U.S. patent application number 11/829656 was filed with the patent office on 2008-09-04 for human cytokine receptor.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Zhi Chen, Steven D. Hughes, Wayne Kindsvogel, Scott R. Presnell, Wenfeng Xu.
Application Number | 20080214785 11/829656 |
Document ID | / |
Family ID | 23068131 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080214785 |
Kind Code |
A1 |
Presnell; Scott R. ; et
al. |
September 4, 2008 |
HUMAN CYTOKINE RECEPTOR
Abstract
Cytokines and their receptors have proven usefulness in both
basic research and as therapeutics. The present invention provides
a new human cytokine receptor designated as "Zcytor16."
Inventors: |
Presnell; Scott R.; (Tacoma,
WA) ; Xu; Wenfeng; (Mukilteo, WA) ;
Kindsvogel; Wayne; (Seattle, WA) ; Chen; Zhi;
(Seattle, WA) ; Hughes; Steven D.; (Seattle,
WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
23068131 |
Appl. No.: |
11/829656 |
Filed: |
July 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11829011 |
Jul 26, 2007 |
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11829656 |
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10968432 |
Oct 19, 2004 |
7265203 |
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11829011 |
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10104919 |
Mar 22, 2002 |
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10968432 |
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60279222 |
Mar 27, 2001 |
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Current U.S.
Class: |
530/351 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
31/00 20180101; A61P 17/06 20180101; A61P 29/00 20180101; A61K
38/00 20130101; A61P 35/00 20180101; A61P 37/08 20180101; A61P
39/02 20180101; A61P 19/02 20180101; C07K 14/715 20130101; A61P
31/04 20180101 |
Class at
Publication: |
530/351 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Claims
1. A heterodimeric receptor complex comprising an isolated soluble
cytokine receptor polypeptide, wherein the isolated soluble
cytokine receptor polypeptide comprises amino acid residues 28 to
231 of SEQ ID NO:2, and wherein the soluble cytokine receptor
polypeptide binds IL-TIF (SEQ ID NO:15) or antagonizes IL-TIF
activity, and further comprising a soluble zcytor11
polypeptide.
2. An isolated polypeptide according to claim 1, wherein the
heterodimeric receptor complex further comprises an affinity tag,
chemical moiety, toxin, or label.
3. A heterodimeric receptor complex comprising an isolated soluble
cytokine receptor polypeptide comprising amino acid residues 28 to
231 of SEQ ID NO:2, and a soluble zcytor11 polypeptide.
4. An isolated heterodimeric soluble receptor complex comprising a
first soluble cytokine receptor polypeptide and a second soluble
cytokine receptor polypeptide, wherein the first soluble cytokine
receptor polypeptide comprises amino acid residues 28 to 231 of SEQ
ID NO:2, and wherein the second soluble cytokine receptor
polypeptide comprises a soluble zcytor11 receptor polypeptide.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/829,011, filed on Jul. 26, 2007, which is a divisional of
U.S. application Ser. No. 10/968,432, filed Oct. 19, 2004, which is
a continuation of U.S. application Ser. No. 10/104,919, filed Mar.
22, 2002, which claims benefit of Provisional Application
60/279,222, filed on Mar. 27, 2001, all of which are incorporated
herein by reference. Under 35 U.S.C. .sctn. 119(e)(1), this
application claims benefit of said Provisional Application.
TECHNICAL FIELD
[0002] The present invention relates generally to a new protein
expressed by human cells. In particular, the present invention
relates to a novel gene that encodes a receptor, designated as
"Zcytor16," and to nucleic acid molecules encoding Zcytor16
polypeptides, and antibodies to the polypeptide.
BACKGROUND OF THE INVENTION
[0003] Cytokines are soluble, small proteins that mediate a variety
of biological effects, including the regulation of the growth and
differentiation of many cell types (see, for example, Arai et al,
Annu. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol
3:311 (1991); Paul and Seder, Cell 76:241 (1994)). Proteins that
constitute the cytokine group include interleukins, interferons,
colony stimulating factors, tumor necrosis factors, and other
regulatory molecules. For example, human interleukin-17 is a
cytokine which stimulates the expression of interleukin-6,
intracellular adhesion molecule 1, interleukin-8, granulocyte
macrophage colony-stimulating factor, and prostaglandin E2
expression, and plays a role in the preferential maturation of
CD34+ hematopoietic precursors into neutrophils (Yao et al, J.
Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593
(1996)).
[0004] Receptors that bind cytokines are typically composed of one
or more integral membrane proteins that bind the cytokine with high
affinity and transduce this binding event to the cell through the
cytoplasmic portions of the certain receptor subunits. Cytokine
receptors have been grouped into several classes on the basis of
similarities in their extracellular ligand binding domains. For
example, the receptor chains responsible for binding and/or
transducing the effect of interferons are members of the class II
cytokine receptor family, based upon a characteristic 200 residue
extracellular domain.
[0005] The demonstrated in vivo activities of cytokines and their
receptors illustrate the clinical potential of, and need for, other
cytokines, cytokine receptors, cytokine agonists, and cytokine
antagonists.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention provides a novel receptor, designated
"Zcytor16." The present invention also provides Zcytor16
polypeptides and Zcytor16 fusion proteins, as well as nucleic acid
molecules encoding such polypeptides and proteins, and methods for
using these nucleic acid molecules and amino acid sequences.
DETAILED DESCRIPTION OF THE INVENTION
1. Overview
[0007] An illustrative nucleotide sequence that encodes Zcytor16 is
provided by SEQ ID NO:1, and SEQ ID NO:37. The encoded polypeptide
has the following amino acid sequence: MMPKHCFLGF LISFFLTGVA
GTQSTHESLK PQRVQFQSRN FHNILQWQPG RALTGNSSVY FVQYKIYGQR QWKNKEDCWG
TQELSCDLTS ETSDIQEPYY GRVRAASAGS YSEWSMTPRF TPWWETKIDP PVMNITQVNG
SLLVILHAPN LPYRYQKEKN VSIEDYYELL YRVFIINNSL EKEQKVYEGA HRAVEIEALT
PHSSYCVVAE IYQPMLDRRS QRSEERCVEI P (SEQ ID NO:2). The 231 amino
acid polypeptide represents the extracellular domain, also called a
cytokine-binding domain, of a new class II cytokine receptor.
Features of the Zcytor16 polypeptide include putative signal
sequences at amino acid residues 1 to 21, or 1 to 22 of SEQ ID NO:2
(also shown in SEQ ID NO:38), and a mature soluble receptor
polypeptide from residues 23 to 231 or 23 to 231 of SEQ ID NO:2.
The receptor has two fibronectin III domains, also called
immunoglobulin superfamily (Ig) domains, characteristic of the
class II cytokine receptor family that comprise amino acid residues
32 to 123 (fibronectin III domain I), and 132 to 230 (fibronectin
III domain II) of SEQ ID NO:2, and a linker that resides between
the Ig domains (i.e., at amino acid residues 128-131 of SEQ ID
NO:2). Thus molecules of the present invention include polypeptides
that include a cytokine binding domain comprising amino acids 32 to
230 of SEQ ID NO:2. Moreover, additional variants of the zcytor16
polypeptide include polypeptides that comprise amino acid residues
28 to 123, 23 to 123, 22 to 123, or 32 to 127, 28 to 127, 23 to
127, 22 to 127, (fibronectin III domain I), and 132 to 230 or 231
(fibronectin III domain II) of SEQ ID NO:2, and a linker that
resides between the Ig domains (i.e., at amino acid residues
124-131, or 128-131 of SEQ ID NO:2). Thus molecules of the present
invention include polypeptides that include a cytokine binding
domain comprising amino acids 22, 23, or 28 to 132 to 230 or 231 of
SEQ ID NO:2. In addition, zcytor16 contains conserved motifs and
residues characteristic of class II cytokines: an SXWS (SEQ ID
NO:39) motif from residue 220-223 of SEQ ID NO:2; conserved
Tryptophan residues at residues 47, 72, and 114 of SEQ ID NO:2; and
conserved Cysteine residues at residues 78, 86, 206, and 227 of SEQ
ID NO:2. The Zcytor16 gene is expressed in monocytes, lymphoid,
placenta, spleen, tonsil and other tissues, and resides in human
chromosome 6q23-q24. The full-length mRNA, and consequently the
cDNA, is shown in SEQ ID NO:37 that, upstream of the initiating
methionine (nucleotide 237 of SEQ ID NO:37), includes a 5'
untranslated region (UTR) (nucleotides 1-236 of SEQ ID NO:37) that
includes a stop codon (TGA) embedded in a consensus Kozak sequence
typical of translation in vertebrates. Moreover, the 3' UTR
(nucleotides 933-1610 of SEQ ID NO:37) contains an mRNA stability
motif (e.g., nucleotides 1458-1465 of SEQ ID NO:37) and a polyA
tail.
[0008] As described below, the present invention provides isolated
polypeptides comprising an amino acid sequence that is at least
70%, at least 80%, or at least 90% identical to a reference amino
acid sequence of SEQ ID NO:2 selected from the group consisting of:
(a) amino acid residues 28 to 127; (b) amino acid residues 132 to
231; (c) amino acid residues 28 to 231; (d) amino acid residues 23
to 230; (e) amino acid residues 23 to 231; (f) amino acid residues
22 to 230; (g) amino acid residues 22 to 231; and (h) amino acid
residues 1 to 231, wherein the isolated polypeptide specifically
binds with an antibody that specifically binds with a polypeptide
consisting of the amino acid sequence of SEQ ID NO:2. Illustrative
polypeptides include polypeptides comprising either amino acid
residues 22 to 231 of SEQ ID NO:2 or amino acid residues 28 to 231
of SEQ ID NO:2. Moreover, the present invention also provides
isolated polypeptides as disclosed above that bind IL-TIF (e.g.,
human IL-TIF polypeptide sequence as shown in SEQ ID NO:15). The
human IL-TIF polynucleotide sequence is shown in SEQ ID NO: 14. The
mouse IL-TIF polynucleotide sequence is shown in SEQ ID NO:42, and
corresponding polypeptide is shown in SEQ ID NO:43.
[0009] The present invention also provides isolated polypeptides
comprising at least 15 contiguous amino acid residues of an amino
acid sequence of SEQ ID NO:2 selected from the group consisting of:
(a) amino acid residues 28 to 127; (b) amino acid residues 132 to
231; (c) amino acid residues 28 to 231; (d) amino acid residues 23
to 230; (e) amino acid residues 23 to 231; (f) amino acid residues
22 to 230; (g) amino acid residues 22 to 231; and (h) amino acid
residues 1 to 231. Illustrative polypeptides include polypeptides
that either comprise, or consist of, amino acid residues (a) to
(h). Moreover, the present invention also provides isolated
polypeptides as disclosed above that bind IL-TIF.
[0010] The present invention also includes variant Zcytor16
polypeptides, wherein the amino acid sequence of the variant
polypeptide shares an identity with amino acid residues 22 to 231,
or 28 to 231 of SEQ ID NO:2 selected from the group consisting of
at least 70% identity, at least 80% identity, at least 90%
identity, at least 95% identity, or greater than 95% identity, and
wherein any difference between the amino acid sequence of the
variant polypeptide and the corresponding amino acid sequence of
SEQ ID NO:2 is due to one or more conservative amino acid
substitutions. Moreover, the present invention also provides
isolated polypeptides as disclosed above that bind IL-TIF.
[0011] The present invention further provides antibodies and
antibody fragments that specifically bind with such polypeptides.
Exemplary antibodies include polyclonal antibodies, murine
monoclonal antibodies, humanized antibodies derived from murine
monoclonal antibodies, and human monoclonal antibodies.
Illustrative antibody fragments include F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, scFv, and minimal recognition units. The present
invention further includes compositions comprising a carrier and a
peptide, polypeptide, or antibody described herein.
[0012] The present invention also provides isolated nucleic acid
molecules that encode a Zcytor16 polypeptide, wherein the nucleic
acid molecule is selected from the group consisting of: (a) a
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO:3, (b) a nucleic acid molecule encoding an amino acid sequence
that comprises either amino acid residues 22 to 231 of SEQ ID NO:2
or amino acid residues 28 to 231 of SEQ ID NO:2, and (c) a nucleic
acid molecule that remains hybridized following stringent wash
conditions to a nucleic acid molecule comprising the nucleotide
sequence of nucleotides 64 or 82, to 690 or 693 of SEQ ID NO:1, or
the complement of the nucleotide sequence of nucleotides 64 or 82,
to 690 or 693 of SEQ ID NO:1. Illustrative nucleic acid molecules
include those in which any difference between the amino acid
sequence encoded by nucleic acid molecule (c) and the corresponding
amino acid sequence of SEQ ID NO:2 is due to a conservative amino
acid substitution. The present invention further contemplates
isolated nucleic acid molecules that comprise nucleotides 64, 67,
82, or 94 to 690 or 693 of SEQ ID NO:1. Moreover, the present
invention also provides isolated polynucleotides that encode
polypeptides as disclosed above that bind IL-TIF.
[0013] The present invention also includes vectors and expression
vectors comprising such nucleic acid molecules. Such expression
vectors may comprise a transcription promoter, and a transcription
terminator, wherein the promoter is operably linked with the
nucleic acid molecule, and wherein the nucleic acid molecule is
operably linked with the transcription terminator. The present
invention further includes recombinant host cells and recombinant
viruses comprising these vectors and expression vectors.
Illustrative host cells include bacterial, yeast, fungal, insect,
mammalian, and plant cells. Recombinant host cells comprising such
expression vectors can be used to produce Zcytor16 polypeptides by
culturing such recombinant host cells that comprise the expression
vector and that produce the Zcytor16 protein, and, optionally,
isolating the Zcytor16 protein from the cultured recombinant host
cells.
[0014] In addition, the present invention provides pharmaceutical
compositions comprising a pharmaceutically acceptable carrier and
at least one of such an expression vector or recombinant virus
comprising such expression vectors. The present invention further
includes pharmaceutical compositions, comprising a pharmaceutically
acceptable carrier and a polypeptide described herein.
[0015] The present invention also contemplates methods for
detecting the presence of Zcytor16 RNA in a biological sample,
comprising the steps of (a) contacting a Zcytor16 nucleic acid
probe under hybridizing conditions with either (i) test RNA
molecules isolated from the biological sample, or (ii) nucleic acid
molecules synthesized from the isolated RNA molecules, wherein the
probe has a nucleotide sequence comprising a portion of the
nucleotide sequence of SEQ ID NO:1, or its complement, and (b)
detecting the formation of hybrids of the nucleic acid probe and
either the test RNA molecules or the synthesized nucleic acid
molecules, wherein the presence of the hybrids indicates the
presence of Zcytor16 RNA in the biological sample. For example,
suitable probes consist of the following nucleotide sequences of
SEQ ID NO:1: nucleotides 64, 67, 82 or 94 to 690 or 693;
nucleotides 64, 67, 82 or 94 to 369 or 381; 394 to 690 or 693; and
nucleotides 1 to 690 or 693. Other suitable probes consist of the
complement of these nucleotide sequences, or a portion of the
nucleotide sequences or their complements.
[0016] The present invention further provides methods for detecting
the presence of Zcytor16 polypeptide in a biological sample,
comprising the steps of: (a) contacting the biological sample with
an antibody or an antibody fragment that specifically binds with a
polypeptide consisting of the amino acid sequence of SEQ ID NO:2,
wherein the contacting is performed under conditions that allow the
binding of the antibody or antibody fragment to the biological
sample, and (b) detecting any of the bound antibody or bound
antibody fragment. Such an antibody or antibody fragment may
further comprise a detectable label selected from the group
consisting of radioisotope, fluorescent label, chemiluminescent
label, enzyme label, bioluminescent label, and colloidal gold.
[0017] The present invention also provides kits for performing
these detection methods. For example, a kit for detection of
Zcytor16 gene expression may comprise a container that comprises a
nucleic acid molecule, wherein the nucleic acid molecule is
selected from the group consisting of (a) a nucleic acid molecule
comprising the nucleotide sequence of nucleotides 64, 67, 82, or 94
to 693 of SEQ ID NO:1, (b) a nucleic acid molecule comprising the
complement of nucleotides 64, 67, 82, or 94 to 693 of the
nucleotide sequence of SEQ ID NO:1, (c) a nucleic acid molecule
that is a fragment of (a) consisting of at least eight nucleotides,
and (d) a nucleic acid molecule that is a fragment of (b)
consisting of at least eight nucleotides. Such a kit may also
comprise a second container that comprises one or more reagents
capable of indicating the presence of the nucleic acid molecule. On
the other hand, a kit for detection of Zcytor16 protein may
comprise a container that comprises an antibody, or an antibody
fragment, that specifically binds with a polypeptide consisting of
the amino acid sequence of SEQ ID NO:2.
[0018] The present invention also contemplates anti-idiotype
antibodies, or anti-idiotype antibody fragments, that specifically
bind an antibody or antibody fragment that specifically binds a
polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
An exemplary anti-idiotype antibody binds with an antibody that
specifically binds a polypeptide consisting of amino acid residues
22 to 231, or 28 to 231 of SEQ ID NO:2.
[0019] The present invention also provides isolated nucleic acid
molecules comprising a nucleotide sequence that encodes a Zcytor16
secretion signal sequence and a nucleotide sequence that encodes a
biologically active polypeptide, wherein the Zcytor16 secretion
signal sequence comprises an amino acid sequence of residues 1 to
21, of SEQ ID NO:2. Illustrative biologically active polypeptides
include Factor VIIa, proinsulin, insulin, follicle stimulating
hormone, tissue type plasminogen activator, tumor necrosis factor,
interleukin, colony stimulating factor, interferon, erythropoietin,
and thrombopoietin. Moreover, the present invention provides fusion
proteins comprising a Zcytor16 secretion signal sequence and a
polypeptide, wherein the Zcytor16 secretion signal sequence
comprises an amino acid sequence of residues 1 to 21, of SEQ ID
NO:2.
[0020] The present invention also provides fusion proteins,
comprising a Zcytor16 polypeptide and an immunoglobulin moiety. In
such fusion proteins, the immunoglobulin moiety may be an
immunoglobulin heavy chain constant region, such as a human F.sub.c
fragment. The present invention further includes isolated nucleic
acid molecules that encode such fusion proteins.
[0021] The present invention also provides monomeric, homodimeric,
heterodimeric and multimeric receptors comprising a zcytor16
extracellular domain. Such receptors are soluble or membrane bound,
and act as antagonists of the zcytor16 ligand, IL-TIF (e.g., the
human IL-TIF as shown in SEQ ID NO:15). In a preferred embodiment,
such receptors are soluble receptors comprising at least one
zcytor16 extracellular domain polypeptide comprising amino acids
22-231, or 22-210 of SEQ ID NO:2. The present invention further
includes isolated nucleic acid molecules that encode such receptor
polypeptides.
[0022] The present invention also provides polyclonal and
monoclonal antibodies to monomeric, homodimeric, heterodimeric and
multimeric receptors comprising a zcytor16 extracellular domain
such as those described above. Moreover, such antibodies can be
used antagonize the binding to the zcytor16 ligand, IL-TIF (SEQ ID
NO:15), to the zcytor16 receptor.
[0023] The present invention also provides a method for detecting a
genetic abnormality in a patient, comprising: obtaining a genetic
sample from a patient; producing a first reaction product by
incubating the genetic sample with a polynucleotide comprising at
least 14 contiguous nucleotides of SEQ ID NO:1 or the complement of
SEQ ID NO:1, under conditions wherein said polynucleotide will
hybridize to complementary polynucleotide sequence; visualizing the
first reaction product; and comparing said first reaction product
to a control reaction product from a wild type patient, wherein a
difference between said first reaction product and said control
reaction product is indicative of a genetic abnormality in the
patient.
[0024] The present invention also provides a method for detecting a
cancer in a patient, comprising: obtaining a tissue or biological
sample from a patient; incubating the tissue or biological sample
with an antibody as described above under conditions wherein the
antibody binds to its complementary polypeptide in the tissue or
biological sample; visualizing the antibody bound in the tissue or
biological sample; and comparing levels of antibody bound in the
tissue or biological sample from the patient to a normal control
tissue or biological sample, wherein an increase in the level of
antibody bound to the patient tissue or biological sample relative
to the normal control tissue or biological sample is indicative of
a cancer in the patient.
[0025] The present invention also provides a method for detecting a
cancer in a patient, comprising: obtaining a tissue or biological
sample from a patient; labeling a polynucleotide comprising at
least 14 contiguous nucleotides of SEQ ID NO:1 or the complement of
SEQ ID NO:1; incubating the tissue or biological sample with under
conditions wherein the polynucleotide will hybridize to
complementary polynucleotide sequence; visualizing the labeled
polynucleotide in the tissue or biological sample; and comparing
the level of labeled polynucleotide hybridization in the tissue or
biological sample from the patient to a normal control tissue or
biological sample, wherein an increase in the labeled
polynucleotide hybridization to the patient tissue or biological
sample relative to the normal control tissue or biological sample
is indicative of a cancer in the patient.
[0026] These and other aspects of the invention will become evident
upon reference to the following detailed description. In addition,
various references are identified below and are incorporated by
reference in their entirety.
2. Definitions
[0027] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0028] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), oligonucleotides, fragments generated by
the polymerase chain reaction (PCR), and fragments generated by any
of ligation, scission, endonuclease action, and exonuclease action.
Nucleic acid molecules can be composed of monomers that are
naturally-occurring nucleotides (such as DNA and RNA), or analogs
of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally-occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0029] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence. For example, the sequence 5' ATGCACGGG 3' is
complementary to 5' CCCGTGCAT 3'.
[0030] The term "contig" denotes a nucleic acid molecule that has a
contiguous stretch of identical or complementary sequence to
another nucleic acid molecule. Contiguous sequences are said to
"overlap" a given stretch of a nucleic acid molecule either in
their entirety or along a partial stretch of the nucleic acid
molecule.
[0031] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons as
compared to a reference nucleic acid 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).
[0032] The term "structural gene" refers to a nucleic acid molecule
that is transcribed into messenger RNA (mRNA), which is then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0033] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0034] A "nucleic acid molecule construct" is a nucleic acid
molecule, either single- or double-stranded, that has been modified
through human intervention to contain segments of nucleic acid
combined and juxtaposed in an arrangement not existing in
nature.
[0035] "Linear DNA" denotes non-circular DNA molecules having free
5' and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0036] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0037] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, CAAT sequences, differentiation-specific elements (DSEs;
McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclic AMP response
elements (CREs), serum response elements (SREs; Treisman, Seminars
in Cancer Biol. 1.47 (1990)), glucocorticoid response elements
(GREs), and binding sites for other transcription factors, such as
CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye
et al., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response
element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and
octamer factors (see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1
(1994)). If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing
agent if the promoter is a constitutive promoter. Repressible
promoters are also known.
[0038] A "core promoter" contains essential nucleotide sequences
for promoter function, including the TATA box and start of
transcription. By this definition, a core promoter may or may not
have detectable activity in the absence of specific sequences that
may enhance the activity or confer tissue specific activity.
[0039] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter. For example, a
regulatory element may contain a nucleotide sequence that binds
with cellular factors enabling transcription exclusively or
preferentially in particular cells, tissues, or organelles. These
types of regulatory elements are normally associated with genes
that are expressed in a "cell-specific," "tissue-specific," or
"organelle-specific" manner.
[0040] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0041] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0042] 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."
[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] A peptide or polypeptide encoded by a non-host DNA molecule
is a "heterologous" peptide or polypeptide.
