala Glu Thr Thr Tyr Trp Ile Ser Pro Gln Gly Ile Pro Glu
Thr100 105 110Lys Val Gln Asp Met Asp Cys Val Tyr Tyr Asn Trp Gln
Tyr Leu Leu115 120 125Cys Ser Trp Lys Pro Gly Ile Gly Val Leu Leu
Asp Thr Asn Tyr Asn130 135 140Leu Phe Tyr Trp Tyr Glu Gly Leu Asp
His Ala Leu Gln Cys Val Asp145 150 155 160Tyr Ile Lys Ala Asp Gly
Gln Asn Ile Gly Cys Arg Phe Pro Tyr Leu165 170 175Glu Ala Ser Asp
Tyr Lys Asp Phe Tyr Ile Cys Val Asn Gly Ser Ser180 185 190Glu Asn
Lys Pro Ile Arg Ser Ser Tyr Phe Thr Phe Gln Leu Gln Asn195 200
205Ile Val Lys Pro Leu Pro Pro Val Tyr Leu Thr Phe Thr Arg Glu
Ser210 215 220Ser Cys Glu Ile Lys Leu Lys Trp Ser Ile Pro Leu Gly
Pro Ile Pro225 230 235 240Ala Arg Cys Phe Asp Tyr Glu Ile Glu Ile
Arg Glu Asp Asp Thr Thr245 250 255Leu Val Thr Ala Thr Val Glu Asn
Glu Thr Tyr Thr Leu Lys Thr Thr260 265 270Asn Glu Thr Arg Gln Leu
Cys Phe Val Val Arg Ser Lys Val Asn Ile275 280 285Tyr Cys Ser Asp
Asp Gly Ile Trp Ser Glu Trp Ser Asp Lys Gln Cys290 295 300Trp Glu
Gly Glu Asp Leu Ser Lys Lys Thr Leu Leu Arg305 310 31585519DNAHomo
sapiensCDS(1)...(519) 85atc acg tgc cct ccc ccc atg tcc gtg gaa cac
gca gac atc tgg gtc 48Ile Thr Cys Pro Pro Pro Met Ser Val Glu His
Ala Asp Ile Trp Val1 5 10 15aag agc tac agc ttg tac tcc agg gag cgg
tac att tgt aac tct ggt 96Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg
Tyr Ile Cys Asn Ser Gly20 25 30ttc aag cgt aaa gcc ggc acg tcc agc
ctg acg gag tgc gtg ttg aac 144Phe Lys Arg Lys Ala Gly Thr Ser Ser
Leu Thr Glu Cys Val Leu Asn35 40 45aag gcc acg aat gtc gcc cac tgg
aca acc ccc agt ctc aaa tgc att 192Lys Ala Thr Asn Val Ala His Trp
Thr Thr Pro Ser Leu Lys Cys Ile50 55 60aga gac cct gcc ctg gtt cac
caa agg cca gcg cca ccc tcc aca gta 240Arg Asp Pro Ala Leu Val His
Gln Arg Pro Ala Pro Pro Ser Thr Val65 70 75 80acg acg gca ggg gtg
acc cca cag cca gag agc ctc tcc cct tct gga 288Thr Thr Ala Gly Val
Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly85 90 95aaa gag ccc gca
gct tca tct ccc agc tca aac aac aca gcg gcc aca 336Lys Glu Pro Ala
Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr100 105 110aca gca
gct att gtc ccg ggc tcc cag ctg atg cct tca aaa tca cct 384Thr Ala
Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro115 120
125tcc aca gga acc aca gag ata agc agt cat gag tcc tcc cac ggc acc
432Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly
Thr130 135 140ccc tct cag aca aca gcc aag aac tgg gaa ctc aca gca
tcc gcc tcc 480Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala
Ser Ala Ser145 150 155 160cac cag ccg cca ggt gtg tat cca cag ggc
cac agc gac 519His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser
Asp165 17086173PRTHomo sapiens 86Ile Thr Cys Pro Pro Pro Met Ser
Val Glu His Ala Asp Ile Trp Val1 5 10 15Lys Ser Tyr Ser Leu Tyr Ser
Arg Glu Arg Tyr Ile Cys Asn Ser Gly20 25 30Phe Lys Arg Lys Ala Gly
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn35 40 45Lys Ala Thr Asn Val
Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile50 55 60Arg Asp Pro Ala
Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val65 70 75 80Thr Thr
Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly85 90 95Lys
Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr100 105
110Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser
Pro115 120 125Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser
His Gly Thr130 135 140Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu
Thr Ala Ser Ala Ser145 150 155 160His Gln Pro Pro Gly Val Tyr Pro
Gln Gly His Ser Asp165 170
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References