[0045] An "integrated genetic element" is a segment of DNA that has
been incorporated into a chromosome of a host cell after that
element is introduced into the cell through human manipulation.
Within the present invention, integrated genetic elements are most
commonly derived from linearized plasmids that are introduced into
the cells by electroporation or other techniques. Integrated
genetic elements are passed from the original host cell to its
progeny.
[0046] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage, that has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide tetracycline resistance
or ampicillin resistance.
[0047] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcription promoter, a gene, and a
transcription terminator. Gene expression is usually placed under
the control of a promoter, and such a gene is said to be "operably
linked to" the promoter. Similarly, a regulatory element and a core
promoter are operably linked if the regulatory element modulates
the activity of the core promoter.
[0048] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector. In the present context, an example of a recombinant host is
a cell that produces Zcytor16 from an expression vector. In
contrast, Zcytor16 can be produced by a cell that is a "natural
source" of Zcytor16, and that lacks an expression vector.
[0049] "Integrative transformants" are recombinant host cells, in
which heterologous DNA has become integrated into the genomic DNA
of the cells.
[0050] A "fusion protein" is a hybrid protein expressed by a
nucleic acid molecule comprising nucleotide sequences of at least
two genes. For example, a fusion protein can comprise at least part
of a Zcytor16 polypeptide fused with a polypeptide that binds an
affinity matrix. Such a fusion protein provides a means to isolate
large quantities of Zcytor16 using affinity chromatography.
[0051] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule termed a "ligand." This interaction
mediates the effect of the ligand on the cell. 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). Membrane-bound receptors are characterized by a
multi-domain structure comprising an extracellular ligand-binding
domain and an intracellular effector domain that is typically
involved in signal transduction. In certain membrane-bound
receptors, the extracellular ligand-binding domain and the
intracellular effector domain are located in separate polypeptides
that comprise the complete functional receptor.
[0052] In general, the binding of ligand to receptor results in a
conformational change in the receptor that causes an interaction
between the effector domain and other molecule(s) in the cell,
which in turn leads to an alteration in the metabolism of the cell.
Metabolic events that are often linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, increases in cyclic AMP production, mobilization
of cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids.
[0053] A "soluble receptor" is a receptor polypeptide that is not
bound to a cell membrane. Soluble receptors are most commonly
ligand-binding receptor polypeptides that lack transmembrane and
cytoplasmic domains, and other linkage to the cell membrane such as
via glycophosphoinositol (gpi). 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 or
translated from alternatively spliced mRNAs. Soluble receptors can
be monomeric, homodimeric, heterodimeric, or multimeric, with
multimeric receptors generally not comprising more than 9 subunits,
preferably not comprising more than 6 subunits, and most preferably
not comprising more than 3 subunits. 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. Soluble receptors of class I and class II cytokine
receptors generally comprise the extracellular cytokine binding
domain free of a transmembrane domain and intracellular domain. For
example, representative soluble receptors include a soluble
receptor for CRF2-4 (Genbank Accession No. Z17227) as shown in SEQ
ID NO:35; a soluble receptor for IL-10R (Genbank Accession No.s
U00672 and NM.sub.--001558) as shown in SEQ ID NO:36; and a soluble
receptor for zcytor11 (U.S. Pat. No. 5,965,704) as shown in SEQ ID
NO:34. It is well within the level of one of skill in the art to
delineate what sequences of a known class I or class II cytokine
sequence comprise the extracellular cytokine binding domain free of
a transmembrane domain and intracellular domain. Moreover, one of
skill in the art using the genetic code can readily determine
polynucleotides that encode such soluble receptor polypeptides.
[0054] The term "secretory signal sequence" denotes a DNA sequence
that encodes a peptide (a "secretory peptide") that, as a component
of a larger polypeptide, directs the larger polypeptide through a
secretory pathway of a cell in which it is synthesized. The larger
polypeptide is commonly cleaved to remove the secretory peptide
during transit through the secretory pathway.
[0055] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, or greater than 99%
pure. One way to show that a particular protein preparation
contains an isolated polypeptide is by the appearance of a single
band following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis of the protein preparation and Coomassie Brilliant
Blue staining of the gel. However, 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.
[0056] 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.
[0057] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0058] 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 polypeptide encoded by a
splice variant of an mRNA transcribed from a gene.
[0059] As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, co-stimulatory
molecules, hematopoietic factors, and synthetic analogs of these
molecules.
[0060] 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 less than 10.sup.9 M.sup.-1.
[0061] An "anti-idiotype antibody" is an antibody that binds with
the variable region domain of an immunoglobulin. In the present
context, an anti-idiotype antibody binds with the variable region
of an anti-Zcytor16 antibody, and thus, an anti-idiotype antibody
mimics an epitope of Zcytor16.
[0062] An "antibody fragment" is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. For example, an anti-Zcytor16
monoclonal antibody fragment binds with an epitope of Zcytor16.
[0063] The term "antibody fragment" also includes a synthetic or a
genetically engineered polypeptide that binds to a specific
antigen, such as polypeptides consisting of the light chain
variable region, "Fv" fragments consisting of the variable regions
of the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker ("scFv proteins"), and minimal recognition
units consisting of the amino acid residues that mimic the
hypervariable region.
[0064] A "chimeric antibody" is a recombinant protein that contains
the variable domains and complementary determining regions derived
from a rodent antibody, while the remainder of the antibody
molecule is derived from a human antibody.
[0065] "Humanized antibodies" are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain.
[0066] As used herein, a "therapeutic agent" is a molecule or atom
which is conjugated to an antibody moiety to produce a conjugate
which is useful for therapy. Examples of therapeutic agents include
drugs, toxins, immunomodulators, chelators, boron compounds,
photoactive agents or dyes, and radioisotopes.
[0067] A "detectable label" is a molecule or atom which can be
conjugated to an antibody moiety to produce a molecule useful for
diagnosis. Examples of detectable labels include chelators,
photoactive agents, radioisotopes, fluorescent agents, paramagnetic
ions, or other marker moieties.
[0068] 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 (1985)), substance P, FLAG
peptide (Hopp et al., Biotechnology 6:1204 (1988)), streptavidin
binding peptide, or other antigenic epitope or binding domain. See,
in general, Ford et al., Protein Expression and Purification 2:95
(1991). DNA molecules encoding affinity tags are available from
commercial suppliers (e.g., Pharmacia Biotech, Piscataway,
N.J.).
[0069] A "naked antibody" is an entire antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic
agent. Naked antibodies include both polyclonal and monoclonal
antibodies, as well as certain recombinant antibodies, such as
chimeric and humanized antibodies.
[0070] As used herein, the term "antibody component" includes both
an entire antibody and an antibody fragment.
[0071] An "immunoconjugate" is a conjugate of an antibody component
with a therapeutic agent or a detectable label.
[0072] As used herein, the term "antibody fusion protein" refers to
a recombinant molecule that comprises an antibody component and a
Zcytor16 polypeptide component. Examples of an antibody fusion
protein include a protein that comprises a Zcytor16 extracellular
domain, and either an Fc domain or an antigen-biding region.
[0073] A "target polypeptide" or a "target peptide" is an amino
acid sequence that comprises at least one epitope, and that is
expressed on a target cell, such as a tumor cell, or a cell that
carries an infectious agent antigen. T cells recognize peptide
epitopes presented by a major histocompatibility complex molecule
to a target polypeptide or target peptide and typically lyse the
target cell or recruit other immune cells to the site of the target
cell, thereby killing the target cell.
[0074] An "antigenic peptide" is a peptide which will bind a major
histocompatibility complex molecule to form an MHC-peptide complex
which is recognized by a T cell, thereby inducing a cytotoxic
lymphocyte response upon presentation to the T cell. Thus,
antigenic peptides are capable of binding to an appropriate major
histocompatibility complex molecule and inducing a cytotoxic T
cells response, such as cell lysis or specific cytokine release
against the target cell which binds or expresses the antigen. The
antigenic peptide can be bound in the context of a class I or class
II major histocompatibility complex molecule, on an antigen
presenting cell or on a target cell.
[0075] In eukaryotes, RNA polymerase II catalyzes the transcription
of a structural gene to produce mRNA. A nucleic acid molecule can
be designed to contain an RNA polymerase II template in which the
RNA transcript has a sequence that is complementary to that of a
specific mRNA. The RNA transcript is termed an "anti-sense RNA" and
a nucleic acid molecule that encodes the anti-sense RNA is termed
an "anti-sense gene." Anti-sense RNA molecules are capable of
binding to mRNA molecules, resulting in an inhibition of mRNA
translation.
[0076] An "anti-sense oligonucleotide specific for Zcytor16" or a
"Zcytor16 anti-sense oligonucleotide" is an oligonucleotide having
a sequence (a) capable of forming a stable triplex with a portion
of the Zcytor16 gene, or (b) capable of forming a stable duplex
with a portion of an mRNA transcript of the Zcytor16 gene.
[0077] A "ribozyme" is a nucleic acid molecule that contains a
catalytic center. The term includes RNA enzymes, self-splicing
RNAs, self-cleaving RNAs, and nucleic acid molecules that perform
these catalytic functions. A nucleic acid molecule that encodes a
ribozyme is termed a "ribozyme gene."
[0078] An "external guide sequence" is a nucleic acid molecule that
directs the endogenous ribozyme, RNase P, to a particular species
of intracellular mRNA, resulting in the cleavage of the mRNA by
RNase P. A nucleic acid molecule that encodes an external guide
sequence is termed an "external guide sequence gene."
[0079] The term "variant Zcytor16 gene" refers to nucleic acid
molecules that encode a polypeptide having an amino acid sequence
that is a modification of SEQ ID NO:2. Such variants include
naturally-occurring polymorphisms of Zcytor16 genes, as well as
synthetic genes that contain conservative amino acid substitutions
of the amino acid sequence of SEQ ID NO:2. Additional variant forms
of Zcytor16 genes are nucleic acid molecules that contain
insertions or deletions of the nucleotide sequences described
herein. A variant Zcytor16 gene can be identified, for example, by
determining whether the gene hybridizes with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1, or its
complement, under stringent conditions.
[0080] Alternatively, variant Zcytor16 genes can be identified by
sequence comparison. Two amino acid sequences have "100% amino acid
sequence identity" if the amino acid residues of the two amino acid
sequences are the same when aligned for maximal correspondence.
Similarly, two nucleotide sequences have "100% nucleotide sequence
identity" if the nucleotide residues of the two nucleotide
sequences are the same when aligned for maximal correspondence.
Sequence comparisons can be performed using standard software
programs such as those included in the LASERGENE bioinformatics
computing suite, which is produced by DNASTAR (Madison, Wis.).
Other methods for comparing two nucleotide or amino acid sequences
by determining optimal alignment are well-known to those of skill
in the art (see, for example, Peruski and Peruski, The Internet and
the New Biology Tools for Genomic and Molecular Research (ASM
Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and
Computer Databases of Nucleic Acids and Proteins," in Methods in
Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and
Bishop (ed.), Guide to Human Genome Computing, 2nd Edition
(Academic Press, Inc. 1998)). Particular methods for determining
sequence identity are described below.
[0081] Regardless of the particular method used to identify a
variant Zcytor16 gene or variant Zcytor16 polypeptide, a variant
gene or polypeptide encoded by a variant gene may be functionally
characterized the ability to bind specifically to an anti-Zcytor16
antibody. A variant Zcytor16 gene or variant Zcytor16 polypeptide
may also be functionally characterized the ability to bind to its
ligand, IL-TIF, using a biological or biochemical assay described
herein.
[0082] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0083] 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.
[0084] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0085] The present invention includes functional fragments of
Zcytor16 genes. Within the context of this invention, a "functional
fragment" of a Zcytor16 gene refers to a nucleic acid molecule that
encodes a portion of a Zcytor16 polypeptide which is a domain
described herein or at least specifically binds with an
anti-Zcytor16 antibody.
[0086] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are 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%.
3. Production of Zcytor16 Polynucleotides or Genes
[0087] Nucleic acid molecules encoding a human Zcytor16 gene can be
obtained by screening a human cDNA or genomic library using
polynucleotide probes based upon SEQ ID NO:1 or SEQ ID NO:37. These
techniques are standard and well-established.
[0088] As an illustration, a nucleic acid molecule that encodes a
human Zcytor16 gene can be isolated from a cDNA library. In this
case, the first step would be to prepare the cDNA library by
isolating RNA from a tissue, such as tonsil tissue, using methods
well-known to those of skill in the art. In general, RNA isolation
techniques must provide a method for breaking cells, a means of
inhibiting RNase-directed degradation of RNA, and a method of
separating RNA from DNA, protein, and polysaccharide contaminants.
For example, total RNA can be isolated by freezing tissue in liquid
nitrogen, grinding the frozen tissue with a mortar and pestle to
lyse the cells, extracting the ground tissue with a solution of
phenol/chloroform to remove proteins, and separating RNA from the
remaining impurities by selective precipitation with lithium
chloride (see, for example, Ausubel et al. (eds.), Short Protocols
in Molecular Biology, 3.sup.rd Edition, pages 4-1 to 4-6 (John
Wiley & Sons 1995) ["Ausubel (1995)"]; Wu et al., Methods in
Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu
(1997)"]).
[0089] Alternatively, total RNA can be isolated by extracting
ground tissue with guanidinium isothiocyanate, extracting with
organic solvents, and separating RNA from contaminants using
differential centrifugation (see, for example, Chirgwin et al.,
Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu
(1997) at pages 33-41).
[0090] In order to construct a cDNA library, poly(A).sup.+ RNA must
be isolated from a total RNA preparation. Poly(A).sup.+ RNA can be
isolated from total RNA using the standard technique of
oligo(dT)-cellulose chromatography (see, for example, Aviv and
Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972); Ausubel (1995) at
pages 4-11 to 4-12).
[0091] Double-stranded cDNA molecules are synthesized from
poly(A).sup.+ RNA using techniques well-known to those in the art.
(see, for example, Wu (1997) at pages 41-46). Moreover,
commercially available kits can be used to synthesize
double-stranded cDNA molecules. For example, such kits are
available from Life Technologies, Inc. (Gaithersburg, Md.),
CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Promega
Corporation (Madison, Wis.) and STRATAGENE (La Jolla, Calif.).
[0092] Various cloning vectors are appropriate for the construction
of a cDNA library. For example, a cDNA library can be prepared in a
vector derived from bacteriophage, such as a .lamda.gt10 vector.
See, for example, Huynh et al., "Constructing and Screening cDNA
Libraries in .lamda.gt10 and .lamda. gt11," in DNA Cloning: A
Practical Approach Vol. I, Glover (ed.), page 49 (IRL Press, 1985);
Wu (1997) at pages 47-52.
[0093] Alternatively, double-stranded cDNA molecules can be
inserted into a plasmid vector, such as a pBLUESCRIPT vector
(STRATAGENE; La Jolla, Calif.), a LAMDAGEM-4 (Promega Corp.) or
other commercially available vectors. Suitable cloning vectors also
can be obtained from the American Type Culture Collection
(Manassas, Va.).
[0094] To amplify the cloned cDNA molecules, the cDNA library is
inserted into a prokaryotic host, using standard techniques. For
example, a cDNA library can be introduced into competent E. coli
DH5 or DH10B cells, which can be obtained, for example, from Life
Technologies, Inc. or GIBCO BRL (Gaithersburg, Md.).
[0095] A human genomic library can be prepared by means well known
in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6;
Wu (1997) at pages 307-327). Genomic DNA can be isolated by lysing
tissue with the detergent Sarkosyl, digesting the lysate with
proteinase K, clearing insoluble debris from the lysate by
centrifugation, precipitating nucleic acid from the lysate using
isopropanol, and purifying resuspended DNA on a cesium chloride
density gradient.
[0096] DNA fragments that are suitable for the production of a
genomic library can be obtained by the random shearing of genomic
DNA or by the partial digestion of genomic DNA with restriction
endonucleases. Genomic DNA fragments can be inserted into a vector,
such as a bacteriophage or cosmid vector, in accordance with
conventional techniques, such as the use of restriction enzyme
digestion to provide appropriate termini, the use of alkaline
phosphatase treatment to avoid undesirable joining of DNA
molecules, and ligation with appropriate ligases. Techniques for
such manipulation are well known in the art (see, for example,
Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages
307-327).
[0097] Alternatively, human genomic libraries can be obtained from
commercial sources such as Research Genetics (Huntsville, Ala.) and
the American Type Culture Collection (Manassas, Va.).
[0098] A library containing cDNA or genomic clones can be screened
with one or more polynucleotide probes based upon SEQ ID NO:1,
using standard methods (see, for example, Ausubel (1995) at pages
6-1 to 6-11).
[0099] Nucleic acid molecules that encode a human Zcytor16 gene can
also be obtained using the polymerase chain reaction (PCR) with
oligonucleotide primers having nucleotide sequences that are based
upon the nucleotide sequences of the Zcytor16 gene, as described
herein. General methods for screening libraries with PCR are
provided by, for example, Yu et al., "Use of the Polymerase Chain
Reaction to Screen Phage Libraries," in Methods in Molecular
Biology, Vol. 15. PCR Protocols: Current Methods and Applications,
White (ed.), pages 211-215 (Humana Press, Inc. 1993). Moreover,
techniques for using PCR to isolate related genes are described by,
for example, Preston, "Use of Degenerate Oligonucleotide Primers
and the Polymerase Chain Reaction to Clone Gene Family Members," in
Methods in Molecular Biology, Vol 15: PCR Protocols: Current
Methods and Applications, White (ed.), pages 317-337 (Humana Press,
Inc. 1993).
[0100] Anti-Zcytor16 antibodies, produced as described below, can
also be used to isolate DNA sequences that encode human Zcytor16
genes from cDNA libraries. For example, the antibodies can be used
to screen .lamda.gt11 expression libraries, or the antibodies can
be used for immunoscreening following hybrid selection and
translation (see, for example, Ausubel (1995) at pages 6-12 to
6-16; Margolis et al., "Screening .lamda. expression libraries with
antibody and protein probes," in DNA Cloning 2: Expression Systems,
2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University
Press 1995)).
[0101] As an alternative, a Zcytor16 gene can be obtained by
synthesizing nucleic acid molecules using mutually priming long
oligonucleotides and the nucleotide sequences described herein
(see, for example, Ausubel (1995) at pages 8-8 to 8-9). Established
techniques using the polymerase chain reaction provide the ability
to synthesize DNA molecules at least two kilobases in length (Adang
et al., Plant Molec. Biol 21:1131 (1993), Bambot et al., PCR
Methods and Applications 2:266 (1993), Dillon et al., "Use of the
Polymerase Chain Reaction for the Rapid Construction of Synthetic
Genes," in Methods in Molecular Biology, Vol 15: PCR Protocols:
Current Methods and Applications, White (ed.), pages 263-268,
(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.
4:299 (1995)).
[0102] The nucleic acid molecules of the present invention can also
be synthesized with "gene machines" using protocols such as 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 genes (60 to 80 base pairs) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 base pairs), however, special
strategies may be required, 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. For reviews on polynucleotide synthesis,
see, for example, Glick and Pasternak, Molecular Biotechnology,
Principles and Applications of Recombinant DNA (ASM Press 1994),
Itakura et al., Annu. Rev. Biochem. 53:323 (1984), and Climie et
al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).
[0103] The sequence of a Zcytor16 cDNA or Zcytor16 genomic fragment
can be determined using standard methods. Zcytor16 polynucleotide
sequences disclosed herein can also be used as probes or primers to
clone 5' non-coding regions of a Zcytor16 gene. Promoter elements
from a Zcytor16 gene can be used to direct the expression of
heterologous genes in, for example, tonsil tissue of transgenic
animals or patients treated with gene therapy. The identification
of genomic fragments containing a Zcytor16 promoter or regulatory
element can be achieved using well-established techniques, such as
deletion analysis (see, generally, Ausubel (1995)).
[0104] Cloning of 5' flanking sequences also facilitates production
of Zcytor16 proteins by "gene activation," as disclosed in U.S.
Pat. No. 5,641,670. Briefly, expression of an endogenous Zcytor16
gene in a cell is altered by introducing into the Zcytor16 locus a
DNA construct comprising at least a targeting sequence, a
regulatory sequence, an exon, and an unpaired splice donor site.
The targeting sequence is a Zcytor16 5' non-coding sequence that
permits homologous recombination of the construct with the
endogenous Zcytor16 locus, whereby the sequences within the
construct become operably linked with the endogenous Zcytor16
coding sequence. In this way, an endogenous Zcytor16 promoter can
be replaced or supplemented with other regulatory sequences to
provide enhanced, tissue-specific, or otherwise regulated
expression.
4. Production of Zcytor16 Gene Variants
[0105] The present invention provides a variety of nucleic acid
molecules, including DNA and RNA molecules, that encode the
Zcytor16 polypeptides disclosed herein. Those skilled in the art
will readily recognize that, in view of the degeneracy of the
genetic code, considerable sequence variation is possible among
these polynucleotide molecules. SEQ ID NO:3 is a degenerate
nucleotide sequence that encompasses all nucleic acid molecules
that encode the Zcytor16 polypeptide of SEQ ID NO:2. Those skilled
in the art will recognize that the degenerate sequence of SEQ ID
NO:3 also provides all RNA sequences encoding SEQ ID NO:2, by
substituting U for T. Moreover, the present invention also provides
isolated soluble monomeric, homodimeric, heterodimeric and
multimeric receptor polypeptides that comprise at least one
zcytor16 receptor subunit that is substantially homologous to the
receptor polypeptide of SEQ ID NO:3. Thus, the present invention
contemplates Zcytor16 polypeptide-encoding nucleic acid molecules
comprising nucleotide 1 to nucleotide 693 of SEQ ID NO:1, and their
RNA equivalents.
[0106] Table 1 sets forth the one-letter codes used within SEQ ID
NO:3 to denote degenerate nucleotide positions. "Resolutions" are
the nucleotides denoted by a code letter. "Complement" indicates
the code for the complementary nucleotide(s). For example, the code
Y denotes either C or T, and its complement R denotes A or G, A
being complementary to T, and G being complementary to C.
TABLE-US-00001 TABLE 1 Nucleotide Resolution Complement Resolution
A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T
K G|T M A|C S C|G S C|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G
V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T
[0107] The degenerate codons used in SEQ ID NO:3, encompassing all
possible codons for a given amino acid, are set forth in Table
2.
TABLE-US-00002 TABLE 2 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
[0108] 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 an amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequences of SEQ ID NO:2.
Variant sequences can be readily tested for functionality as
described herein.
[0109] Different species can exhibit "preferential codon usage." In
general, see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas
et al. Curr. Biol 6:315 (1996), Wain-Hobson et al., Gene 13:355
(1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids
Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp
and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr.
Opin. Biotechnol 6:494 (1995), and Makrides, Microbiol Rev. 60:512
(1996). 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 sequences
disclosed herein serve as a template for optimizing expression of
polynucleotides in various cell types and species commonly used in
the art and disclosed herein. Sequences containing preferential
codons can be tested and optimized for expression in various
species, and tested for functionality as disclosed herein.
[0110] The present invention further provides variant polypeptides
and nucleic acid molecules that represent counterparts 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
Zcytor16 polypeptides from other mammalian species, including
mouse, porcine, ovine, bovine, canine, feline, equine, and other
primate polypeptides. Orthologs of human Zcytor16 can be cloned
using information and compositions provided by the present
invention in combination with conventional cloning techniques. For
example, a Zcytor16 cDNA can be cloned using mRNA obtained from a
tissue or cell type that expresses Zcytor16 as disclosed herein.
Suitable sources of mRNA can be identified by probing northern
blots with probes designed from the sequences disclosed herein. A
library is then prepared from mRNA of a positive tissue or cell
line.
[0111] A Zcytor16-encoding cDNA can be isolated by a variety of
methods, such as by probing with a complete or partial human cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using the polymerase
chain reaction with primers designed from the representative human
Zcytor16 sequences disclosed herein. In addition, a 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 Zcytor16
polypeptide.
[0112] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 oe SEQ ID NO:37 represents a single allele
of human Zcytor16, and that allelic variation and alternative
splicing are expected to occur. Allelic variants of this sequence
can be cloned by probing cDNA or genomic libraries from different
individuals according to standard procedures. Allelic variants of
the nucleotide sequences disclosed herein, including those
containing silent mutations and those in which mutations result in
amino acid sequence changes, are within the scope of the present
invention, as are proteins which are allelic variants of the amino
acid sequences disclosed herein. cDNA molecules generated from
alternatively spliced mRNAs, which retain the properties of the
Zcytor16 polypeptide are included within the scope of the present
invention, as are polypeptides encoded by such cDNAs and mRNAs.
Allelic variants and splice variants of these sequences can be
cloned by probing cDNA or genomic libraries from different
individuals or tissues according to standard procedures known in
the art.
[0113] Using the methods discussed above, one of ordinary skill in
the art can prepare a variety of polypeptides that comprise a
soluble receptor subunit that is substantially homologous to SEQ ID
NO:1 or SEQ ID NO:2, SEQ ID NO:13, or SEQ ID NO:37 amino acids 22
to 231 or 28-231 of SEQ ID NO:2, or allelic variants thereof and
retain the ligand-binding properties of the wild-type zcytor16
receptor. Such polypeptides may also include additional polypeptide
segments as generally disclosed herein.
[0114] Within certain embodiments of the invention, the isolated
nucleic acid molecules can hybridize under stringent conditions to
nucleic acid molecules comprising nucleotide sequences disclosed
herein. For example, such nucleic acid molecules can hybridize
under stringent conditions to nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, to nucleic acid molecules
consisting of the nucleotide sequence of nucleotides 64, 67, 82, or
94 to 693 of SEQ ID NO:1, or to nucleic acid molecules comprising a
nucleotide sequence complementary to SEQ ID NO:1 or to nucleotides
64, 67, 82, or 94 to 693 of SEQ ID NO:1, or fragments thereof. 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.
[0115] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Stringent hybridization conditions encompass
temperatures of about 5-25.degree. C. below the T.sub.m of the
hybrid and a hybridization buffer having up to 1 M Na.sup.+. Higher
degrees of stringency at lower temperatures can be achieved with
the addition of formamide which reduces the T.sub.m of the hybrid
about 1.degree. C. for each 1% formamide in the buffer solution.
Generally, such stringent conditions include temperatures of
20-70.degree. C. and a hybridization buffer containing up to
6.times.SSC and 0-50% formamide. A higher degree of stringency can
be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having up to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 1.times.SSC and 0-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0116] The above conditions are meant to serve as a guide and it is
well within the abilities of one skilled in the art to adapt these
conditions for use with a particular polynucleotide hybrid. The
T.sub.m for a specific target sequence is the temperature (under
defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those conditions
which influence the T.sub.m include, the size and base pair content
of the polynucleotide probe, the ionic strength of the
hybridization solution, and the presence of destabilizing agents in
the hybridization solution. Numerous equations for calculating
T.sub.m are known in the art, and are specific for DNA, RNA and
DNA-RNA hybrids and polynucleotide probe sequences of varying
length (see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989);
Ausubel et al., (eds.), Current Protocols in Molecular Biology
(John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide
to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and
Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence
analysis software such as OLIGO 6.0 (LSR; Long Lake, Minn.) and
Primer Premier 4.0 (Premier Biosoft International; Palo Alto,
Calif.), as well as sites on the Internet, are available tools for
analyzing a given sequence and calculating T.sub.m based on user
defined criteria. Such programs can also analyze a given sequence
under defined conditions and identify suitable probe sequences.
Typically, hybridization of longer polynucleotide sequences, >50
base pairs, is performed at temperatures of about 20-25.degree. C.
below the calculated T.sub.m, For smaller probes, <50 base
pairs, hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below. This allows for the maximum rate of
hybridization for DNA-DNA and DNA-RNA hybrids.
[0117] The length of the polynucleotide sequence influences the
rate and stability of hybrid formation. Smaller probe sequences,
<50 base pairs, reach equilibrium with complementary sequences
rapidly, but may form less stable hybrids. Incubation times of
anywhere from minutes to hours can be used to achieve hybrid
formation. Longer probe sequences come to equilibrium more slowly,
but form more stable complexes even at lower temperatures.
Incubations are allowed to proceed overnight or longer. Generally,
incubations are carried out for a period equal to three times the
calculated Cot time. Cot time, the time it takes for the
polynucleotide sequences to reassociate, can be calculated for a
particular sequence by methods known in the art.
[0118] The base pair composition of polynucleotide sequence will
effect the thermal stability of the hybrid complex, thereby
influencing the choice of hybridization temperature and the ionic
strength of the hybridization buffer. A-T pairs are less stable
than G-C pairs in aqueous solutions containing sodium chloride.
Therefore, the higher the G-C content, the more stable the hybrid.
Even distribution of G and C residues within the sequence also
contribute positively to hybrid stability. In addition, the base
pair composition can be manipulated to alter the T.sub.m of a given
sequence. For example, 5-methyldeoxycytidine can be substituted for
deoxycytidine and 5-bromodeoxuridine can be substituted for
thymidine to increase the T.sub.m, whereas
7-deazz-2'-deoxyguanosine can be substituted for guanosine to
reduce dependence on T.sub.m.
[0119] The ionic concentration of the hybridization buffer also
affects the stability of the hybrid. Hybridization buffers
generally contain blocking agents such as Denhardt's solution
(Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA,
tRNA, milk powders (BLOTTO), heparin or SDS, and a Na.sup.+ source,
such as SSC (1.times.SSC: 0.15 M sodium chloride, 15 mM sodium
citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). Typically, hybridization
buffers contain from between 10 mM-1 M Na.sup.+. The addition of
destabilizing or denaturing agents such as formamide,
tetralkylammonium salts, guanidinium cations or thiocyanate cations
to the hybridization solution will alter the T.sub.m of a hybrid.
Typically, formamide is used at a concentration of up to 50% to
allow incubations to be carried out at more convenient and lower
temperatures. Formamide also acts to reduce non-specific background
when using RNA probes.
[0120] As an illustration, a nucleic acid molecule encoding a
variant Zcytor16 polypeptide can be hybridized with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID
NO:37 (or its complement) at 42.degree. C. overnight in a solution
comprising 50% formamide, 5.times.SSC, 50 mM sodium phosphate (pH
7.6), 5.times.Denhardt's solution (100.times.Denhardt's solution:
2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v)
bovine serum albumin), 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA. One of skill in the art can
devise variations of these hybridization conditions. For example,
the hybridization mixture can be incubated at a higher temperature,
such as about 65.degree. C., in a solution that does not contain
formamide. Moreover, premixed hybridization solutions are available
(e.g., EXPRESSHYB Hybridization Solution from CLONTECH
Laboratories, Inc.), and hybridization can be performed according
to the manufacturer's instructions.
[0121] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. As an illustration, nucleic acid molecules
encoding a variant Zcytor16 polypeptide remain hybridized with a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1
(or its complement) under stringent washing conditions, in which
the wash stringency is equivalent to 0.5.times.-2.times.SSC with
0.1% SDS at 55-65.degree. C., including 0.5.times.SSC with 0.1% SDS
at 55.degree. C., or 2.times.SSC with 0.1% SDS at 65.degree. C. One
of skill in the art can readily devise equivalent conditions, for
example, by substituting SSPE for SSC in the wash solution.
[0122] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. For example, nucleic acid
molecules encoding a variant Zcytor16 polypeptide remain hybridized
with a nucleic acid molecule having the nucleotide sequence of SEQ
ID NO:1 (or its complement) under highly stringent washing
conditions, in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C.,
including 0.1.times.SSC with 0.1% SDS at 50.degree. C., or
0.2.times.SSC with 0.1% SDS at 65.degree. C.
[0123] The present invention also provides isolated Zcytor16
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:2, or their orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides having at least 70%, at least 80%, at least 90%, at
least 95% or greater than 95% sequence identity to the sequences
shown in SEQ ID NO:2, or their orthologs.
[0124] The present invention also contemplates Zcytor16 variant
nucleic acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NO:2, and a hybridization
assay, as described above. Such Zcytor16 variants include nucleic
acid molecules (1) that remain hybridized with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1 or SEQ ID
NO:37 (or its complement) under stringent washing conditions, in
which the wash stringency is equivalent to 0.5.times.-2.times.SSC
with 0.1% SDS at 55-65.degree. C., and (2) that encode a
polypeptide having at least 70%, at least 80%, at least 90%, at
least 95% or greater than 95% sequence identity to the amino acid
sequence of SEQ ID NO:2. Alternatively, Zcytor16 variants can be
characterized as nucleic acid molecules (1) that remain hybridized
with a nucleic acid molecule having the nucleotide sequence of SEQ
ID NO:1 or SEQ ID NO:37 (or its complement) under highly stringent
washing conditions, in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C., and (2)
that encode a polypeptide having at least 70%, at least 80%, at
least 90%, at least 95% or greater than 95% sequence identity to
the amino acid sequence of SEQ ID NO:2.
[0125] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). 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])(100).
TABLE-US-00003 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4
R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0
2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3
-3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3
1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3
-3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1
-2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1
-1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2
-3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2
-2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0126] 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 Zcytor16 variant. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990). Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:2) 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. Illustrative
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0127] 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 parameters set as described above.
[0128] The present invention includes nucleic acid molecules that
encode a polypeptide having a conservative amino acid change,
compared with an amino acid sequence disclosed herein. For example,
variants can be obtained that contain one or more amino acid
substitutions of SEQ ID NO:2, in which an alkyl amino acid is
substituted for an alkyl amino acid in a Zcytor16 amino acid
sequence, an aromatic amino acid is substituted for an aromatic
amino acid in a Zcytor16 amino acid sequence, a sulfur-containing
amino acid is substituted for a sulfur-containing amino acid in a
Zcytor16 amino acid sequence, a hydroxy-containing amino acid is
substituted for a hydroxy-containing amino acid in a Zcytor16 amino
acid sequence, an acidic amino acid is substituted for an acidic
amino acid in a Zcytor16 amino acid sequence, a basic amino acid is
substituted for a basic amino acid in a Zcytor16 amino acid
sequence, or a dibasic monocarboxylic amino acid is substituted for
a dibasic monocarboxylic amino acid in a Zcytor16 amino acid
sequence. Among the common amino acids, for example, a
"conservative amino acid substitution" is illustrated by a
substitution among amino acids within each of the following groups:
(1) glycine, alanine, valine, leucine, and isoleucine, (2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,
(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)
lysine, arginine and histidine. The BLOSUM62 table 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 above), 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).
[0129] Particular variants of Zcytor16 are characterized by having
at least 70%, at least 80%, at least 90%, at least 95% or greater
than 95% sequence identity to the corresponding amino acid sequence
(e.g., SEQ ID NO:2), wherein the variation in amino acid sequence
is due to one or more conservative amino acid substitutions.
[0130] Conservative amino acid changes in a Zcytor16 gene can be
introduced, for example, by substituting nucleotides for the
nucleotides recited in SEQ ID NO:1. Such "conservative amino acid"
variants can be obtained by oligonucleotide-directed mutagenesis,
linker-scanning mutagenesis, mutagenesis using the polymerase chain
reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22;
and McPherson (ed.), Directed Mutagenesis: A Practical Approach
(IRL Press 1991)). A variant Zcytor16 polypeptide can be identified
by the ability to specifically bind anti-Zcytor16 antibodies.
[0131] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is typically carried out in a
cell-free system comprising an E. coli S30 extract and commercially
available enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722 (1991), Ellman et al., Methods Enzymol 202:301
(1991), Chung et al., Science 259:806 (1993), and Chung et al.,
Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0132] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993)).
[0133] 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 Zcytor16 amino acid residues.
[0134] 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 (1989), Bass et
al., Proc. Nat'l 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 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).
[0135] Although sequence analysis can be used to further define the
Zcytor16 ligand binding region, amino acids that play a role in
Zcytor16 binding activity (such as binding of zcytor16 to ligand
IL-TIF, or to an anti-zcytor16 antibody) 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 (1992), Smith et al., J. Mol. Biol. 224:899 (1992), and
Wlodaver et al., FEBS Lett. 309:59 (1992).
[0136] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204, and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover,
Zcytor16 labeled with biotin or FITC can be used for expression
cloning of Zcytor16 ligands.
[0137] Variants of the disclosed Zcytor16 nucleotide and
polypeptide sequences can also be generated through DNA shuffling
as disclosed by Stemmer, Nature 370:389 (1994), Stemmer, Proc.
Nat'l Acad. Sci. USA 91:10747 (1994), and international publication
No. WO 97/20078. Briefly, variant DNA molecules are generated by in
vitro homologous recombination by random fragmentation of a parent
DNA followed by reassembly using PCR, resulting in randomly
introduced point mutations. This technique can be modified by using
a family of parent DNA molecules, such as allelic variants or DNA
molecules from different species, to introduce additional
variability into the process. Selection or screening for the
desired activity, followed by additional iterations of mutagenesis
and assay provides for rapid "evolution" of sequences by selecting
for desirable mutations while simultaneously selecting against
detrimental changes.
[0138] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-Zcytor16 antibodies, 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.
[0139] The present invention also includes "functional fragments"
of Zcytor16 polypeptides and nucleic acid molecules encoding such
functional fragments. Routine deletion analyses of nucleic acid
molecules can be performed to obtain functional fragments of a
nucleic acid molecule that encodes a Zcytor16 polypeptide. As an
illustration, DNA molecules having the nucleotide sequence of SEQ
ID NO:1 can be digested with Bal31 nuclease to obtain a series of
nested deletions. The fragments are then inserted into expression
vectors in proper reading frame, and the expressed polypeptides are
isolated and tested for the ability to bind anti-Zcytor16
antibodies. One alternative to exonuclease digestion is to use
oligonucleotide-directed mutagenesis to introduce deletions or stop
codons to specify production of a desired fragment. Alternatively,
particular fragments of a Zcytor16 gene can be synthesized using
the polymerase chain reaction.
[0140] This general approach is exemplified by 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, Vol 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).
[0141] Analysis of the particular sequences disclosed herein
provide a set of illustrative functional fragments presented in
Table 4. The nucleotides encoding additional human zcytor16
functional variant domains described herein, not show in Table 4,
can be determined with reference to SEQ ID NO:1 or SEQ ID NO:37.
Such functional fragments include for example, the following
nucleotide sequences of SEQ ID NO:1: nucleotides 64, 67, 82 or 94
to 690 or 693; nucleotides 64, 67, 82 or 94 to 369 or 381; 394 to
690 or 693; and nucleotides 1 to 690 or 693, and amino acid
sequences encoded thereby, e.g, such as those shown in SEQ ID NO:2
respectively.
TABLE-US-00004 TABLE 4 Amino acid residues Nucleotides Zcytor16
Feature (SEQ ID NO: 2) (SEQ ID NO: 1) First Ig Domain 32-123 94-369
Second Ig Domain 132-230 394-690 Both Ig Domains 32-230 94-690
[0142] The present invention also contemplates functional fragments
of a Zcytor16 gene that have amino acid changes, compared with an
amino acid sequence disclosed herein. A variant Zcytor16 gene can
be identified on the basis of structure by determining the level of
identity with disclosed nucleotide and amino acid sequences, as
discussed above. An alternative approach to identifying a variant
gene on the basis of structure is to determine whether a nucleic
acid molecule encoding a potential variant Zcytor16 gene can
hybridize to a nucleic acid molecule comprising a nucleotide
sequence, such as SEQ ID NO:1 or SEQ ID NO:37.
[0143] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a Zcytor16
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0144] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein.
[0145] Antigenic epitope-bearing peptides and polypeptides can
contain at least four to ten amino acids, at least ten to fifteen
amino acids, or about 15 to about 30 amino acids of an amino acid
sequence disclosed herein. Such epitope-bearing peptides and
polypeptides can be produced by fragmenting a Zcytor16 polypeptide,
or by chemical peptide synthesis, as described herein. Moreover,
epitopes can be selected by phage display of random peptide
libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol.
5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616
(1996)). Standard methods for identifying epitopes and producing
antibodies from small peptides that comprise an epitope are
described, for example, by Mole, "Epitope Mapping," in Methods in
Molecular Biology, Vol 10, Manson (ed.), pages 105-116 (The Humana
Press, Inc. 1992), Price, "Production and Characterization of
Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering, and Clinical Application, Ritter and
Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and
Coligan et al. (eds.), Current Protocols in Immunology, pages
9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons
1997).
[0146] For any Zcytor16 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above. Moreover,
those of skill in the art can use standard software to devise
Zcytor16 variants based upon the nucleotide and amino acid
sequences described herein. Accordingly, the present invention
includes a computer-readable medium encoded with a data structure
that provides at least one of the following sequences: SEQ ID NO:1,
SEQ ID NO:2, and SEQ ID NO:3. Suitable forms of computer-readable
media include magnetic media and optically-readable media. Examples
of magnetic media include a hard or fixed drive, a random access
memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk
cache, and a ZIP disk. Optically readable media are exemplified by
compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW),
and CD-recordable), and digital versatile/video discs (DVD) (e.g.,
DVD-ROM, DVD-RAM, and DVD+RW).
6. Production of Zcytor16 Polypeptides
[0147] The polypeptides of the present invention, including
full-length polypeptides; soluble monomeric, homodimeric,
heterodimeric and multimeric receptors; full-length receptors;
receptor fragments (e.g. ligand-binding fragments), functional
fragments, and fusion proteins, can be produced in recombinant host
cells following conventional techniques. To express a Zcytor16
gene, a nucleic acid molecule encoding the polypeptide must be
operably linked to regulatory sequences that control
transcriptional expression in an expression vector and then,
introduced into a host cell. In addition to transcriptional
regulatory sequences, such as promoters and enhancers, expression
vectors can include translational regulatory sequences and a marker
gene which is suitable for selection of cells that carry the
expression vector.
[0148] Expression vectors that are suitable for production of a
foreign protein in eukaryotic cells typically contain (1)
prokaryotic DNA elements coding for a bacterial replication origin
and an antibiotic resistance marker to provide for the growth and
selection of the expression vector in a bacterial host; (2)
eukaryotic DNA elements that control initiation of transcription,
such as a promoter; and (3) DNA elements that control the
processing of transcripts, such as a transcription
termination/polyadenylation sequence. As discussed above,
expression vectors can also include nucleotide sequences encoding a
secretory sequence that directs the heterologous polypeptide into
the secretory pathway of a host cell. For example, a Zcytor16
expression vector may comprise a Zcytor16 gene and a secretory
sequence derived from any secreted gene.
[0149] Zcytor16 proteins of the present invention may be expressed
in mammalian cells. Examples of suitable mammalian host cells
include African green monkey kidney cells (Vero; ATCC CRL 1587),
human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster
kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314),
canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary
cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin et al., Som. Cell
Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC
CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;
ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC
CRL 1650) and murine embryonic cells (1H-3T3; ATCC CRL 1658).
[0150] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from viral sources, such as
adenovirus, bovine papilloma virus, simian virus, or the like, in
which the regulatory signals are associated with a particular gene
which has a high level of expression. Suitable transcriptional and
translational regulatory sequences also can be obtained from
mammalian genes, such as actin, collagen, myosin, and
metallothionein genes.
[0151] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl Genet. 1:273
(1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355
(1982)), the SV40 early promoter (Benoist et al., Nature 290:304
(1981)), the Rous sarcoma virus promoter (Gorman et al., Proc.
Nat'l Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter
(Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor
virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein
Engineering: Principles and Practice, Cleland et al. (eds.), pages
163-181 (John Wiley & Sons, Inc. 1996)).
[0152] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
Zcytor16 gene expression in mammalian cells if the prokaryotic
promoter is regulated by a eukaryotic promoter (Zhou et al., Mol
Cell Biol. 10:4529 (1990), and Kaufman et al., Nucl Acids Res.
19:4485 (1991)).
[0153] In certain embodiments, a DNA sequence encoding a Zcytor16
monomeric or homodimeric soluble receptor polypeptide, or a DNA
sequence encoding an additional subunit of a heterodimeric or
multimeric Zcytor16 soluble receptor, e.g., CRF2-4 or IL10R,
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. Multiple
components of a soluble receptor complex can be co-transfected on
individual expression vectors or be contained in a single
expression vector. Such techniques of expressing multiple
components of protein complexes are well known in the art.
[0154] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
The transfected cells can be selected and propagated to provide
recombinant host cells that comprise the expression vector stably
integrated in the host cell genome. Techniques for introducing
vectors into eukaryotic cells and techniques for selecting such
stable transformants using a dominant selectable marker are
described, for example, by Ausubel (1995) and by Murray (ed.), Gene
Transfer and Expression Protocols (Humana Press 1991).
[0155] For example, one suitable selectable marker is a gene that
provides resistance to the antibiotic neomycin. In this case,
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 suitable 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. Alternatively, markers that introduce an altered phenotype,
such as green fluorescent protein, or cell surface proteins such as
CD4, CD8, Class I MHC, placental alkaline phosphatase may be used
to sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0156] Zcytor16 polypeptides can also be produced by cultured
mammalian cells using a viral delivery system. Exemplary viruses
for this purpose include adenovirus, retroviruses, herpesvirus,
vaccinia virus and adeno-associated virus (AAV). Adenovirus, a
double-stranded DNA virus, is currently the best studied gene
transfer vector for delivery of heterologous nucleic acid (for a
review, see Becker et al., Meth. Cell Biol 43:161 (1994), and
Douglas and Curiel, Science & Medicine 4.44 (1997)). Advantages
of the adenovirus system include the accommodation of relatively
large DNA inserts, the ability to grow to high-titer, the ability
to infect a broad range of mammalian cell types, and flexibility
that allows use with a large number of available vectors containing
different promoters.
[0157] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. An
option is to delete the essential E1 gene from the viral vector,
which results in the inability to replicate unless the E1 gene is
provided by the host cell. Adenovirus vector-infected human 293
cells (ATCC Nos. CRL-1573, 45504, 45505), for example, 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 (1994)).
[0158] Zcytor16 can also be expressed in other higher eukaryotic
cells, such as avian, fungal, insect, yeast, or plant cells. The
baculovirus system provides an efficient means to introduce cloned
Zcytor16 genes into insect cells. Suitable expression vectors are
based upon the Autographa californica multiple nuclear polyhedrosis
virus (AcMNPV), and contain well-known promoters such as Drosophila
heat shock protein (hsp) 70 promoter, Autographa californica
nuclear polyhedrosis virus immediate-early gene promoter (ie-1) and
the delayed early 39K promoter, baculovirus p10 promoter, and the
Drosophila metallothionein promoter. A second method of making
recombinant baculovirus utilizes a transposon-based system
described by Luckow (Luckow, et al., J. Virol. 67:4566 (1993)).
This system, which utilizes transfer vectors, is sold in the
BAC-to-BAC kit (Life Technologies, Rockville, Md.). This system
utilizes a transfer vector, PFASTBAC (Life Technologies) containing
a Tn7 transposon to move the DNA encoding the Zcytor16 polypeptide
into a baculovirus genome maintained in E. coli as a large plasmid
called a "bacmid." See, Hill-Perkins and Possee, J. Gen. Virol.
71:971 (1990), Bonning, et al., J. Gen. Virol 75:1551 (1994), and
Chazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (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
Zcytor16 polypeptide, for example, a Glu-Glu epitope tag
(Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952 (1985)). Using
a technique known in the art, a transfer vector containing a
Zcytor16 gene 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 then isolated using common techniques.
[0159] The illustrative PFASTBAC vector can be modified to a
considerable degree. For example, the polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins (see, for example,
Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et
al., J. Gen. Virol 75:1551 (1994), and Chazenbalk and Rapoport, J.
Biol. Chem. 270:1543 (1995). In such transfer vector constructs, a
short or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed which replace the
native Zcytor16 secretory signal sequences with secretory signal
sequences derived from insect proteins. For example, a secretory
signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), or
baculovirus gp67 (PharMingen: San Diego, Calif.) can be used in
constructs to replace the native Zcytor16 secretory signal
sequence.
[0160] The recombinant virus or bacmid is used to transfect host
cells. Suitable insect host cells include cell lines derived from
IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such
as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21 (Invitrogen Corporation;
San Diego, Calif.), as well as Drosophila Schneider-2 cells, and
the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
can be used to grow and to 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. When recombinant virus is used, the cells are typically
grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3.
[0161] Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus Vectors," in Methods in Molecular Biology, Volume 7.
Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168
(The Humana Press, Inc. 1991), by Patel et al., "The baculovirus
expression system," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University
Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by
Richardson (ed.), Baculovirus Expression Protocols (The Humana
Press, Inc. 1995), and by Lucknow, "Insect Cell Expression
Technology," in Protein Engineering: Principles and Practice,
Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
[0162] Fungal cells, including yeast cells, can also be used to
express the genes described herein. Yeast species of particular
interest in this regard include Saccharomyces cerevisiae, Pichia
pastoris, and Pichia methanolica. Suitable promoters for expression
in yeast include promoters from GAL1 (galactose), PGK
(phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1
(alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
Many yeast cloning vectors have been designed and are readily
available. These vectors include YIp-based vectors, such as YIp5,
YRp vectors, such as YRp17, YEp vectors such as YEp13 and YCp
vectors, such as YCp19. 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 suitable 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. Additional 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.
[0163] 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 (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.
[0164] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23 (1998), and in international publication Nos.
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, the promoter and terminator in the
plasmid can 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 suitable 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), and 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, host cells can be used
in which both methanol utilization genes (AUG1 and AUG2) are
deleted. For production of secreted proteins, host cells can be
deficient in vacuolar protease genes (PEP4 and PRB1).
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. P. methanolica cells can be transformed 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.
[0165] Expression vectors can also be introduced into plant
protoplasts, intact plant tissues, or isolated plant cells. Methods
for introducing expression vectors into plant tissue include the
direct infection or co-cultivation of plant tissue with
Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA
injection, electroporation, and the like. See, for example, Horsch
et al., Science 227:1229 (1985), Klein et al., Biotechnology 10:268
(1992), and Miki et al., "Procedures for Introducing Foreign DNA
into Plants," in Methods in Plant Molecular Biology and
Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,
1993).
[0166] Alternatively, Zcytor16 genes can be expressed in
prokaryotic host cells. Suitable promoters that can be used to
express Zcytor16 polypeptides in a prokaryotic host are well-known
to those of skill in the art and include promoters capable of
recognizing the T4, T3, Sp6 and T7 polymerases, the P.sub.R and
P.sub.L promoters of bacteriophage lambda, the trp, recA, heat
shock, lacUV5, tac, lpp-lacSpr, phoA, and lacZ promoters of E.
coli, promoters of B. subtilis, the promoters of the bacteriophages
of Bacillus, Streptomyces promoters, the int promoter of
bacteriophage lambda, the bla promoter of pBR322, and the CAT
promoter of the chloramphenicol acetyl transferase gene.
Prokaryotic promoters have been reviewed by Glick, J. Ind.
Microbiol. 1:277 (1987), Watson et al., Molecular Biology of the
Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al.
(1995).
[0167] Suitable prokaryotic hosts include E. coli and Bacillus
subtilus. Suitable strains of E. coli include BL21(DE3),
BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF',
DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109,
JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for
example, Brown (ed.), Molecular Biology Labfax (Academic Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL
Press 1985)).
[0168] When expressing a Zcytor16 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.
[0169] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et al., "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995), and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), page 101 (John Wiley & Sons, Inc. 1996)).
[0170] Standard methods for introducing expression vectors into
bacterial, yeast, insect, and plant cells are provided, for
example, by Ausubel (1995).
[0171] General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Established methods for
isolating recombinant proteins from a baculovirus system are
described by Richardson (ed.), Baculovirus Expression Protocols
(The Humana Press, Inc. 1995).
[0172] As an alternative, polypeptides of the present invention can
be synthesized by exclusive solid phase synthesis, partial solid
phase methods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill
in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149
(1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd
Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept.
Prot. 3:3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach (IRL Press 1989), Fields and Colowick,
"Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289
(Academic Press 1997), and Lloyd-Williams et al., Chemical
Approaches to the Synthesis of Peptides and Proteins (CRC Press,
Inc. 1997)). Variations in total chemical synthesis strategies,
such as "native chemical ligation" and "expressed protein ligation"
are also standard (see, for example, Dawson et al., Science 266:776
(1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997),
Dawson, Methods Enzymol 287: 34 (1997), Muir et al, Proc. Nat'l
Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol.
Chem. 273:16205 (1998)).
[0173] Peptides and polypeptides of the present invention comprise
at least six, at least nine, or at least 15 contiguous amino acid
residues of SEQ ID NO:2. As an illustration, polypeptides can
comprise at least six, at least nine, or at least 15 contiguous
amino acid residues of any of the following amino acid sequences of
SEQ ID NO:2: amino acid residues amino acid residues 22 to 230 or
231, amino acid residues 23 to 230 or 231, amino acid residues 28
to 230 or 231, or amino acids 32 to 230 or 231; amino acid residues
22, 23, 28 or 32 to 127; amino acid residues 22, 23, 28 or 32 to
123, and amino acid residues 132 to 230 or 231. Within certain
embodiments of the invention, the polypeptides comprise 20, 30, 40,
50, 100, or more contiguous residues of these amino acid sequences.
Nucleic acid molecules encoding such peptides and polypeptides are
useful as polymerase chain reaction primers and probes.
[0174] Moreover, zcytor16 polypeptides can be expressed as
monomers, homodimers, heterodimers, or multimers within higher
eukaryotic cells. Such cells can be used to produce zcytor16
monomeric, homodimeric, heterodimeric and multimeric receptor
polypeptides that comprise at least one zcytor16 polypeptide
("zcytor16-comprising receptors" or "zcytor16-comprising receptor
polypeptides"), or can be used as assay cells in screening systems.
Within one aspect of the present invention, a polypeptide of the
present invention comprising the zcytor16 extracellular domain is
produced by a cultured cell, and the cell is used to screen for
ligands for the receptor, including the natural ligand, IL-TIF, as
well as agonists and antagonists of the natural ligand. To
summarize this approach, a cDNA or gene encoding the receptor is
combined with other genetic elements required for its expression
(e.g., a transcription promoter), and the resulting expression
vector is inserted into a host cell. Cells that express the DNA and
produce functional receptor are selected and used within a variety
of screening systems. Each component of the monomeric, homodimeric,
heterodimeric and multimeric receptor complex can be expressed in
the same cell. Moreover, the components of the monomeric,
homodimeric, heterodimeric and multimeric receptor complex can also
be fused to a transmembrane domain or other membrane fusion moiety
to allow complex assembly and screening of transfectants as
described above.
[0175] Mammalian cells suitable for use in expressing Zcytor16
receptors and transducing a receptor-mediated signal include cells
that express other receptor subunits that may form a functional
complex with Zcytor16. These subunits may include those of the
interferon receptor family or of other class II or class I cytokine
receptors, e.g., CRF2-4 (Genbank Accession No. Z17227), IL-10R
(Genbank Accession No.s U00672 and NM.sub.--001558), zcytor11 (U.S.
Pat. No. 5,965,704), zcytor7 (U.S. Pat. No. 5,945,511), and IL-9R.
It is also preferred to use a cell from the same species as the
receptor to be expressed. Within a preferred embodiment, the cell
is dependent upon an exogenously supplied hematopoietic growth
factor for its proliferation. Preferred cell lines of this type are
the human TF-1 cell line (ATCC number CRL-2003) and the AML-193
cell line (ATCC number CRL-9589), which are GM-CSF-dependent human
leukemic cell lines and BaF3 (Palacios and Steinmetz, Cell 41:
727-734, (1985)) which is an IL-3 dependent murine pre-B cell line.
Other cell lines include BHK, COS-1 and CHO cells.
[0176] Suitable host cells can be engineered to produce the
necessary receptor subunits or other cellular component needed for
the desired cellular response. This approach is advantageous
because cell lines can be engineered to express receptor subunits
from any species, thereby overcoming potential limitations arising
from species specificity. Species orthologs 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
GM-CSF or IL-3, can thus be engineered to become dependent upon
another cytokine that acts through the zcytor16 receptor, such as
IL-TIF.
[0177] Cells expressing functional receptor 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 a target cell. One such assay is a cell proliferation
assay. Cells are cultured in the presence or absence of a test
compound, and cell proliferation is detected by, for example,
measuring incorporation of tritiated thymidine or by colorimetric
assay based on the metabolic breakdown of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
(Mosman, J. Immunol. Meth. 65: 55-63, (1983)). An alternative assay
format uses cells that are further engineered to express a reporter
gene. The reporter gene is linked to a promoter element that is
responsive to the receptor-linked pathway, and the assay detects
activation of transcription of the reporter gene. A preferred
promoter element in this regard is a serum response element, or
SRE. See, e.g., 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. 269:29094-29101, (1994); Schenborn and
Goiffin, Promega.sub.--Notes 41:11, 1993). Luciferase activity
assay kits are commercially available from, for example, Promega
Corp., Madison, Wis. Target cell lines of this type can be used to
screen libraries of chemicals, cell-conditioned culture media,
fungal broths, soil samples, water samples, and the like. For
example, a bank of cell-conditioned media samples can be assayed on
a target cell to identify cells that produce ligand. Positive cells
are then used to produce a cDNA library in a mammalian expression
vector, which is divided into pools, transfected into host cells,
and expressed. Media samples from the transfected cells are then
assayed, with subsequent division of pools, re-transfection,
subculturing, and re-assay of positive cells to isolate a cloned
cDNA encoding the ligand.
[0178] A natural ligand for the Zcytor16 receptor can also be
identified by mutagenizing a cell line expressing the full-length
receptor or receptor fusion (e.g., comprising the zcytor16
extracellular domain fused to the transmembrane and signaling
domain of another cytokine receptor) and culturing it under
conditions that select for autocrine growth. See WIPO publication
WO 95/21930. Within a typical procedure, IL-3 dependent BaF3 cells
expressing Zcytor16 and the necessary additional subunits are
mutagenized, such as with 2-ethylmethanesulfonate (EMS). The cells
are then allowed to recover in the presence of IL-3, then
transferred to a culture medium lacking IL-3 and IL-4. Surviving
cells are screened for the production of a zcytor16 ligand (e.g.,
IL-TIF), such as by adding soluble receptor to the culture medium
or by assaying conditioned media on wild-type BaF3 cells and BaF3
cells expressing the receptor. Using this method, cells and tissues
expressing IL-TIF can be identified.
[0179] Moreover several IL-TIF responsive cell lines are known
(Dumontier et al., J. Immunol. 164:1814-1819, 2000; Dumoutier, L.
et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; Xie M H et
al., J. Biol. Chem. 275: 31335-31339, 2000; Kotenko S V et al., JBC
in press), as well as those that express the IL-TIF receptor
subunit zcytor11. For example, the following cells are responsive
to IL-TIF: TK-10 (Xie M H et al., supra.) (human renal carcinoma);
SW480 (ATCC No. CCL-228) (human colon adenocarcinoma); HepG2 (ATCC
No. HB-8065) (human hepatoma); PC12 (ATCC No. CRL-1721) (murine
neuronal cell model; rat pheochromocytoma); and MES13 (ATCC No.
CRL-1927) (murine kidney mesangial cell line). In addition, some
cell lines express zcytor11 (IL-TIF receptor) are also candidates
for responsive cell lines to IL-TIF: A549 (ATCC No. CCL-185) (human
lung carcinoma); G-361 (ATCC No. CRL-1424) (human melanoma); and
Caki-1 (ATCC No. HTB-46) (human renal carcinoma). These cells can
be used in assays to assess the functionality of zcytor16 as an
IL-TIF antagonist or anti-inflammatory factor.
[0180] An additional screening approach provided by the present
invention includes the use of hybrid receptor polypeptides. These
hybrid polypeptides fall into two general classes. Within the first
class, the intracellular domain of Zcytor16, 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 (e.g., TPO in the case the mpl receptor
extracellular domain is used) and assayed for a response. This
system provides a means for analyzing signal transduction mediated
by Zcytor16 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 Zcytor16 monomeric,
homodimeric, heterodimeric and multimeric receptors of the present
invention.
[0181] A second class of hybrid receptor polypeptides comprise the
extracellular (ligand-binding) domain of Zcytor16 (approximately
residues 23 to 231 of SEQ ID NO:2; SEQ ID NO:13) with an
intracellular domain of a second receptor, preferably a
hematopoietic cytokine receptor, and a transmembrane domain. Hybrid
zcytor11 monomers, homodimers, heterodimers and multimers of the
present invention receptors of this second class are expressed in
cells known to be capable of responding to signals transduced by
the second receptor. Together, these two classes of hybrid
receptors enable the identification of a responsive cell type for
the development of an assay for detecting IL-TIF. Moreover, such
cells can be used in the presence of IL-TIF to assay the soluble
receptor antagonists of the present invention in a competition-type
assay. In such assay, a decrease in the proliferation or signal
transduction activity of IL-TIF in the presence of a soluble
receptor of the present invention demonstrates antagonistic
activity. Moreover IL-TIF-soluble receptor binding assays can also
be used to assess whether a soluble receptor antagonizes IL-TIF
activity.
7. Production of Zcytor16 Fusion Proteins and Conjugates
[0182] One general class of Zcytor16 analogs are variants having an
amino acid sequence that is a mutation of the amino acid sequence
disclosed herein. Another general class of Zcytor16 analogs is
provided by anti-idiotype antibodies, and fragments thereof, as
described below. Moreover, recombinant antibodies comprising
anti-idiotype variable domains can be used as analogs (see, for
example, Monfardini et al., Proc. Assoc. Am. Physicians 108:420
(1996)). Since the variable domains of anti-idiotype Zcytor16
antibodies mimic Zcytor16, these domains can provide Zcytor16
binding activity. Methods of producing anti-idiotypic catalytic
antibodies are known to those of skill in the art (see, for
example, Joron et al., Ann. N Y Acad. Sci. 672:216 (1992),
Friboulet et al., Appl Biochem. Biotechnol 47:229 (1994), and
Avalle et al., Ann. N Y Acad. Sci. 864:118 (1998)).
[0183] Another approach to identifying Zcytor16 analogs is provided
by the use of combinatorial libraries. Methods for constructing and
screening phage display and other combinatorial libraries are
provided, for example, by Kay et al., Phage Display of Peptides and
Proteins (Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384,
Kay, et. al., U.S. Pat. No. 5,747,334, and Kauffman et al., U.S.
Pat. No. 5,723,323.
[0184] Zcytor16 polypeptides have both in vivo and in vitro uses.
As an illustration, a soluble form of Zcytor16 can be added to cell
culture medium to inhibit the effects of the Zcytor16 ligand
produced by the cultured cells.
[0185] Fusion proteins of Zcytor16 can be used to express Zcytor16
in a recombinant host, and to isolate the produced Zcytor16. As
described below, particular Zcytor16 fusion proteins also have uses
in diagnosis and therapy. One type of fusion protein comprises a
peptide that guides a Zcytor16 polypeptide from a recombinant host
cell. To direct a Zcytor16 polypeptide into the secretory pathway
of a eukaryotic host cell, a secretory signal sequence (also known
as a signal peptide, a leader sequence, prepro sequence or pre
sequence) is provided in the Zcytor16 expression vector. While the
secretory signal sequence may be derived from Zcytor16, a suitable
signal sequence may also be derived from another secreted protein
or synthesized de novo. The secretory signal sequence is operably
linked to a Zcytor16-encoding sequence such that 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 nucleotide sequence encoding the polypeptide of interest,
although certain secretory signal sequences may be positioned
elsewhere in the nucleotide 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).
[0186] Although the secretory signal sequence of Zcytor16 or
another protein produced by mammalian cells (e.g., tissue-type
plasminogen activator signal sequence, as described, for example,
in U.S. Pat. No. 5,641,655) is useful for expression of Zcytor16 in
recombinant mammalian hosts, a yeast signal sequence is preferred
for expression in yeast cells. Examples of suitable yeast signal
sequences are those derived from yeast mating pheromone
.alpha.-factor (encoded by the MF.alpha.1 gene), invertase (encoded
by the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene).
See, for example, Romanos et al., "Expression of Cloned Genes in
Yeast," in DNA Cloning 2: A Practical Approach, 2.sup.nd Edition,
Glover and Hames (eds.), pages 123-167 (Oxford University Press
1995).
[0187] Zcytor16 monomeric, homodimeric, heterodimeric and
multimeric receptor polypeptides can be prepared by expressing a
truncated DNA encoding the extracellular domain, for example, a
polypeptide which contains SEQ ID NO:13, amino acids 22-210 of SEQ
ID NO:2, or the corresponding region of a non-human receptor. It is
preferred that the extracellular domain polypeptides be prepared in
a form substantially free of transmembrane and intracellular
polypeptide segments. To direct the export of the receptor domain
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. To facilitate purification of the secreted receptor
domain, a C-terminal extension, such as a poly-histidine tag,
substance P, Flag.TM. peptide (Hopp et al., Biotechnology
6:1204-1210, (1988); available from Eastman Kodak Co., New Haven,
Conn.) or another polypeptide or protein for which an antibody or
other specific binding agent is available, can be fused to the
receptor polypeptide. Moreover, heterodimeric and multimeric
non-zcytor16 subunit extracellular cytokine binding domains are a
also prepared as above.
[0188] In an alternative approach, a receptor extracellular domain
of zcytor16 or other class I or II cytokine receptor component can
be expressed as a fusion with immunoglobulin heavy chain constant
regions, typically an F.sub.c fragment, which contains two constant
region domains and a hinge region but lacks the variable region
(See, Sledziewski, A Z et al., U.S. Pat. Nos. 6,018,026 and
5,750,375). The soluble zcytor16, soluble zcytor16/CRF2-4
heterodimers, and monomeric, homodimeric, heterodimeric and
multimeric polypeptides of the present invention include such
fusions. 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 closed 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 Zcytor16-Ig chimera is added to a sample containing the
ligand (e.g., cell-conditioned culture media or tissue extracts)
under conditions that facilitate receptor-ligand binding (typically
near-physiological temperature, pH, and ionic strength). The
chimera-ligand complex is then separated by the mixture using
protein A, which is immobilized on a solid support (e.g., insoluble
resin beads). The ligand is then eluted using conventional chemical
techniques, such as with a salt or pH gradient. In the alternative,
the chimera itself can be bound to a solid support, with binding
and elution carried out as above. The chimeras may be used in vivo
to regulate inflammatory responses including acute phase responses
such as serum amyloid A (SAA), C-reactive protein (CRP), and the
like. Chimeras with high binding affinity are administered
parenterally (e.g., by intramuscular, subcutaneous or intravenous
injection). Circulating molecules bind ligand and are cleared from
circulation by normal physiological processes. For use in assays,
the chimeras are bound to a support via the F.sub.c region and used
in an ELISA format.
[0189] 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 zcytor16
receptor or soluble zcytor16 heterodimeric polypeptide, such as
soluble zcytor16/CRF2-4, 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 zcytor16 receptor
or immunoglobulin-soluble zcytor16 heterodimeric or multimeric
polypeptide, such as immunoglobulin-soluble zcytor16/CRF2-4 fusions
can be expressed in genetically engineered cells to produce a
variety of multimeric zcytor16 receptor analogs. Auxiliary domains
can be fused to soluble zcytor16 receptor or soluble zcytor16
heterodimeric or multimeric polypeptides, such as soluble
zcytor16/CRF2-4 to target them to specific cells, tissues, or
macromolecules (e.g., collagen, or cells expressing the zcytor16
ligand, IL-TIF). A zcytor16 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.
[0190] In bacterial cells, it is often desirable to express a
heterologous protein as a fusion protein to decrease toxicity,
increase stability, and to enhance recovery of the expressed
protein. For example, Zcytor16 can be expressed as a fusion protein
comprising a glutathione S-transferase polypeptide. Glutathione
S-transferease fusion proteins are typically soluble, and easily
purifiable from E. coli lysates on immobilized glutathione columns.
In similar approaches, a Zcytor16 fusion protein comprising a
maltose binding protein polypeptide can be isolated with an amylose
resin column, while a fusion protein comprising the C-terminal end
of a truncated Protein A gene can be purified using IgG-Sepharose.
Established techniques for expressing a heterologous polypeptide as
a fusion protein in a bacterial cell are described, for example, by
Williams et al., "Expression of Foreign Proteins in E. coli Using
Plasmid Vectors and Purification of Specific Polyclonal
Antibodies," in DNA Cloning 2: A Practical Approach, 2.sup.nd
Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University
Press 1995). In addition, commercially available expression systems
are available. For example, the PINPOINT Xa protein purification
system (Promega Corporation; Madison, Wis.) provides a method for
isolating a fusion protein comprising a polypeptide that becomes
biotinylated during expression with a resin that comprises
avidin.
[0191] Peptide tags that are useful for isolating heterologous
polypeptides expressed by either prokaryotic or eukaryotic cells
include polyHistidine tags (which have an affinity for
nickel-chelating resin), c-myc tags, calmodulin binding protein
(isolated with calmodulin affinity chromatography), substance P,
the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu
tag, and the FLAG tag (which binds with anti-FLAG antibodies). See,
for example, Luo et al., Arch. Biochem. Biophys. 329:215 (1996),
Morganti et al., Biotechnol Appl Biochem. 23:67 (1996), and Zheng
et al., Gene 186:55 (1997). Nucleic acid molecules encoding such
peptide tags are available, for example, from Sigma-Aldrich
Corporation (St. Louis, Mo.).
[0192] The present invention also contemplates that the use of the
secretory signal sequence contained in the Zcytor16 polypeptides of
the present invention to direct other polypeptides into the
secretory pathway. A signal fusion polypeptide can be made wherein
a secretory signal sequence derived from amino acid residues 1 to
21, or 1 to 22, of SEQ ID NO:2 is operably linked to another
polypeptide using methods known in the art and disclosed herein.
The secretory signal sequence contained in the fusion polypeptides
of the present invention is preferably fused amino-terminally to an
additional peptide to direct the additional peptide into the
secretory pathway. Such constructs have numerous applications known
in the art. For example, these novel secretory signal sequence
fusion constructs can direct the secretion of an active component
of a normally non-secreted protein, such as a receptor. Such
fusions may be used in a transgenic animal or in a cultured
recombinant host to direct peptides through the secretory pathway.
With regard to the latter, exemplary polypeptides include
pharmaceutically active molecules such as Factor VIIa, proinsulin,
insulin, follicle stimulating hormone, tissue type plasminogen
activator, tumor necrosis factor, interleukins (e.g., interleukin-1
(IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, and IL-15), colony stimulating factors
(e.g., granulocyte-colony stimulating factor (G-CSF) and
granulocyte macrophage-colony stimulating factor (GM-CSF)),
interferons (e.g., interferons-.alpha., -.beta., -.gamma.,
-.omega., -.delta., and -.tau.), the stem cell growth factor
designated "S1 factor," erythropoietin, and thrombopoietin. The
Zcytor16 secretory signal sequence contained in the fusion
polypeptides of the present invention is preferably fused
amino-terminally to an additional peptide to direct the additional
peptide into the secretory pathway. Fusion proteins comprising a
Zcytor16 secretory signal sequence can be constructed using
standard techniques.
[0193] Another form of fusion protein comprises a Zcytor16
polypeptide and an immunoglobulin heavy chain constant region,
typically an F.sub.c fragment, which contains two or three constant
region domains and a hinge region but lacks the variable region. As
an illustration, Chang et al., U.S. Pat. No. 5,723,125, describe a
fusion protein comprising a human interferon and a human
immunoglobulin Fc fragment. The C-terminal of the interferon is
linked to the N-terminal of the Fc fragment by a peptide linker
moiety. An example of a peptide linker is a peptide comprising
primarily a T cell inert sequence, which is immunologically inert.
An exemplary peptide linker has the amino acid sequence: GGSGG
SGGGG SGGGG S (SEQ ID NO:4). In this fusion protein, an
illustrative Fc moiety is a human .gamma.4 chain, which is stable
in solution and has little or no complement activating activity.
Accordingly, the present invention contemplates a Zcytor16 fusion
protein that comprises a Zcytor16 moiety and a human Fc fragment,
wherein the C-terminus of the Zcytor16 moiety is attached to the
N-terminus of the Fc fragment via a peptide linker, such as a
peptide consisting of the amino acid sequence of SEQ ID NO:4. The
Zcytor16 moiety can be a Zcytor16 molecule or a fragment thereof.
For example, a fusion protein can comprise amino acid residues 22
to 231, or 28 to 231 of SEQ ID NO:2 and an Fc fragment (e.g., a
human Fc fragment).
[0194] In another variation, a Zcytor16 fusion protein comprises an
IgG sequence, a Zcytor16 moiety covalently joined to the
aminoterminal end of the IgG sequence, and a signal peptide that is
covalently joined to the aminoterminal of the Zcytor16 moiety,
wherein the IgG sequence consists of the following elements in the
following order: a hinge region, a CH.sub.2 domain, and a CH.sub.3
domain. Accordingly, the IgG sequence lacks a CH.sub.1 domain. The
Zcytor16 moiety displays a Zcytor16 activity, as described herein,
such as the ability to bind with a Zcytor16 ligand. This general
approach to producing fusion proteins that comprise both antibody
and nonantibody portions has been described by LaRochelle et al.,
EP 742830 (WO 95/21258).
[0195] Fusion proteins comprising a Zcytor16 moiety and an Fc
moiety can be used, for example, as an in vitro assay tool. For
example, the presence of a Zcytor16 ligand in a biological sample
can be detected using a Zcytor16-immunoglobulin fusion protein, in
which the Zcytor16 moiety is used to bind the ligand, and a
macromolecule, such as Protein A or anti-Fc antibody, is used to
bind the fusion protein to a solid support. Such systems can be
used to identify agonists and antagonists that interfere with the
binding of a Zcytor16 ligand to its receptor.
[0196] Other examples of antibody fusion proteins include
polypeptides that comprise an antigen-binding domain and a Zcytor16
fragment that contains a Zcytor16 extracellular domain. Such
molecules can be used to target particular tissues for the benefit
of Zcytor16 binding activity.
[0197] The present invention further provides a variety of other
polypeptide fusions. For example, part or all of a domain(s)
conferring a biological function can be swapped between Zcytor16 of
the present invention with the functionally equivalent domain(s)
from another member of the cytokine receptor family. Polypeptide
fusions can be expressed in recombinant host cells to produce a
variety of Zcytor16 fusion analogs. A Zcytor16 polypeptide can be
fused to two or more moieties or domains, 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, for example, Tuan et al., Connective Tissue Research
34:1 (1996).
[0198] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. General methods for
enzymatic and chemical cleavage of fusion proteins are described,
for example, by Ausubel (1995) at pages 16-19 to 16-25.
[0199] Zcytor16 polypeptides can be used to identify and to isolate
Zcytor16 ligands. For example, proteins and peptides of the present
invention can be immobilized on a column and used to bind ligands
from a biological sample that is run over the column (Hermanson et
al. (eds.), Immobilized Affinity Ligand Techniques, pages 195-202
(Academic Press 1992)). As such, zcytor16 polypeptides of the
present invention can be used to identify and isolate IL-TIF for
either diagnostic, or production purposes.
[0200] The activity of a Zcytor16 polypeptide can also be observed
by a silicon-based biosensor microphysiometer, which measures the
extracellular acidification rate or proton excretion associated
with receptor binding and subsequent physiologic cellular
responses. An exemplary device is the CYTOSENSOR Microphysiometer
manufactured by Molecular Devices, Sunnyvale, Calif. A variety of
cellular responses, such as cell proliferation, ion transport,
energy production, inflammatory response, regulatory and receptor
activation, and the like, can be measured by this method (see, for
example, McConnell et al., Science 257:1906 (1992), Pitchford et
al., Meth. Enzymol 228:84 (1997), Arimilli et al., J. Immunol.
Meth. 212:49 (1998), Van Liefde et al., Eur. J. Pharmacol 346:87
(1998)). The microphysiometer can be used for assaying eukaryotic,
prokaryotic, adherent, or non-adherent cells. By measuring
extracellular acidification changes in cell media over time, the
microphysiometer directly measures cellular responses to various
stimuli, including agonists, ligands, or antagonists of
Zcytor16.
[0201] For example, the microphysiometer is used to measure
responses of an Zcytor16-expressing eukaryotic cell, compared to a
control eukaryotic cell that does not express Zcytor16 polypeptide.
Suitable cells responsive to Zcytor16-modulating stimuli include
recombinant host cells comprising a Zcytor16 expression vector, and
cells that naturally express Zcytor16. Extracellular acidification
provides one measure for a Zcytor16-modulated cellular response. In
addition, this approach can be used to identify ligands, agonists,
and antagonists of Zcytor16 ligand, IL-TIF. For example, a molecule
can be identified as an agonist of Zcytor16 ligand by providing
cells that express a Zcytor16 polypeptide, culturing a first
portion of the cells in the absence of the test compound, culturing
a second portion of the cells in the presence of the test compound,
and determining whether the second portion exhibits a cellular
response, in comparison with the first portion.
[0202] Alternatively, a solid phase system can be used to identify
a Zcytor16 ligand, or an agonist or antagonist of a Zcytor16
ligand. For example, a Zcytor16 polypeptide or Zcytor16 fusion
protein, or zcytor16 monomeric, homodimeric, heterodimeric or
multimeric soluble receptor can be immobilized onto the surface of
a receptor chip of a commercially available biosensor instrument
(BIACORE, Biacore AB; Uppsala, Sweden). The use of this instrument
is disclosed, for example, by Karlsson, Immunol. Methods 145:229
(1991), and Cunningham and Wells, J. Mol. Biol 234:554 (1993).
[0203] In brief a Zcytor16 polypeptide or fusion protein is
covalently attached, using amine or sulfhydryl chemistry, to
dextran fibers that are attached to gold film within a flow cell. A
test sample is then passed through the cell. If a ligand is present
in the sample, it will bind to the immobilized polypeptide or
fusion protein, 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. This system can also be
used to examine antibody-antigen interactions, and the interactions
of other complement/anti-complement pairs.
[0204] Zcytor16 binding domains can be further characterized 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 of Zcytor16 ligand agonists. See, for
example, de Vos et al., Science 255:306 (1992), Smith et al., J.
Mol. Biol 224:899 (1992), and Wlodaver et al., FEBS Lett. 309:59
(1992).
[0205] The present invention also contemplates chemically modified
Zcytor16 compositions, in which a Zcytor16 polypeptide is linked
with a polymer. Illustrative Zcytor16 polypeptides are soluble
polypeptides that lack a functional transmembrane domain, such as a
polypeptide consisting of amino acid residues 22 to 231, or 28 to
231 of SEQ ID NO:2. Typically, the polymer is water soluble so that
the Zcytor16 conjugate does not precipitate in an aqueous
environment, such as a physiological environment. An example of a
suitable polymer is one that has been modified to have a single
reactive group, such as an active ester for acylation, or an
aldehyde for alkylation, In this way, the degree of polymerization
can be controlled. An example of a reactive aldehyde is
polyethylene glycol propionaldehyde, or mono-(C1-C10) alkoxy, or
aryloxy derivatives thereof (see, for example, Harris, et al., U.S.
Pat. No. 5,252,714). The polymer may be branched or unbranched.
Moreover, a mixture of polymers can be used to produce Zcytor16
conjugates.
[0206] Zcytor16 conjugates used for therapy can comprise
pharmaceutically acceptable water-soluble polymer moieties.
Suitable water-soluble polymers include polyethylene glycol (PEG),
monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG,
poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG
propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol,
dextran, cellulose, or other carbohydrate-based polymers. Suitable
PEG may have a molecular weight from about 600 to about 60,000,
including, for example, 5,000, 12,000, 20,000 and 25,000. A
Zcytor16 conjugate can also comprise a mixture of such
water-soluble polymers.
[0207] One example of a Zcytor16 conjugate comprises a Zcytor16
moiety and a polyalkyl oxide moiety attached to the N-terminus of
the Zcytor16 moiety. PEG is one suitable polyalkyl oxide. As an
illustration, Zcytor16 can be modified with PEG, a process known as
"PEGylation." PEGylation of Zcytor16 can be carried out by any of
the PEGylation reactions known in the art (see, for example, EP 0
154 316, Delgado et al., Critical Reviews in Therapeutic Drug
Carrier Systems 9:249 (1992), Duncan and Spreafico, Clin.
Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol
68:1 (1998)). For example, PEGylation can be performed by an
acylation reaction or by an alkylation reaction with a reactive
polyethylene glycol molecule. In an alternative approach, Zcytor16
conjugates are formed by condensing activated PEG, in which a
terminal hydroxy or amino group of PEG has been replaced by an
activated linker (see, for example, Karasiewicz et al., U.S. Pat.
No. 5,382,657).
[0208] PEGylation by acylation typically requires reacting an
active ester derivative of PEG with a Zcytor16 polypeptide. An
example of an activated PEG ester is PEG esterified to
N-hydroxysuccinimide. As used herein, the term "acylation" includes
the following types of linkages between Zcytor16 and a water
soluble polymer: amide, carbamate, urethane, and the like. Methods
for preparing PEGylated Zcytor16 by acylation will typically
comprise the steps of (a) reacting a Zcytor16 polypeptide with PEG
(such as a reactive ester of an aldehyde derivative of PEG) under
conditions whereby one or more PEG groups attach to Zcytor16, and
(b) obtaining the reaction product(s). Generally, the optimal
reaction conditions for acylation reactions will be determined
based upon known parameters and desired results. For example, the
larger the ratio of PEG:Zcytor16, the greater the percentage of
polyPEGylated Zcytor16 product.
[0209] The product of PEGylation by acylation is typically a
polyPEGylated Zcytor16 product, wherein the lysine .epsilon.-amino
groups are PEGylated via an acyl linking group. An example of a
connecting linkage is an amide. Typically, the resulting Zcytor16
will be at least 95% mono-, di-, or tri-pegylated, although some
species with higher degrees of PEGylation may be formed depending
upon the reaction conditions. PEGylated species can be separated
from unconjugated Zcytor16 polypeptides using standard purification
methods, such as dialysis, ultrafiltration, ion exchange
chromatography, affinity chromatography, and the like.
[0210] PEGylation by alkylation generally involves reacting a
terminal aldehyde derivative of PEG with Zcytor16 in the presence
of a reducing agent. PEG groups can be attached to the polypeptide
via a --CH.sub.2--NH group.
[0211] Derivatization via reductive alkylation to produce a
monoPEGylated product takes advantage of the differential
reactivity of different types of primary amino groups available for
derivatization. Typically, the reaction is performed at a pH that
allows one to take advantage of the pKa differences between the
.epsilon.-amino groups of the lysine residues and the .alpha.-amino
group of the N-terminal residue of the protein. By such selective
derivatization, attachment of a water-soluble polymer that contains
a reactive group such as an aldehyde, to a protein is controlled.
The conjugation with the polymer occurs predominantly at the
N-terminus of the protein without significant modification of other
reactive groups such as the lysine side chain amino groups. The
present invention provides a substantially homogenous preparation
of Zcytor16 monopolymer conjugates.
[0212] Reductive alkylation to produce a substantially homogenous
population of monopolymer Zcytor16 conjugate molecule can comprise
the steps of: (a) reacting a Zcytor16 polypeptide with a reactive
PEG under reductive alkylation conditions at a pH suitable to
permit selective modification of the .alpha.-amino group at the
amino terminus of the Zcytor16, and (b) obtaining the reaction
product(s). The reducing agent used for reductive alkylation should
be stable in aqueous solution and able to reduce only the Schiff
base formed in the initial process of reductive alkylation.
Illustrative reducing agents include sodium borohydride, sodium
cyanoborohydride, dimethylamine borane, trimethylamine borane, and
pyridine borane.
[0213] For a substantially homogenous population of monopolymer
Zcytor16 conjugates, the reductive alkylation reaction conditions
are those that permit the selective attachment of the water-soluble
polymer moiety to the N-terminus of Zcytor16. Such reaction
conditions generally provide for pKa differences between the lysine
amino groups and the .alpha.-amino group at the N-terminus. The pH
also affects the ratio of polymer to protein to be used. In
general, if the pH is lower, a larger excess of polymer to protein
will be desired because the less reactive the N-terminal
.alpha.-group, the more polymer is needed to achieve optimal
conditions. If the pH is higher, the polymer:Zcytor16 need not be
as large because more reactive groups are available. Typically, the
pH will fall within the range of 3 to 9, or 3 to 6. This method can
be employed for making zcytor16-comprising homodimeric,
heterodimeric or multimeric soluble receptor conjugates.
[0214] Another factor to consider is the molecular weight of the
water-soluble polymer. Generally, the higher the molecular weight
of the polymer, the fewer number of polymer molecules which may be
attached to the protein. For PEGylation reactions, the typical
molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to
about 50 kDa, or about 12 kDa to about 25 kDa. The molar ratio of
water-soluble polymer to Zcytor16 will generally be in the range of
1:1 to 100:1. Typically, the molar ratio of water-soluble polymer
to Zcytor16 will be 1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1
for monoPEGylation.
[0215] General methods for producing conjugates comprising a
polypeptide and water-soluble polymer moieties are known in the
art. See, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657,
Greenwald et al., U.S. Pat. No. 5,738,846, Nieforth et al., Clin.
Pharmacol. Ther. 59:636 (1996), Monkarsh et al., Anal. Biochem.
247:434 (1997)). This method can be employed for making
zcytor16-comprising homodimeric, heterodimeric or multimeric
soluble receptor conjugates.
[0216] The present invention contemplates compositions comprising a
peptide or polypeptide described herein. Such compositions can
further comprise a carrier. The carrier can be a conventional
organic or inorganic carrier. Examples of carriers include water,
buffer solution, alcohol, propylene glycol, macrogol, sesame oil,
corn oil, and the like.
8. Isolation of Zcytor16 Polypeptides
[0217] The polypeptides of the present invention can be purified to
at least about 80% purity, to at least about 90% purity, to at
least about 95% purity, or greater than 95% purity with respect to
contaminating macromolecules, particularly other proteins and
nucleic acids, and free of infectious and pyrogenic agents. The
polypeptides of the present invention may also be purified to a
pharmaceutically pure state, which is greater than 99.9% pure. In
certain preparations, purified polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin.
[0218] Fractionation and/or conventional purification methods can
be used to obtain preparations of Zcytor16 purified from natural
sources (e.g., tonsil tissue), synthetic Zcytor16 polypeptides, and
recombinant Zcytor16 polypeptides and fusion Zcytor16 polypeptides
purified from recombinant host cells. In general, 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 suitable. 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.
[0219] 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. Selection of a particular
method for polypeptide isolation and purification 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 1988), and
Doonan, Protein Purification Protocols (The Humana Press 1996).
[0220] Additional variations in Zcytor16 isolation and purification
can be devised by those of skill in the art. For example,
anti-Zcytor16 antibodies, obtained as described below, can be used
to isolate large quantities of protein by immunoaffinity
purification.
[0221] The polypeptides of the present invention can also be
isolated by exploitation of particular 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
(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 (M. Deutscher,
(ed.), Meth. Enzymol. 182:529 (1990)). 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, the ligand-binding properties of zcytor16
extracellular domain can be exploited for purification, for
example, of zcytor16-comprising soluble receptors; for example, by
using affinity chromatography wherein IL-TIF ligand is bound to a
column and the zcytor16-comprising receptor is bound and
subsequently eluted using standard chromatography methods.
[0222] Zcytor16 polypeptides or fragments thereof may also be
prepared through chemical synthesis, as described above. Zcytor16
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.
9. Production of Antibodies to Zcytor16 Proteins
[0223] Antibodies to Zcytor16 can be obtained, for example, using
the product of a Zcytor16 expression vector or Zcytor16 isolated
from a natural source as an antigen. Particularly useful
anti-Zcytor16 antibodies "bind specifically" with Zcytor16.
Antibodies are considered to be specifically binding if the
antibodies exhibit at least one of the following two properties:
(1) antibodies bind to Zcytor16 with a threshold level of binding
activity, and (2) antibodies do not significantly cross-react with
polypeptides related to Zcytor16.
[0224] With regard to the first characteristic, antibodies
specifically bind if they bind to a Zcytor16 polypeptide, peptide
or epitope with a binding affinity (K.sub.a) of 10.sup.6 M.sup.-1
or greater, preferably 10.sup.7 M.sup.-1 or greater, more
preferably 10.sup.8 M.sup.-1 or greater, and most preferably
10.sup.9 M.sup.-1 or greater. The binding affinity of an antibody
can be readily determined by one of ordinary skill in the art, for
example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci.
51:660 (1949)). With regard to the second characteristic,
antibodies do not significantly cross-react with related
polypeptide molecules, for example, if they detect Zcytor16, but
not presently known polypeptides using a standard Western blot
analysis. Examples of known related polypeptides include known
cytokine receptors.
[0225] Anti-Zcytor16 antibodies can be produced using antigenic
Zcytor16 epitope-bearing peptides and polypeptides. Antigenic
epitope-bearing peptides and polypeptides of the present invention
contain a sequence of at least nine, or between 15 to about 30
amino acids contained within SEQ ID NO:2 or another amino acid
sequence disclosed herein. However, peptides or polypeptides
comprising a larger portion of an amino acid sequence of the
invention, containing from 30 to 50 amino acids, or any length up
to and including the entire amino acid sequence of a polypeptide of
the invention, also are useful for inducing antibodies that bind
with Zcytor16. It is desirable that the amino acid sequence of the
epitope-bearing peptide is selected to provide substantial
solubility in aqueous solvents (i.e., the sequence includes
relatively hydrophilic residues, while hydrophobic residues are
typically avoided). Moreover, amino acid sequences containing
proline residues may be also be desirable for antibody
production.
[0226] As an illustration, potential antigenic sites in Zcytor16
were identified using the Jameson-Wolf method, Jameson and Wolf,
CABIOS 4:181, (1988), as implemented by the PROTEAN program
(version 3.14) of LASERGENE (DNASTAR; Madison, Wis.). Default
parameters were used in this analysis.
[0227] The Jameson-Wolf method predicts potential antigenic
determinants by combining six major subroutines for protein
structural prediction. Briefly, the Hopp-Woods method, Hopp et al.,
Proc. Nat'l Acad. Sci. USA 78:3824 (1981), was first used to
identify amino acid sequences representing areas of greatest local
hydrophilicity (parameter: seven residues averaged). In the second
step, Emini's method, Emini et al., J. Virology 55:836 (1985), was
used to calculate surface probabilities (parameter: surface
decision threshold (0.6)=1). Third, the Karplus-Schultz method,
Karplus and Schultz, Naturwissenschaften 72:212 (1985), was used to
predict backbone chain flexibility (parameter: flexibility
threshold (0.2)=1). In the fourth and fifth steps of the analysis,
secondary structure predictions were applied to the data using the
methods of Chou-Fasman, Chou, "Prediction of Protein Structural
Classes from Amino Acid Composition," in Prediction of Protein
Structure and the Principles of Protein Conformation, Fasman (ed.),
pages 549-586 (Plenum Press 1990), and Garnier-Robson, Garnier et
al., J. Mol. Biol. 120:97 (1978) (Chou-Fasman parameters:
conformation table=64 proteins; .alpha. region threshold=103;
.beta. region threshold=105; Garnier-Robson parameters: .alpha. and
.beta. decision constants=0). In the sixth subroutine, flexibility
parameters and hydropathy/solvent accessibility factors were
combined to determine a surface contour value, designated as the
"antigenic index." Finally, a peak broadening function was applied
to the antigenic index, which broadens major surface peaks by
adding 20, 40, 60, or 80% of the respective peak value to account
for additional free energy derived from the mobility of surface
regions relative to interior regions. This calculation was not
applied, however, to any major peak that resides in a helical
region, since helical regions tend to be less flexible.
[0228] The results of this analysis indicated that the following
amino acid sequences of SEQ ID NO:2 would provide suitable
antigenic peptides: amino acids 24 to 42 ("antigenic peptide 1"),
amino acids 24 to 33 ("antigenic peptide 2"), 37 to 42 ("antigenic
peptide 3"), amino acids 48 to 55 ("antigenic peptide 4"), amino
acids 68 to 81 ("antigenic peptide 5"), amino acids 88 to 97
("antigenic peptide 6"), amino acids 126 to 132 ("antigenic peptide
7"), amino acids 156 to 165 ("antigenic peptide 8"), amino acids
178 to 185 ("antigenic peptide 9"), and amino acids 216 to 227
("antigenic peptide 10"). The present invention contemplates the
use of any one of antigenic peptides 1 to 10 to generate antibodies
to Zcytor16. The present invention also contemplates polypeptides
comprising at least one of antigenic peptides 1 to 10.
[0229] Moreover, suitable antigens also include the zcytor16
polypeptides comprising a zcytor16 cytokine binding, or
extracellular domain disclosed above in combination with another
class I or II cytokine extracellular domain, such as those that
form soluble zcytor16 heterodimeric or multimeric polypeptides,
such as soluble zcytor16/CRF2-4, zcytor16/zcytor11,
zcytor16/zcytor7, and the like.
[0230] Polyclonal antibodies to recombinant Zcytor16 protein or to
Zcytor16 isolated from natural sources can be prepared using
methods well-known to those of skill in the art. See, for example,
Green et al., "Production of Polyclonal Antisera," in
Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press
1992), and Williams et al., "Expression of foreign proteins in E.
coli using plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995). The
immunogenicity of a Zcytor16 polypeptide can 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
Zcytor16 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.
[0231] Although polyclonal antibodies are typically raised in
animals such as horses, cows, dogs, chicken, rats, mice, rabbits,
guinea pigs, goats, or sheep, an anti-Zcytor16 antibody of the
present invention may also be derived from a subhuman primate
antibody. General techniques for raising diagnostically and
therapeutically useful antibodies in baboons may be found, for
example, in Goldenberg et al., international patent publication No.
WO 91/11465, and in Losman et al., Int. J. Cancer 46:310
(1990).
[0232] Alternatively, monoclonal anti-Zcytor16 antibodies can be
generated. Rodent mono-clonal antibodies to specific antigens may
be obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) ["Coligan"], Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)).
[0233] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a Zcytor16 gene product,
verifying the presence of antibody production by removing a serum
sample, removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0234] In addition, an anti-Zcytor16 antibody of the present
invention may be derived from a human monoclonal antibody. Human
monoclonal antibodies are obtained from transgenic mice that have
been engineered to produce specific human antibodies in response to
antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies specific for human antigens,
and the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described, for example, by Green et al., Nature Genet.
7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et
al., Int. Immun. 6:579 (1994).
[0235] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0236] For particular uses, it may be desirable to prepare
fragments of anti-Zcytor16 antibodies. Such antibody fragments can
be obtained, for example, by proteolytic hydrolysis of the
antibody. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. As an
illustration, antibody fragments can be produced by enzymatic
cleavage of antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent to produce 3.5 S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al.,
in Methods in Enzymology Vol 1, page 422 (Academic Press 1967), and
by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0237] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0238] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437 (1992)).
[0239] The Fv fragments may comprise V.sub.H and V.sub.L chains
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are connected by an oligonucleotide. The structural
gene is inserted into an expression vector which is subsequently
introduced into a host cell, such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science
242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et
al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
[0240] As an illustration, a scFV can be obtained by exposing
lymphocytes to Zcytor16 polypeptide in vitro, and selecting
antibody display libraries in phage or similar vectors (for
instance, through use of immobilized or labeled Zcytor16 protein or
peptide). Genes encoding polypeptides having potential Zcytor16
polypeptide binding domains can be obtained by screening random
peptide libraries displayed on phage (phage display) or on
bacteria, such as E. coli. Nucleotide sequences encoding the
polypeptides can be obtained in a number of ways, such as through
random mutagenesis and random polynucleotide synthesis. These
random peptide display libraries can be used to screen for peptides
which interact with a known target which can be a protein or
polypeptide, such as a ligand or receptor, a biological or
synthetic macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide display
libraries are known in the art (Ladner et al., U.S. Pat. No.
5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,
U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698,
and Kay et al., Phage Display of Peptides and Proteins (Academic
Press, Inc. 1996)) and random peptide display libraries and kits
for screening such libraries are available commercially, for
instance from CLONTECH Laboratories, Inc. (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
Zcytor16 sequences disclosed herein to identify proteins which bind
to Zcytor16.
[0241] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0242] Alternatively, an anti-Zcytor16 antibody may be derived from
a "humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA
89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer
et al., J. Immun. 150:2844 (1993), Sudhir (ed.), Antibody
Engineering Protocols (Humana Press, Inc. 1995), Kelley,
"Engineering Therapeutic Antibodies," in Protein Engineering
Principles and Practice, Cleland et al. (eds.), pages 399-434 (John
Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Pat. No.
5,693,762 (1997).
[0243] Polyclonal anti-idiotype antibodies can be prepared by
immunizing animals with anti-Zcytor16 antibodies or antibody
fragments, using standard techniques. See, for example, Green et
al., "Production of Polyclonal Antisera," in Methods In Molecular
Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively,
monoclonal anti-idiotype antibodies can be prepared using
anti-Zcytor16 antibodies or antibody fragments as immunogens with
the techniques, described above. As another alternative, humanized
anti-idiotype antibodies or subhuman primate anti-idiotype
antibodies can be prepared using the above-described techniques.
Methods for producing anti-idiotype antibodies are described, for
example, by Irie, U.S. Pat. No. 5,208,146, Greene, et. al., U.S.
Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol
77:1875 (1996).
10. Use of Zcytor16 Nucleotide Sequences to Detect Gene Expression
and Gene Structure
[0244] Nucleic acid molecules can be used to detect the expression
of a Zcytor16 gene in a biological sample. Suitable probe molecules
include double-stranded nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, or a portion thereof, as well
as single-stranded nucleic acid molecules having the complement of
the nucleotide sequence of SEQ ID NO:1, or a portion thereof. Probe
molecules may be DNA, RNA, oligonucleotides, and the like. As used
herein, the term "portion" refers to at least eight nucleotides to
at least 20 or more nucleotides. Illustrative probes bind with
regions of the Zcytor16 gene that have a low sequence similarity to
comparable regions in other cytokine receptor genes.
[0245] In a basic assay, a single-stranded probe molecule is
incubated with RNA, isolated from a biological sample, under
conditions of temperature and ionic strength that promote base
pairing between the probe and target Zcytor16 RNA species. After
separating unbound probe from hybridized molecules, the amount of
hybrids is detected.
[0246] In addition, as zcytor16 is spleen-specific, polynucleotide
probes, anti-zcytor16 antibodies, and detection the presence of
zcytor16 polypeptides in tissue can be used to assess whether
spleen tissue is present, for example, after surgery involving the
excision of a diseased or cancerous spleen. As such, the
polynucleotides, polypeptides, and antibodies of the present
invention can be used as an aid to determine whether all spleen
tissue is excised after surgery, for example, after surgery for
spleen cancer. In such instances, it is especially important to
remove all potentially diseased tissue to maximize recovery from
the cancer, and to minimize recurrence. Preferred embodiments
include fluorescent, radiolabeled, or calorimetrically labeled
antibodies, that can be used in situ.
[0247] Moreover, anti-zcytor16 antibodies and binding fragments can
be used for tagging and sorting cells that specifically-express
Zcytor16, such as mononuclear cells, lymphoid cells, e.g, activated
CD4+ T-cells and CD19+ B-cells, and other described herein. Such
methods of cell tagging and sorting are well known in the art (see,
e.g., "Molecular Biology of the Cell", 3.sup.rd Ed., Albert, B. et
al. (Garland Publishing, London & New York, 1994). One of skill
in the art would recognize the importance of separating cell tissue
types to study cells, and the use of antibodies to separate
specific cell tissue types. Basically, antibodies that bind to the
surface of a cell type are coupled to various matrices such as
collagen, polysaccharide beads, or plastic to form an affinity
surface to which only cells recognized by the antibodies will
adhere. The bound cells are then recovered by conventional
techniques. Other methods involve separating cells by a
fluorescence-activated cell sorter (FACS). In this technique one
labels cells with antibodies that are coupled to a fluorescent dye.
The labeled cells are then separated from unlabeled cells in a FACS
machine. In FACS sorting individual cells traveling in single file
pass through a laser beam and the fluorescence of each cell is
measured. Slightly further down-stream, tiny droplets, most
containing either one or no cells, are formed by a vibrating
nozzle. The droplets containing a single cell are automatically
give a positive or negative charge at the moment of formation,
depending on whether the cell they contain is fluorescent, and then
deflected by a strong electric field into an appropriate container.
Such machines can select 1 cell in 1000 and sort about 5000 cells
each second. This produces a uniform population of cells for cell
culture.
[0248] One of skill in the art would recognize that the antibodies
to the Zcytor16 polypeptides of the present invention are useful,
because not all tissue types express the Zcytor16 receptor and
because it is important that biologists be able to separate
specific cell types for further study and/or therapeutic
re-implantation into the body. This is particularly relevant in
cells such as immune cells, wherein zcytor16 is expressed.
[0249] Moreover, use of Zcytor16 polynucleotide probes,
anti-zcytor16 antibodies, and detection the presence of zcytor16
polypeptides in tissue can be used in the diagnosis and/or
prevention of spontaneous abortions, or to monitor placental health
and function. Since Zcytor16 is expressed in the placenta, it could
play a role in the critical functions of placenta, such as
proliferation or survival of trophoblast cells, and the like. Thus,
zcytor16 could be essential for the function of the placenta, thus
maturation of embryos. Therefore, a supplement of Zcytor16
polypeptide, or anti-zcytor16 antibodies may be beneficial in the
prevention and treatment of certain types of spontaneous abortions,
or premature birth of babies caused by abnormal expression of
Zcytor16 in the placenta, or as a diagnostic to assess the function
of the placenta. For example, as zcytor16 is normally expressed in
placenta, the absence of zcytor16 expression may be indicative of
abnormal placenta function.
[0250] Well-established hybridization methods of RNA detection
include northern analysis and dot/slot blot hybridization (see, for
example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et al. (eds.),
"Analysis of Gene Expression at the RNA Level," in Methods in Gene
Biotechnology, pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid
probes can be detectably labeled with radioisotopes such as
.sup.32P or .sup.35S. Alternatively, Zcytor16 RNA can be detected
with a nonradioactive hybridization method (see, for example, Isaac
(ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes
(Humana Press, Inc. 1993)). Typically, nonradioactive detection is
achieved by enzymatic conversion of chromogenic or chemiluminescent
substrates. Illustrative nonradioactive moieties include biotin,
fluorescein, and digoxigenin.
[0251] Zcytor16 oligonucleotide probes are also useful for in vivo
diagnosis. As an illustration, .sup.18F-labeled oligonucleotides
can be administered to a subject and visualized by positron
emission tomography (Tavitian et al., Nature Medicine 4:467
(1998)).
[0252] Numerous diagnostic procedures take advantage of the
polymerase chain reaction (PCR) to increase sensitivity of
detection methods. Standard techniques for performing PCR are
well-known (see, generally, Mathew (ed.), Protocols in Human
Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR
Protocols: Current Methods and Applications (Humana Press, Inc.
1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press,
Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols
(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR
(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis
(Humana Press, Inc. 1998)).
[0253] PCR primers can be designed to amplify a portion of the
Zcytor16 gene that has a low sequence similarity to a comparable
region in other proteins, such as other cytokine receptor
proteins.
[0254] One variation of PCR for diagnostic assays is reverse
transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is
isolated from a biological sample, reverse transcribed to cDNA, and
the cDNA is incubated with Zcytor16 primers (see, for example, Wu
et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR,"
in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc.
1997)). PCR is then performed and the products are analyzed using
standard techniques.
[0255] As an illustration, RNA is isolated from biological sample
using, for example, the gunadinium-thiocyanate cell lysis procedure
described above. Alternatively, a solid-phase technique can be used
to isolate mRNA from a cell lysate. A reverse transcription
reaction can be primed with the isolated RNA using random
oligonucleotides, short homopolymers of dT, or Zcytor16 anti-sense
oligomers. Oligo-dT primers offer the advantage that various mRNA
nucleotide sequences are amplified that can provide control target
sequences. Zcytor16 sequences are amplified by the polymerase chain
reaction using two flanking oligonucleotide primers that are
typically 20 bases in length.
[0256] PCR amplification products can be detected using a variety
of approaches. For example, PCR products can be fractionated by gel
electrophoresis, and visualized by ethidium bromide staining.
Alternatively, fractionated PCR products can be transferred to a
membrane, hybridized with a detectably-labeled Zcytor16 probe, and
examined by autoradiography. Additional alternative approaches
include the use of digoxigenin-labeled deoxyribonucleic acid
triphosphates to provide chemiluminescence detection, and the
C-TRAK calorimetric assay.
[0257] Another approach for detection of Zcytor16 expression is
cycling probe technology (CPT), in which a single-stranded DNA
target binds with an excess of DNA-RNA-DNA chimeric probe to form a
complex, the RNA portion is cleaved with RNAase H, and the presence
of cleaved chimeric probe is detected (see, for example, Beggs et
al., J. Clin. Microbiol. 34:2985 (1996), Bekkaoui et al.,
Biotechniques 20:240 (1996)). Alternative methods for detection of
Zcytor16 sequences can utilize approaches such as nucleic acid
sequence-based amplification (NASBA), cooperative amplification of
templates by cross-hybridization (CATCH), and the ligase chain
reaction (LCR) (see, for example, Marshall et al., U.S. Pat. No.
5,686,272 (1997), Dyer et al., J. Virol Methods 60:161 (1996),
Ehricht et al., Eur. J. Biochem. 243:358 (1997), and Chadwick et
al., J. Virol Methods 70:59 (1998)). Other standard methods are
known to those of skill in the art.
[0258] Zcytor16 probes and primers can also be used to detect and
to localize Zcytor16 gene expression in tissue samples. Methods for
such in situ hybridization are well-known to those of skill in the
art (see, for example, Choo (ed.), In Situ Hybridization Protocols
(Humana Press, Inc. 1994), Wu et al. (eds.), "Analysis of Cellular
DNA or Abundance of mRNA by Radioactive In Situ Hybridization
(RISH)," in Methods in Gene Biotechnology, pages 259-278 (CRC
Press, Inc. 1997), and Wu et al. (eds.), "Localization of DNA or
Abundance of mRNA by Fluorescence In Situ Hybridization (RISH)," in
Methods in Gene Biotechnology, pages 279-289 (CRC Press, Inc.
1997)). Various additional diagnostic approaches are well-known to
those of skill in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc.
1996), and Elles, Molecular Diagnosis of Genetic Diseases (Humana
Press, Inc., 1996)). Suitable test samples include blood, urine,
saliva, tissue biopsy, and autopsy material.
[0259] The Zcytor16 gene resides in human chromosome 6q23-q24
(e.g., Example 24). This region is associated with various
disorders, including insulin dependent diabetes mellitus, retinal
cone dystrophy, breast cancer, and Parkinson disease. Moreover,
defects in the zcytor16 locus itself may result in a heritable
human disease states as discussed herein. One of skill in the art
would appreciate that defects in cytokine receptors are known to
cause particular diseases in humans. For example, polymorphisms of
cytokine receptors are associated with pulmonary alveolar
proteinosis, familial periodic fever, and erythroleukemia.
Moreover, growth hormone receptor mutation results in dwarfism
(Amselem, S et al., New Eng. J. Med. 321: 989-995, 1989), IL-2
receptor gamma mutation results in severe combined immunodeficiency
(SCID) (Noguchi, M et al., Cell 73: 147-157, 1993), c-Mpl mutation
results in thrombocytopenia (Ihara, K et al., Proc. Nat. Acad. Sci.
96: 3132-3136, 1999), and severe mycobacterial and Salmonella
infections result in interleukin-12 receptor-deficient patients (de
Jong, R et al., Science 280: 1435-1438, 1998), amongst others.
Thus, similarly, defects in zcytor16 can cause a disease state or
susceptibility to disease or infection. As the Zcytor16 gene is
located at 6q23-q24, zcytor16 polynucleotide probes can be used to
detect chromosome 6q23-q24 loss, trisomy, duplication or
translocation associated with human diseases, such as inflammatory
diseases, chronic inflammation, dysfunction of inflammatory
response, immune cell cancers, bone marrow cancers, spleen cancers,
prostate cancer, thyroid, parathyroid or other cancers, or immune
diseases. Moreover, molecules of the present invention, such as the
polypeptides, antagonists, agonists, polynucleotides and antibodies
of the present invention would aid in the detection, diagnosis
prevention, and treatment associated with a zcytor16 genetic
defect. Thus, Zcytor16 nucleotide sequences can be used in
linkage-based testing for various diseases, and to determine
whether a subject's chromosomes contain a mutation in the Zcytor16
gene. Detectable chromosomal aberrations at the Zcytor16 gene locus
include, but are not limited to, aneuploidy, gene copy number
changes, loss of heterogeneity of 6q23-q24, translocation in
6q23-q24, insertions, deletions, restriction site changes and
rearrangements. Human diseases associated with such detectable
rearrangements are know in the art, for example see, OMIM.TM.,
National Center for Biotechnology Information, National Library of
Medicine, Bethesda, Md. gene map, e.g., translocations, LOH,
trisomy, and the like. Of particular interest are genetic
alterations that inactivate a Zcytor16 gene, or gross chromosomal
alterations in and around the zcytor16 locus.
[0260] Similarly, defects in the Zcytor16 gene itself may result in
a heritable human disease state. Moreover, one of skill in the art
would appreciate that defects in cytokine receptors are known to
cause disease states in humans. For example, growth hormone
receptor mutation results in dwarfism (Amselem, S et al., New Eng.
J. Med. 321: 989-995, 1989), IL-2 receptor gamma mutation results
in severe combined immunodeficiency (SCID) (Noguchi, M et al., Cell
73: 147-157, 1993), c-Mpl mutation results in thrombocytopenia
(Ihara, K et al., Proc. Nat. Acad. Sci. 96: 3132-3136, 1999), and
severe mycobacterial and Salmonella infections result in
interleukin-12 receptor-deficient patients (de Jong, R et al.,
Science 280: 1435-1438, 1998), amongst others. Thus, similarly,
defects in zcytor16 can cause a disease state or susceptibility to
disease or infection. As, zcytor16 is a cytokine receptor within a
chromosomal region where aberrations may be involved in cancer, and
is shown to be expressed in ovarian cancer, the molecules of the
present invention could also be directly involved in cancer
formation or metastasis. As the Zcytor16 gene is located at the
6q23-q24 region zcytor16, polynucleotide probes can be used to
detect chromosome 6q23-q24 loss, loss of heterogeneity (LOH),
trisomy, duplication or translocation associated with human
diseases, such as immune cell cancers, neuroblastoma, bone marrow
cancers, thyroid, parathyroid, prostate, melanoma, or other
cancers, or immune diseases. Moreover, molecules of the present
invention, such as the polypeptides, antagonists, agonists,
polynucleotides and antibodies of the present invention would aid
in the detection, diagnosis prevention, and treatment associated
with a Zcytor16 genetic defect.
[0261] A diagnostic could assist physicians in determining the type
of disease and appropriate associated therapy, or assistance in
genetic counseling. As such, the inventive anti-zcytor16
antibodies, polynucleotides, and polypeptides can be used for the
detection of zcytor16 polypeptide, mRNA or anti-zcytor16
antibodies, thus serving as markers and be directly used for
detecting or genetic diseases or cancers, as described herein,
using methods known in the art and described herein. Further,
zcytor16 polynucleotide probes can be used to detect abnormalities
or genotypes associated with chromosome 6q23-q24 deletions and
translocations associated with human diseases, other translocations
involved with malignant progression of tumors or other 6q23-q24
mutations, which are expected to be involved in chromosome
rearrangements in malignancy; or in other cancers, or in
spontaneous abortion. Similarly, zcytor16 polynucleotide probes can
be used to detect abnormalities or genotypes associated with
chromosome 6q23-q24 trisomy and chromosome loss associated with
human diseases. Thus, zcytor16 polynucleotide probes can be used to
detect abnormalities or genotypes associated with these
defects.
[0262] As discussed above, defects in the Zcytor16 gene itself may
result in a heritable human disease state. For example, zcytor16
expression is elevated in several tissue-specific human cancers, as
described herein. Molecules of the present invention, such as the
polypeptides, antagonists, agonists, polynucleotides and antibodies
of the present invention would aid in the detection, diagnosis
prevention, and treatment associated with a zcytor16 genetic
defect. In addition, zcytor16 polynucleotide probes can be used to
detect allelic differences between diseased or non-diseased
individuals at the zcytor16 chromosomal locus. As such, the
zcytor16 sequences can be used as diagnostics in forensic DNA
profiling.
[0263] In general, the diagnostic methods used in genetic linkage
analysis, to detect a genetic abnormality or aberration in a
patient, are known in the art. Analytical probes will be generally
at least 20 nt in length, although somewhat shorter probes can be
used (e.g., 14-17 nt). PCR primers are at least 5 nt in length,
preferably 15 or more, more preferably 20-30 nt. For gross analysis
of genes, or chromosomal DNA, a zcytor16 polynucleotide probe may
comprise an entire exon or more. Exons are readily determined by
one of skill in the art by comparing zcytor16 sequences (SEQ ID
NO:1) with the human genomic DNA for zcytor16. In general, the
diagnostic methods used in genetic linkage analysis, to detect a
genetic abnormality or aberration in a patient, are known in the
art. Most diagnostic methods comprise the steps of (a) obtaining a
genetic sample from a potentially diseased patient, diseased
patient or potential non-diseased carrier of a recessive disease
allele; (b) producing a first reaction product by incubating the
genetic sample with a zcytor16 polynucleotide probe wherein the
polynucleotide will hybridize to complementary polynucleotide
sequence, such as in RFLP analysis or by incubating the genetic
sample with sense and antisense primers in a PCR reaction under
appropriate PCR reaction conditions; (iii) Visualizing the first
reaction product by gel electrophoresis and/or other known method
such as visualizing the first reaction product with a zcytor16
polynucleotide probe wherein the polynucleotide will hybridize to
the complementary polynucleotide sequence of the first reaction;
and (iv) comparing the visualized first reaction product to a
second control reaction product of a genetic sample from wild type
patient. A difference between the first reaction product and the
control reaction product is indicative of a genetic abnormality in
the diseased or potentially diseased patient, or the presence of a
heterozygous recessive carrier phenotype for a non-diseased
patient, or the presence of a genetic defect in a tumor from a
diseased patient, or the presence of a genetic abnormality in a
fetus or pre-implantation embryo. For example, a difference in
restriction fragment pattern, length of PCR products, length of
repetitive sequences at the zcytor16 genetic locus, and the like,
are indicative of a genetic abnormality, genetic aberration, or
allelic difference in comparison to the normal wild type control.
Controls can be from unaffected family members, or unrelated
individuals, depending on the test and availability of samples.
Genetic samples for use within the present invention include
genomic DNA, mRNA, and cDNA isolated form any tissue or other
biological sample from a patient, such as but not limited to,
blood, saliva, semen, embryonic cells, amniotic fluid, and the
like. The polynucleotide probe or primer can be RNA or DNA, and
will comprise a portion of SEQ ID NO:1, the complement of SEQ ID
NO:1, or an RNA equivalent thereof. Such methods of showing genetic
linkage analysis to human disease phenotypes are well known in the
art. For reference to PCR based methods in diagnostics see see,
generally, Mathew (ed.), Protocols in Human Molecular Genetics
(Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current
Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.),
Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek
and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc.
1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc.
1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc.
1998)).
[0264] Aberrations associated with the Zcytor16 locus can be
detected using nucleic acid molecules of the present invention by
employing molecular genetic techniques, such as restriction
fragment length polymorphism (RFLP) analysis, short tandem repeat
(STR) analysis employing PCR techniques, amplification-refractory
mutation system analysis (ARMS), single-strand conformation
polymorphism (SSCP) detection, RNase cleavage methods, denaturing
gradient gel electrophoresis, fluorescence-assisted mismatch
analysis (FAMA), and other genetic analysis techniques known in the
art (see, for example, Mathew (ed.), Protocols in Human Molecular
Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),
Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana
Press, Inc. 1996), Landegren (ed.), Laboratory Protocols for
Mutation Detection (Oxford University Press 1996), Birren et al.
(eds.), Genome Analysis, Vol. 2: Detecting Genes (Cold Spring
Harbor Laboratory Press 1998), Dracopoli et al. (eds.), Current
Protocols in Human Genetics (John Wiley & Sons 1998), and
Richards and Ward, "Molecular Diagnostic Testing," in Principles of
Molecular Medicine, pages 83-88 (Humana Press, Inc. 1998)).
[0265] The protein truncation test is also useful for detecting the
inactivation of a gene in which translation-terminating mutations
produce only portions of the encoded protein (see, for example,
Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to this
approach, RNA is isolated from a biological sample, and used to
synthesize cDNA. PCR is then used to amplify the Zcytor16 target
sequence and to introduce an RNA polymerase promoter, a translation
initiation sequence, and an in-frame ATG triplet. PCR products are
transcribed using an RNA polymerase, and the transcripts are
translated in vitro with a T7-coupled reticulocyte lysate system.
The translation products are then fractionated by SDS-PAGE to
determine the lengths of the translation products. The protein
truncation test is described, for example, by Dracopoli et al.
(eds.), Current Protocols in Human Genetics, pages 9.11.1-9.11.18
(John Wiley & Sons 1998).
[0266] The present invention also contemplates kits for performing
a diagnostic assay for Zcytor16 gene expression or to detect
mutations in the Zcytor16 gene. Such kits comprise nucleic acid
probes, such as double-stranded nucleic acid molecules comprising
the nucleotide sequence of SEQ ID NO:1, or a portion thereof, as
well as single-stranded nucleic acid molecules having the
complement of the nucleotide sequence of SEQ ID NO:1, or a portion
thereof. Probe molecules may be DNA, RNA, oligonucleotides, and the
like. Kits may comprise nucleic acid primers for performing
PCR.
[0267] Such kits can contain all the necessary elements to perform
a nucleic acid diagnostic assay described above. A kit will
comprise at least one container comprising a Zcytor16 probe or
primer. The kit may also comprise a second container comprising one
or more reagents capable of indicating the presence of Zcytor16
sequences. Examples of such indicator reagents include detectable
labels such as radioactive labels, fluorochromes, chemiluminescent
agents, and the like. A kit may also comprise a means for conveying
to the user that the Zcytor16 probes and primers are used to detect
Zcytor16 gene expression. For example, written instructions may
state that the enclosed nucleic acid molecules can be used to
detect either a nucleic acid molecule that encodes Zcytor16, or a
nucleic acid molecule having a nucleotide sequence that is
complementary to a Zcytor16-encoding nucleotide sequence. The
written material can be applied directly to a container, or the
written material can be provided in the form of a packaging
insert.
11. Use of Anti-Zcytor16 Antibodies to Detect Zcytor16 or
Antagonize Zcytor16 Binding to IL-TIF
[0268] The present invention contemplates the use of anti-Zcytor16
antibodies to screen biological samples in vitro for the presence
of Zcytor16. In one type of in vitro assay, anti-Zcytor16
antibodies are used in liquid phase. For example, the presence of
Zcytor16 in a biological sample can be tested by mixing the
biological sample with a trace amount of labeled Zcytor16 and an
anti-Zcytor16 antibody under conditions that promote binding
between Zcytor16 and its antibody. Complexes of Zcytor16 and
anti-Zcytor16 in the sample can be separated from the reaction
mixture by contacting the complex with an immobilized protein which
binds with the antibody, such as an Fc antibody or Staphylococcus
protein A. The concentration of Zcytor16 in the biological sample
will be inversely proportional to the amount of labeled Zcytor16
bound to the antibody and directly related to the amount of free
labeled Zcytor16. Illustrative biological samples include blood,
urine, saliva, tissue biopsy, and autopsy material.
[0269] Alternatively, in vitro assays can be performed in which
anti-Zcytor16 antibody is bound to a solid-phase carrier. For
example, antibody can be attached to a polymer, such as
aminodextran, in order to link the antibody to an insoluble support
such as a polymer-coated bead, a plate or a tube. Other suitable in
vitro assays will be readily apparent to those of skill in the
art.
[0270] In another approach, anti-Zcytor16 antibodies can be used to
detect Zcytor16 in tissue sections prepared from a biopsy specimen.
Such immunochemical detection can be used to determine the relative
abundance of Zcytor16 and to determine the distribution of Zcytor16
in the examined tissue. General immunochemistry techniques are well
established (see, for example, Ponder, "Cell Marking Techniques and
Their Application," in Mammalian Development: A Practical Approach,
Monk (ed.), pages 115-38 (IRL Press 1987), Coligan at pages
5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley
Interscience 1990), and Manson (ed.), Methods In Molecular Biology,
Vol 10: Immunochemical Protocols (The Humana Press, Inc.
1992)).
[0271] Immunochemical detection can be performed by contacting a
biological sample with an anti-Zcytor16 antibody, and then
contacting the biological sample with a detectably labeled molecule
which binds to the antibody. For example, the detectably labeled
molecule can comprise an antibody moiety that binds to
anti-Zcytor16 antibody. Alternatively, the anti-Zcytor16 antibody
can be conjugated with avidin/streptavidin (or biotin) and the
detectably labeled molecule can comprise biotin (or
avidin/streptavidin). Numerous variations of this basic technique
are well-known to those of skill in the art.
[0272] Alternatively, an anti-Zcytor16 antibody can be conjugated
with a detectable label to form an anti-Zcytor16 immunoconjugate.
Suitable detectable labels include, for example, a radioisotope, a
fluorescent label, a chemiluminescent label, an enzyme label, a
bioluminescent label or colloidal gold. Methods of making and
detecting such detectably-labeled immunoconjugates are well-known
to those of ordinary skill in the art, and are described in more
detail below.
[0273] The detectable label can be a radioisotope that is detected
by autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C.
[0274] Anti-Zcytor16 immunoconjugates can also be labeled with a
fluorescent compound. The presence of a fluorescently-labeled
antibody is determined by exposing the immunoconjugate to light of
the proper wavelength and detecting the resultant fluorescence.
Fluorescent labeling compounds include fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine.
[0275] Alternatively, anti-Zcytor16 immunoconjugates can be
detectably labeled by coupling an antibody component to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged immunoconjugate is determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of chemiluminescent labeling compounds
include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt and an oxalate ester.
[0276] Similarly, a bioluminescent compound can be used to label
anti-Zcytor16 immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0277] Alternatively, anti-Zcytor16 immunoconjugates can be
detectably labeled by linking an anti-Zcytor16 antibody component
to an enzyme. When the anti-Zcytor16-enzyme conjugate is incubated
in the presence of the appropriate substrate, the enzyme moiety
reacts with the substrate to produce a chemical moiety which can be
detected, for example, by spectrophotometric, fluorometric or
visual means. Examples of enzymes that can be used to detectably
label polyspecific immunoconjugates include .beta.-galactosidase,
glucose oxidase, peroxidase and alkaline phosphatase.
[0278] Those of skill in the art will know of other suitable labels
which can be employed in accordance with the present invention. The
binding of marker moieties to anti-Zcytor16 antibodies can be
accomplished using standard techniques known to the art. Typical
methodology in this regard is described by Kennedy et al., Clin.
Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1
(1977), Shih et al., Int'l J. Cancer 46:1101 (1990), Stein et al.,
Cancer Res. 50:1330 (1990), and Coligan, supra.
[0279] Moreover, the convenience and versatility of immunochemical
detection can be enhanced by using anti-Zcytor16 antibodies that
have been conjugated with avidin, streptavidin, and biotin (see,
for example, Wilchek et al. (eds.), "Avidin-Biotin Technology,"
Methods In Enzymology, Vol 184 (Academic Press 1990), and Bayer et
al., "Immunochemical Applications of Avidin-Biotin Technology," in
Methods In Molecular Biology, Vol 10, Manson (ed.), pages 149-162
(The Humana Press, Inc. 1992).
[0280] Methods for performing immunoassays are well-established.
See, for example, Cook and Self, "Monoclonal Antibodies in
Diagnostic Immunoassays," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 180-208, (Cambridge University Press, 1995), Perry, "The Role
of Monoclonal Antibodies in the Advancement of Immunoassay
Technology," in Monoclonal Antibodies: Principles and Applications,
Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and
Diamandis, Immunoassay (Academic Press, Inc. 1996).
[0281] The present invention also contemplates kits for performing
an immunological diagnostic assay for Zcytor16 gene expression.
Such kits comprise at least one container comprising an
anti-Zcytor16 antibody, or antibody fragment. A kit may also
comprise a second container comprising one or more reagents capable
of indicating the presence of Zcytor16 antibody or antibody
fragments. Examples of such indicator reagents include detectable
labels such as a radioactive label, a fluorescent label, a
chemiluminescent label, an enzyme label, a bioluminescent label,
colloidal gold, and the like. A kit may also comprise a means for
conveying to the user that Zcytor16 antibodies or antibody
fragments are used to detect Zcytor16 protein. For example, written
instructions may state that the enclosed antibody or antibody
fragment can be used to detect Zcytor16. The written material can
be applied directly to a container, or the written material can be
provided in the form of a packaging insert.
[0282] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to soluble zcytor16 monomeric receptor or soluble zcytor16
homodimeric, heterodimeric or multimeric polypeptides, and
selection of antibody display libraries in phage or similar vectors
(for instance, through use of immobilized or labeled soluble
zcytor16 monomeric receptor or soluble zcytor16 homodimeric,
heterodimeric or multimeric polypeptides). Genes encoding
polypeptides having potential binding domains such as soluble
zcytor16 monomeric receptor or soluble zcytor16 homodimeric,
heterodimeric or multimeric 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 zcytor16 monomeric
receptor or soluble zcytor16 homodimeric, heterodimeric or
multimeric polypeptide sequences disclosed herein to identify
proteins which bind to zcytor16-comprising receptor polypeptides.
These "binding polypeptides," which interact with soluble
zcytor16-comprising receptor 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 IL-TIF 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 zcytor16-comprising
receptor polypeptides; for detecting or quantitating soluble or
non-soluble zcytor16-comprising receptors as marker of underlying
pathology or disease. These binding polypeptides can also act as
"antagonists" to block soluble or membrane-bound zcytor16 monomeric
receptor or zcytor16 homodimeric, heterodimeric or multimeric
polypeptide binding (e.g. to ligand) and signal transduction in
vitro and in vivo. Again, these binding polypeptides serve as
anti-zcytor16 monomeric receptor or anti-zcytor16 homodimeric,
heterodimeric or multimeric polypeptides and are useful for
inhibiting IL-TIF activity, as well as receptor activity or
protein-binding. Antibodies raised to the natural receptor
complexes of the present invention may be preferred embodiments, as
they may act more specifically against the IL-TIF, 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 IL-TIF proliferation, signal trap,
luciferase or binding assays in the presence of IL-TIF and
zcytor16-comprising soluble receptors, and other biological or
biochemical assays described herein.
[0283] Antibodies to monomeric zcytor16 receptor or zcytor16
homodimeric, heterodimeric or multimeric zcytor16-containing
receptors may be used for tagging cells that express zcytor16
receptors; for isolating soluble zcytor16-comprising receptor
polypeptides by affinity purification; for diagnostic assays for
determining circulating levels of soluble zcytor16-comprising
receptor polypeptides; for detecting or quantitating soluble
zcytor16-comprising receptors as marker of underlying pathology or
disease; in analytical methods employing FACS; for screening
expression libraries; for generating anti-idiotypic antibodies that
can act as IL-TIF agonists; and as neutralizing antibodies or as
antagonists to block zcytor16 receptor function, or to block IL-TIF
activity in vitro and in vivo. Suitable direct tags or labels
include radionuclides, enzymes, substrates, cofactors, biotin,
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 zcytor16-comprising receptor
polypeptides, or fragments thereof may be used in vitro to detect
denatured or non-denatured zcytor16-comprising receptor
polypeptides or fragments thereof in assays, for example, Western
Blots or other assays known in the art.
[0284] Antibodies to soluble zcytor16 receptor or soluble zcytor16
homodimeric, heterodimeric or multimeric receptor polypeptides 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
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 zcytor16 ligand, IL-TIF.
[0285] 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 zcytor16 receptor or soluble zcytor16 homodimeric,
heterodimeric or multimeric receptor 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
zcytor16-comprising soluble or membrane-bound receptor). More
specifically, antibodies to soluble zcytor16-comprising receptor
polypeptides, 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
zcytor16-comprising receptor such as zcytor16-expressing cancers,
or certain disease states.
[0286] Suitable detectable molecules may be directly or indirectly
attached to polypeptides that bind zcytor16-comprising receptor
polypeptides, such as "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.
[0287] 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.
[0288] In another embodiment, zcytor16 binding polypeptide-cytokine
or antibody-cytokine fusion proteins can be used for enhancing in
vivo killing of target tissues (for example, spleen, pancreatic,
blood, lymphoid, colon, and bone marrow cancers), if the binding
polypeptide-cytokine or anti-zcytor16 receptor antibody targets the
hyperproliferative cell (See, generally, Hornick et al., Blood
89:4437-47, 1997). The described fusion proteins enable targeting
of a cytokine to a desired site of action, thereby providing an
elevated local concentration of cytokine. Suitable anti-zcytor16
monomer, homodimer, heterodimer or multimer 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.
[0289] Alternatively, zcytor16 receptor binding polypeptides or
antibody fusion proteins described herein can be used for enhancing
in vivo killing of target tissues by directly stimulating a
zcytor16 receptor-modulated apoptotic pathway, resulting in cell
death of hyperproliferative cells expressing zcytor16-comprising
receptors.
12. Therapeutic Uses of Polypeptides Having Zcytor16 Activity
[0290] Amino acid sequences having Zcytor16 activity can be used to
modulate the immune system by binding Zcytor16 ligand, and thus,
preventing the binding of Zcytor16 ligand with endogenous Zcytor16
receptor. Zcytor16 antagonists, such as anti-Zcytor16 antibodies,
can also be used to modulate the immune system by inhibiting the
binding of Zcytor16 ligand with the endogenous Zcytor16 receptor.
Accordingly, the present invention includes the use of proteins,
polypeptides, and peptides having Zcytor16 activity (such as
Zcytor16 polypeptides, Zcytor16 analogs (e.g., anti-Zcytor16
anti-idiotype antibodies), and Zcytor16 fusion proteins) to a
subject which lacks an adequate amount of this polypeptide, or
which produces an excess of Zcytor16 ligand. Zcytor16 antagonists
(e.g., anti-Zcytor16 antibodies) can be also used to treat a
subject which produces an excess of either Zcytor16 ligand or
Zcytor16. Suitable subjects include mammals, such as humans.
[0291] Moreover, we have shown that the zcytor16 receptor binds a
ligand called T-cell inducible Factor (IL-TIF) (SEQ ID NO:15;
Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000;
mouse IL-TIF sequence is shown in Dumontier et al., J. Immunol.
164:1814-1819, 2000). Moreover, commonly owned zcytor11 (U.S. Pat.
No. 5,965,704) and CRF2-4 receptor also bind IL-TIF (See, WIPO
publication WO 00/24758; Dumontier et al., J. Immunol.
164:1814-1819, 2000; Spencer, S D et al., J. Exp. Med. 187:571-578,
1998; Gibbs, V C and Pennica Gene 186:97-101, 1997 (CRF2-4 cDNA);
Xie, M H et al., J. Biol. Chem. 275: 31335-31339, 2000; and
Kotenko, S V et al., J. Biol. Chem. manuscript in press
M007837200). Moreover, IL-10.beta. receptor may be involved as a
receptor for IL-TIF, and it is believed to be synonymous with
CRF2-4 (Dumoutier, L. et al., Proc. Nat'l. Acad. Sci.
97:10144-10149, 2000; Liu Y et al., J. Immunol. 152; 1821-1829,
1994 (IL-10R cDNA). Within preferred embodiments, the soluble
receptor form of zcytor16, residues 22-231 of SEQ ID NO:2, (SEQ ID
NO:13) is a monomer, homodimer, heterodimer, or multimer that
antagonizes the effects of IL-TIF in vivo. Antibodies and binding
polypeptides to such zcytor16 monomer, homodimer, heterodimer, or
multimers also serve as antagonists of zcytor16 activity.
[0292] IL-TIF has been shown to be induced in the presence of IL-9,
and is suspected to be involved in promoting Th1-type immune
responses, and inflammation. IL-9 stimulates proliferation,
activation, differentiation and/or induction of immune function in
a variety of ways and is implicated in asthma, lung mastocytosis,
and other diseases, as well as activates STAT pathways. Antagonists
of IL-TIF or IL-9 function can have beneficial use against such
human diseases. The present invention provides such novel
antagonists of IL-TIF.
[0293] IL-TIF has been show to be involved in up-regulate the
production of acute phase reactants, such as serum amyloid A (SAA),
.alpha.1-antichymotrypsin, and haptoglobin, and that IL-TIF
expression is increased upon injection of lipopolysaccharide (LPS)
in vivo suggesting that IL-TIF is involved in inflammatory response
(Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149,
2000). Production of acute phase proteins, such as SAA, is
considered s short-term survival mechanism where inflammation is
beneficial; however, maintenance of acute phase proteins for longer
periods contributes to chronic inflammation and can be harmful to
human health. For review, see Uhlar, C M and Whitehead, A S, Eur.
J. Biochem. 265:501-523, 1999, and Baumann H. and Gauldie, J.
Immunology Today 15:74-80, 1994. Moreover, the acute phase protein
SAA is implicated in the pathogenesis of several chronic
inflammatory diseases, is implicated in atherosclerosis and
rheumatoid arthritis, and is the precursor to the amyloid A protein
deposited in amyloidosis (Uhlar, C M and Whitehead, supra.). Thus,
as IL-TIF acts as a pro-inflammatory molecule and induces
production of SAA, antagonists would be useful in treating
inflammatory disease and other diseases associated with acute phase
response proteins induced by IL-TIF. Such antagonists are provided
by the present invention. For example, method of reducing
IL-TIF-induced or IL-9 induced inflammation comprises administering
to a mammal with inflammation an amount of a composition of soluble
zcytor16-comprising receptor sufficient to reduce inflammation.
Moreover, a method of suppressing an inflammatory response in a
mammal with inflammation can comprise: (1) determining a level of
serum amyloid A protein; (2) administering a composition comprising
a soluble zcytor16 cytokine receptor polypeptide as described
herein in an acceptable pharmaceutical vehicle; (3) determining a
post administration level of serum amyloid A protein; (4) comparing
the level of serum amyloid A protein in step (1) to the level of
serum amyloid A protein in step (3), wherein a lack of increase or
a decrease in serum amyloid A protein level is indicative of
suppressing an inflammatory response.
[0294] The receptors of the present invention include at least one
zcytor16 receptor subunit. A second receptor polypeptide included
in the heterodimeric soluble receptor belongs to the receptor
subfamily that includes Interleukin-10 receptor, the interferons
(e.g., interferon-gamma alpha and beta chains and the
interferon-alpha/beta receptor alpha and beta chains), zcytor7,
zcytor11, and CRF2-4. A second soluble receptor polypeptide
included in a heterodimeric soluble receptor can also include a
zcytor11 soluble receptor subunit, disclosed in the commonly owned
U.S. Pat. No. 5,965,704; an IL-10R subunit, such as IL-10R.alpha.;
or a zcytor7 soluble receptor subunit, disclosed in the commonly
owned U.S. Pat. No. 5,945,511. The zcytor11 receptor in conjunction
with CRF2-4 and IL-10 Receptor was shown to signal JAK-STAT pathway
in response to IL-TIF (Xie et al., supra.; Kotenko et al., supra.).
According to the present invention, in addition to a monomeric or
homodimeric zcytor16 receptor polypeptide, a heterodimeric soluble
zcytor16 receptor, as exemplified by an embodiment comprising a
soluble zcytor16 receptor+soluble CRF2-4 receptor heterodimer
(zcytor16/CRF2-4), can act as an antagonist of the IL-TIF. Other
embodiments include soluble heterodimers comprising zcytor16/IL-1
OR, zcytor16/IL-9R, zcytor16/zcytor11, zcytor16/zcytor7, and other
class II receptor subunits, as well as multimeric receptors
including but not limited to zcytor16/CRF2-4/zcytor11 or
zcytor16/CRF2-4/IL-10R.
[0295] Analysis of the tissue distribution of the mRNA
corresponding zcytor16 cDNA showed that mRNA level was highest in
placenta and spleen, and the ligand to which zcytor16 binds
(IL-TIF) is implicated in inducing inflammatory response including
induction of the acute-phase response (Dumoutier, L. et al., Proc.
Nat'l. Acad. Sci. 97:10144-10149, 2000). Thus, particular
embodiments of the present invention are directed toward use of
soluble zcytor16 heterodimers as antagonists in inflammatory and
immune diseases or conditions such as pancreatitis, type I diabetes
(IDDM), pancreatic cancer, pancreatitis, Graves Disease,
inflammatory bowel disease (IBD), Crohn's Disease, colon and
intestinal cancer, diverticulosis, autoimmune disease, sepsis,
organ or bone marrow transplant; inflammation due to trauma, sugery
or infection; amyloidosis; splenomegaly; graft versus host disease;
and where inhibition of inflammation, immune suppression, reduction
of proliferation of hematopoietic, immune, inflammatory or lymphoid
cells, macrophages, T-cells (including Th1 and Th2 cells),
suppression of immune response to a pathogen or antigen, or other
instances where inhibition of IL-TIF or IL-9 cytokine production is
desired.
[0296] Moreover, antibodies or binding polypeptides that bind
zcytor16 polypeptides, monomers, homodimers, heterodimers and
multimers described herein and/or zcytor16 polypeptides, monomers,
homodimers, heterodimers and multimers themselves are useful
to:
[0297] 1) Antagonize or block signaling via the IL-TIF receptors in
the treatment of acute inflammation, inflammation as a result of
trauma, tissue injury, surgery, sepsis or infection, and chronic
inflammatory diseases such as asthma, inflammatory bowel disease
(IBD), chronic colitis, splenomegaly, rheumatoid arthritis,
recurrent acute inflammatory episodes (e.g., tuberculosis), and
treatment of amyloidosis, and atherosclerosis, Castleman's Disease,
asthma, and other diseases associated with the induction of
acute-phase response.
[0298] 2) Antagonize or block signaling via the IL-TIF receptors in
the treatment of autoimmune diseases such as IDDM, multiple
sclerosis (MS), systemic Lupus erythematosus (SLE), myasthenia
gravis, rheumatoid arthritis, and IBD to prevent or inhibit
signaling in immune cells (e.g. lymphocytes, monocytes, leukocytes)
via zcytor16 (Hughes C et al., J. Immunol. 153: 3319-3325, 1994).
Alternatively antibodies, such as monoclonal antibodies (MAb) to
zcytor16-comprising receptors, can also be used as an antagonist to
deplete unwanted immune cells to treat autoimmune disease. Asthma,
allergy and other atopic disease may be treated with an MAb
against, for example, soluble zcytor16 soluble receptors or
zcytor16/CRF2-4 heterodimers, to inhibit the immune response or to
deplete offending cells. Blocking or inhibiting signaling via
zcytor16, using the polypeptides and antibodies of the present
invention, may also benefit diseases of the pancreas, kidney,
pituitary and neuronal cells. IDDM, NIDDM, pancreatitis, and
pancreatic carcinoma may benefit. Zcytor16 may serve as a target
for MAb therapy of cancer where an antagonizing MAb inhibits cancer
growth and targets immune-mediated killing. (Holliger P, and
Hoogenboom, H: Nature Biotech. 16: 1015-1016, 1998). Mabs to
soluble zcytor16 monomers, homodimers, heterodimers and multimers
may also be useful to treat nephropathies such as
glomerulosclerosis, membranous neuropathy, amyloidosis (which also
affects the kidney among other tissues), renal arteriosclerosis,
glomerulonephritis of various origins, fibroproliferative diseases
of the kidney, as well as kidney dysfunction associated with SLE,
IDDM, type II diabetes (NIDDM), renal tumors and other
diseases.
[0299] 3) Agonize or initiate signaling via the IL-TIF receptors in
the treatment of autoimmune diseases such as IDDM, MS, SLE,
myasthenia gravis, rheumatoid arthritis, and IBD. Anti-soluble
zcytor16, anti-soluble zcytor16/CRF2-4 heterodimers and multimer
monoclonal antibodies may signal lymphocytes or other immune cells
to differentiate, alter proliferation, or change production of
cytokines or cell surface proteins that ameliorate autoimmunity.
Specifically, modulation of a T-helper cell response to an
alternate pattern of cytokine secretion may deviate an autoimmune
response to ameliorate disease (Smith J A et al., J. Immunol.
160:4841-4849, 1998). Similarly, agonistic Anti-soluble zcytor16,
anti-solublezcytor16/CRF2-4 heterodimers and multimer monoclonal
antibodies may be used to signal, deplete and deviate immune cells
involved in asthma, allergy and atopoic disease. Signaling via
zcytor16 may also benefit diseases of the pancreas, kidney,
pituitary and neuronal cells. IDDM, NIDDM, pancreatitis, and
pancreatic carcinoma may benefit. Zcytor16 may serve as a target
for MAb therapy of pancreatic cancer where a signaling MAb inhibits
cancer growth and targets immune-mediated killing (Tutt, A L et
al., J. Immunol. 161: 3175-3185, 1998). Similarly renal cell
carcinoma may be treated with monoclonal antibodies to
zcytor16-comprising soluble receptors of the present invention.
[0300] Soluble zcytor16 monomeric, homodimeric, heterodimeric and
multimeric polypeptides described herein can be used to
neutralize/block IL-TIF activity in the treatment of autoimmune
disease, atopic disease, NIDDM, pancreatitis and kidney dysfunction
as described above. A soluble form of zcytor16 may be used to
promote an antibody response mediated by Th cells and/or to promote
the production of IL-4 or other cytokines by lymphocytes or other
immune cells.
[0301] The soluble zcytor16-comprising receptors of the present
invention are useful as antagonists of the IL-TIF cytokine. Such
antagonistic effects can be achieved by direct neutralization or
binding of the IL-TIF. In addition to antagonistic uses, the
soluble receptors of the present invention can bind IL-TIF and act
as carrier proteins for the IL-TIF cytokine, in order to transport
the Ligand to different tissues, organs, and cells within the body.
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. For example, in
acute infection or some cancers, benefit may result from induction
of inflammation and local acute phase response proteins by the
action of IL-TIF. Thus, the soluble receptors of the present
invention can be used to specifically direct the action of the
IL-TIF. See, Cosman, D. Cytokine 5: 95-106, 1993; and
Femandez-Botran, R. Exp. Opin. Invest. Drugs 9:497-513, 2000.
[0302] Moreover, the soluble receptors of the present invention can
be used to stabilize the IL-TIF, to increase the bioavailability,
therapeutic longevity, and/or efficacy of the Ligand by stabilizing
the Ligand from degradation or clearance, or by targeting the
ligand to a site of action within the body. For example the
naturally occurring IL-6/soluble IL-6R complex stabilizes IL-6 and
can signal through the gp130 receptor. See, Cosman, D. supra., and
Femandez-Botran, R. supra. Moreover, Zcytor16 may be combined with
a cognate ligand such as IL-TIF to comprise a ligand/soluble
receptor complex. Such complexes may be used to stimulate responses
from cells presenting a companion receptor subunit such as, for
example, zcytor11 or CRF2-4. The cell specificity of
zcytor16/ligand complexes may differ from that seen for the ligand
administered alone. Furthermore the complexes may have distinct
pharmacokinetic properties such as affecting half-life,
dose/response and organ or tissue specificity. ZcytoR16/IL-TIF
complexes thus may have agonist activity to enhance an immune
response or stimulate mesangial cells or to stimulate hepatic
cells. Alternatively only tissues expressing a signaling subunit
the heterodimerizes with the complex may be affected analogous to
the response to IL6/IL6R complexes (Hirota H. et al., Proc. Nat'l.
Acad. Sci. 92:4862-4866, 1995; Hirano, T. in Thomason, A. (Ed.)
"The Cytokine Handbook", 3.sup.rd Ed., p. 208-209). Soluble
receptor/cytokine complexes for IL12 and CNTF display similar
activities.
[0303] Moreover Inflammation is a protective response by an
organism to fend off an invading agent. Inflammation is a cascading
event that involves many cellular and humoral mediators. On one
hand, suppression of inflammatory responses can leave a host
immunocompromised; however, if left unchecked, inflammation can
lead to serious complications including chronic inflammatory
diseases (e.g., rheumatoid arthritis, multiple sclerosis,
inflammatory bowel disease and the like), septic shock and multiple
organ failure. Importantly, these diverse disease states share
common inflammatory mediators. The collective diseases that are
characterized by inflammation have a large impact on human
morbidity and mortality. Therefore it is clear that
anti-inflammatory proteins, such as zcytor16, could have crucial
therapeutic potential for a vast number of human and animal
diseases, from asthma and allergy to autoimmunity and septic
shock.
[0304] 1. Arthritis
[0305] Arthritis, including osteoarthritis, rheumatoid arthritis,
arthritic joints as a result of injury, and the like, are common
inflammatory conditions which would benefit from the therapeutic
use of anti-inflammatory proteins, such as zcytor16 polypeptides of
the present invention. For Example, rheumatoid arthritis (RA) is a
systemic disease that affects the entire body and is one of the
most common forms of arthritis. It is characterized by the
inflammation of the membrane lining the joint, which causes pain,
stiffness, warmth, redness and swelling. Inflammatory cells release
enzymes that may digest bone and cartilage. As a result of
rheumatoid arthritis, the inflamed joint lining, the synovium, can
invade and damage bone and cartilage leading to joint deterioration
and severe pain amongst other physiologic effects. The involved
joint can lose its shape and alignment, resulting in pain and loss
of movement.
[0306] Rheumatoid arthritis (RA) is an immune-mediated disease
particularly characterized by inflammation and subsequent tissue
damage leading to severe disability and increased mortality. A
variety of cytokines are produced locally in the rheumatoid joints.
Numerous studies have demonstrated that IL-1 and TNF-alpha, two
prototypic pro-inflammatory cytokines, play an important role in
the mechanisms involved in synovial inflammation and in progressive
joint destruction. Indeed, the administration of TNF-alpha and IL-1
inhibitors in patients with RA has led to a dramatic improvement of
clinical and biological signs of inflammation and a reduction of
radiological signs of bone erosion and cartilage destruction.
However, despite these encouraging results, a significant
percentage of patients do not respond to these agents, suggesting
that other mediators are also involved in the pathophysiology of
arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002).
One of those mediators could be IL-9 or IL-TIF, and as such a
molecule that binds or inhibits IL-TIF, such as zcytor16
polypeptides, soluble zcytor11/CRF2-4 receptor polypeptides, or
anti IL-TIF antibodies or binding partners, could serve as a
valuable therapeutic to reduce inflammation in rheumatoid
arthritis, and other arthritic diseases.
[0307] There are several animal models for rheumatoid arthritis
known in the art. For example, in the collagen-induced arthritis
(CIA) model, mice develop chronic inflammatory arthritis that
closely resembles human rheumatoid arthritis. Since CIA shares
similar immunological and pathological features with RA, this makes
it an ideal model for screening potential human anti-inflammatory
compounds. The CIA model is a well-known model in mice that depends
on both an immune response, and an inflammatory response, in order
to occur. The immune response comprises the interaction of B-cells
and CD4+ T-cells in response to collagen, which is given as
antigen, and leads to the production of anti-collagen antibodies.
The inflammatory phase is the result of tissue responses from
mediators of inflammation, as a consequence of some of these
antibodies cross-reacting to the mouse's native collagen and
activating the complement cascade. An advantage in using the CIA
model is that the basic mechanisms of pathogenesis are known. The
relevant T-cell and B-cell epitopes on type II collagen have been
identified, and various immunological (e.g., delayed-type
hypersensitivity and anti-collagen antibody) and inflammatory
(e.g., cytokines, chemokines, and matrix-degrading enzymes)
parameters relating to immune-mediated arthritis have been
determined, and can thus be used to assess test compound efficacy
in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20, 1999;
Williams et al., Immunol. 89:9784-788, 1992; Myers et al., Life
Sci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959,
1995).
[0308] The administration of soluble zcytor16 comprising
polypeptides, such as zcytor16-Fc4 or other zcytor16 soluble and
fusion proteins to these CIA model mice was used to evaluate the
use of zcytor16 to ameliorate symptoms and alter the course of
disease. Since the ligand of zcytor16, IL-TIF, induces production
of SAA, which is implicated in the pathogenesis of rheumatoid
arthritis, and zcytor16 was demonstrated to be able to inhibit
IL-TIF and SAA activity in vitro and in vivo, the systemic or local
administration of zcytor16 comprising polypeptides, such as
zcytor16-Fc4 or other zcytor16 soluble and fusion proteins can
potentially suppress the inflammatory response in RA. The injection
of 10 ug zcytor16-Fc (three times a week for 4 weeks) significantly
reduced the disease score (paw score, incident of inflammation or
disease). Other potential therapeutics include Zcytor16
polypeptides, soluble zcytor11/CRF2-4 receptor polypeptides, or
anti IL-TIF antibodies or binding partners, and the like.
[0309] 2. Endotoxemia
[0310] Endotoxemia is a severe condition commonly resulting from
infectious agents such as bacteria and other infectious disease
agents, sepsis, toxic shock syndrome, or in immunocompromised
patients subjected to opportunistic infections, and the like.
Therapeutically useful of anti-inflammatory proteins, such as
zcytor16 polypeptides of the present invention, could aid in
preventing and treating endotoxemia in humans and animals. Zcytor16
polypeptides, soluble zcytor11/CRF2-4 receptor polypeptides, or
anti IL-TIF antibodies or binding partners, could serve as a
valuable therapeutic to reduce inflammation and pathological
effects in endotoxemia.
[0311] Lipopolysaccharide (LPS) induced endotoxemia engages many of
the proinflammatory mediators that produce pathological effects in
the infectious diseases and LPS induced endotoxemia in rodents is a
widely used and acceptable model for studying the pharmacological
effects of potential pro-inflammatory or immunomodulating agents.
LPS, produced in gram-negative bacteria, is a major causative agent
in the pathogenesis of septic shock (Glausner et al., Lancet
338:732, 1991). A shock-like state can indeed be induced
experimentally by a single injection of LPS into animals. Molecules
produced by cells responding to LPS can target pathogens directly
or indirectly. Although these biological responses protect the host
against invading pathogens, they may also cause harm. Thus, massive
stimulation of innate immunity, occurring as a result of severe
Gram-negative bacterial infection, leads to excess production of
cytokines and other molecules, and the development of a fatal
syndrome, septic shock syndrome, which is characterized by fever,
hypotension, disseminated intravascular coagulation, and multiple
organ failure (Dumitru et al. Cell 103:1071-1083, 2000).
[0312] These toxic effects of LPS are mostly related to macrophage
activation leading to the release of multiple inflammatory
mediators. Among these mediators, TNF appears to play a crucial
role, as indicated by the prevention of LPS toxicity by the
administration of neutralizing anti-TNF antibodies (Beutler et al.,
Science 229:869, 1985). It is well established that 1 ug injection
of E. coli LPS into a C57B1/6 mouse will result in significant
increases in circulating IL-6, TNF-alpha, IL-1, and acute phase
proteins (for example, SAA) approximately 2 hours post injection.
The toxicity of LPS appears to be mediated by these cytokines as
passive immunization against these mediators can result in
decreased mortality (Beutler et al., Science 229:869, 1985). The
potential immunointervention strategies for the prevention and/or
treatment of septic shock include anti-TNF mAb, IL-1 receptor
antagonist, LIF, IL-10, and G-CSF.
[0313] The administration of soluble zcytor16 comprising
polypeptides, such as zcytor16-Fc4 or other zcytor16 soluble and
fusion proteins to these LPS-induced model was used to evaluate the
use of zcytor16 to ameliorate symptoms and alter the course of
LPS-induced disease. The model showed induction of IL-TIF by LPS
injection and the potential treatment of disease by zcytor16
polypeptides. Since LPS induces the production of pro-inflammatory
IL-TIF, SAA or other pro-inflammatory factors possibly contributing
to the pathology of endotoxemia, the neutralization of IL-TIF
activity, SAA or other pro-inflammatory factors by its antagonist
zcytor16 polypeptide can be used to reduce the symptoms of
endotoxemia, such as seen in endotoxic shock. Other potential
therapeutics include Zcytor16 polypeptides, soluble zcytor11/CRF2-4
receptor polypeptides, or anti IL-TIF antibodies or binding
partners, and the like.
[0314] 3 Inflammatory Bowel Disease, IBD
[0315] In the United States approximately 500,000 people suffer
from Inflammatory Bowel Disease (IBD) which can affect either colon
and rectum (Ulcerative colitis) or both, small and large intestine
(Crohn's Disease). The pathogenesis of these diseases is unclear,
but they involve chronic inflammation of the affected tissues.
Zcytor16 polypeptides, soluble zcytor11/CRF2-4 receptor
polypeptides, or anti IL-TIF antibodies or binding partners, could
serve as a valuable therapeutic to reduce inflammation and
pathological effects in IBD and related diseases.
[0316] Ulcerative colitis (UC) is an inflammatory disease of the
large intestine, commonly called the colon, characterized by
inflammation and ulceration of the mucosa or innermost lining of
the colon. This inflammation causes the colon to empty frequently,
resulting in diarrhea. Symptoms include loosening of the stool and
associated abdominal cramping, fever and weight loss. Although the
exact cause of UC is unknown, recent research suggests that the
body's natural defenses are operating against proteins in the body
which the body thinks are foreign (an "autoimmune reaction").
Perhaps because they resemble bacterial proteins in the gut, these
proteins may either instigate or stimulate the inflammatory process
that begins to destroy the lining of the colon. As the lining of
the colon is destroyed, ulcers form releasing mucus, pus and blood.
The disease usually begins in the rectal area and may eventually
extend through the entire large bowel. Repeated episodes of
inflammation lead to thickening of the wall of the intestine and
rectum with scar tissue. Death of colon tissue or sepsis may occur
with severe disease. The symptoms of ulcerative colitis vary in
severity and their onset may be gradual or sudden. Attacks may be
provoked by many factors, including respiratory infections or
stress.
[0317] Although there is currently no cure for UC available,
treatments are focused on suppressing the abnormal inflammatory
process in the colon lining. Treatments including corticosteroids
immunosuppressives (eg. azathioprine, mercaptopurine, and
methotrexate) and aminosalicytates are available to treat the
disease. However, the long-term use of immunosuppressives such as
corticosteroids and azathioprine can result in serious side effects
including thinning of bones, cataracts, infection, and liver and
bone marrow effects. In the patients in whom current therapies are
not successful, surgery is an option. The surgery involves the
removal of the entire colon and the rectum.
[0318] There are several animal models that can partially mimic
chronic ulcerative colitis. The most widely used model is the
2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis
model, which induces chronic inflammation and ulceration in the
colon. When TNBS is introduced into the colon of susceptible mice
via intra-rectal instillation, it induces T-cell mediated immune
response in the colonic mucosa, in this case leading to a massive
mucosal inflammation characterized by the dense infiltration of
T-cells and macrophages throughout the entire wall of the large
bowel. Moreover, this histopathologic picture is accompanies by the
clinical picture of progressive weight loss (wasting), bloody
diarrhea, rectal prolapse, and large bowel wall thickening (Neurath
et al. Intern. Rev. Immunol. 19:51-62, 2000).
[0319] Another colitis model uses dextran sulfate sodium (DSS),
which induces an acute colitis manifested by bloody diarrhea,
weight loss, shortening of the colon and mucosal ulceration with
neutrophil infiltration. DSS-induced colitis is characterized
histologically by infiltration of inflammatory cells into the
lamina propria, with lymphoid hyperplasia, focal crypt damage, and
epithelial ulceration. These changes are thought to develop due to
a toxic effect of DSS on the epithelium and by phagocytosis of
lamina propria cells and production of TNF-alpha and IFN-gamma.
Despite its common use, several issues regarding the mechanisms of
DSS about the relevance to the human disease remain unresolved. DSS
is regarded as a T cell-independent model because it is observed in
T cell-deficient animals such as SCID mice.
[0320] The administration of soluble zcytor16 comprising
polypeptides, such as zcytor16-Fc4 or other zcytor16 soluble and
fusion proteins to these TNBS or DSS models can be used to evaluate
the use of zcytor16 to ameliorate symptoms and alter the course of
gastrointestinal disease. We observed the increased expression of
IL-TIF in colon tissues of DSS-mice by RT-PCR, and the synergistic
activity of IL-TIF with IL-1beta on intestinal cell lines. It
indicates IL-TIF may play a role in the inflammatory response in
colitis, and the neutralization of IL-TIF activity by
administrating zcytor16 polypeptides is a potential therapeutic
approach for IBD. Other potential therapeutics include Zcytor16
polypeptides, soluble zcytor11/CRF2-4 receptor polypeptides, or
anti IL-TIF antibodies or binding partners, and the like.
[0321] 4. Psoriasis
[0322] Psoriasis is a chronic skin condition that affects more than
seven million Americans. Psoriasis occurs when new skin cells grow
abnormally, resulting in inflamed, swollen, and scaly patches of
skin where the old skin has not shed quickly enough. Plaque
psoriasis, the most common form, is characterized by inflamed
patches of skin ("lesions") topped with silvery white scales.
Psoriasis may be limited to a few plaques or involve moderate to
extensive areas of skin, appearing most commonly on the scalp,
knees, elbows and trunk. Although it is highly visible, psoriasis
is not a contagious disease. The pathogenesis of the diseases
involves chronic inflammation of the affected tissues. Zcytor16
polypeptides, soluble zcytor11/CRF2-4 receptor polypeptides, or
anti IL-TIF antibodies or binding partners, could serve as a
valuable therapeutic to reduce inflammation and pathological
effects in psoriasis, other inflammatory skin diseases, skin and
mucosal allergies, and related diseases.
[0323] Psoriasis is a T-cell mediated inflammatory disorder of the
skin that can cause considerable discomfort. It is a disease for
which there is no cure and affects people of all ages. Psoriasis
affects approximately two percent of the populations of European
and North America. Although individuals with mild psoriasis can
often control their disease with topical agents, more than one
million patients worldwide require ultraviolet or systemic
immunosuppressive therapy. Unfortunately, the inconvenience and
risks of ultraviolet radiation and the toxicities of many therapies
limit their long-term use. Moreover, patients usually have
recurrence of psoriasis, and in some cases rebound, shortly after
stopping immunosuppressive therapy.
[0324] IL-20 is a novel IL-10 homologue that causes neonatal
lethality with skin abnormalities including aberrant epidermal
differentiation in IL-20 transgenic mice (Blumberg H et al., Cell
104:9-19, 2001) IL-20 receptor is dramatically upregulated in
psoriatic skin. Since IL-TIF shares a receptor subunit (zcytor11)
with IL-20 receptor, and IL-TIF transgenic mice display a similar
phenotype, it is possible that IL-TIF is also involved in the
inflammatory skin diseases such as psoriasis. The administration of
zcytor16 polypeptide, either subcutaneous or topically, may
potential reduce the inflammation and symptom. Other potential
therapeutics include Zcytor16 polypeptides, soluble zcytor11/CRF2-4
receptor polypeptides, or anti IL-TIF antibodies or binding
partners, and the like.
[0325] Zcytor16 homodimeric, heterodimeric and multimeric receptor
polypeptides may also be used within diagnostic systems for the
detection of circulating levels of IL-TIF ligand, and in the
detection of IL-TIF associated with acute phase inflammatory
response. Within a related embodiment, antibodies or other agents
that specifically bind to Zcytor16 soluble receptors of the present
invention can be used to detect circulating receptor polypeptides;
conversely, Zcytor16 soluble receptors themselves can be used to
detect circulating or locally-acting IL-TIF polypeptides. Elevated
or depressed levels of ligand or receptor polypeptides may be
indicative of pathological conditions, including inflammation or
cancer. IL-TIF is known to induce associated acute phase
inflammatory response. Moreover, detection of acute phase proteins
or molecules such as IL-TIF can be indicative of a chronic
inflammatory condition in certain disease states (e.g., rheumatoid
arthritis). Detection of such conditions serves to aid in disease
diagnosis as well as help a physician in choosing proper
therapy.
[0326] Moreover, soluble zcytor16 receptor polypeptides of the
present invention 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 chronic inflammatory conditions or cancers that are
expressing large amounts of bioactive IL-TIF, soluble zcytor16
receptor or soluble zcytor16 heterodimeric and multimeric receptor
polypeptides, such as soluble zcytor16/CRF2-4 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., steroid or
chemotherapy) to enhance the effect of the therapy in reducing
progression and symptoms, and preventing relapse. Moreover, soluble
zcytor16 receptor polypeptides can be used to slow the progression
of cancers that over-express zcytor16 receptors, by binding ligand
in vivo that could otherwise enhance proliferation of those
cancers.
[0327] Moreover, soluble zcytor16 receptor polypeptides of the
present invention can be used in vivo or in diagnostic applications
to detect IL-TIF-expressing inflammation or cancers in vivo or in
tissue samples. For example, the soluble zcytor16 receptors of the
present invention can be conjugated to a radio-label or fluorescent
label as described herein, and used to detect the presence of the
IL-TIF in a tissue sample using an in vitro ligand-receptor type
binding assay, or fluorescent imaging assay. Moreover, radiolabeled
soluble zcytor16 receptors of the present invention could be
administered in vivo to detect Ligand-expressing solid tumors
through a radio-imaging method known in the art.
[0328] Generally, the dosage of administered Zcytor16 (or Zcytor16
analog or fusion protein) will vary depending upon such factors as
the patient's age, weight, height, sex, general medical condition
and previous medical history. Typically, it is desirable to provide
the recipient with a dosage of Zcytor16 polypeptide which is in the
range of from about 1 pg/kg to 10 mg/kg (amount of agent/body
weight of patient), although a lower or higher dosage also may be
administered as circumstances dictate.
[0329] Administration of a Zcytor16 polypeptide to a subject can be
intravenous, intraarterial, intraperitoneal, intramuscular,
subcutaneous, intrapleural, intrathecal, by perfusion through a
regional catheter, or by direct intralesional injection. When
administering therapeutic proteins by injection, the administration
may be by continuous infusion or by single or multiple boluses.
[0330] Additional routes of administration include oral,
mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is
suitable for polyester microspheres, zein microspheres, proteinoid
microspheres, polycyanoacrylate microspheres, and lipid-based
systems (see, for example, DiBase and Morrel, "Oral Delivery of
Microencapsulated Proteins," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The
feasibility of an intranasal delivery is exemplified by such a mode
of insulin administration (see, for example, Hinchcliffe and Illum,
Adv. Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles
comprising Zcytor16 can be prepared and inhaled with the aid of
dry-powder dispersers, liquid aerosol generators, or nebulizers
(e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al.,
Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is illustrated
by the AERX diabetes management system, which is a hand-held
electronic inhaler that delivers aerosolized insulin into the
lungs. Studies have shown that proteins as large as 48,000 kDa have
been delivered across skin at therapeutic concentrations with the
aid of low-frequency ultrasound, which illustrates the feasibility
of trascutaneous administration (Mitragotri et al., Science 269:850
(1995)). Transdermal delivery using electroporation provides
another means to administer a molecule having Zcytor16 binding
activity (Potts et al., Pharm. Biotechnol. 10:213 (1997)).
[0331] A pharmaceutical composition comprising a protein,
polypeptide, or peptide having Zcytor16 binding activity can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the therapeutic proteins are combined
in a mixture with a pharmaceutically acceptable carrier. A
composition is said to be a "pharmaceutically acceptable carrier"
if its administration can be tolerated by a recipient patient.
Sterile phosphate-buffered saline is one example of a
pharmaceutically acceptable carrier. Other suitable carriers are
well-known to those in the art. See, for example, Gennaro (ed.),
Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing
Company 1995).
[0332] For purposes of therapy, molecules having Zcytor16 binding
activity and a pharmaceutically acceptable carrier are administered
to a patient in a therapeutically effective amount. A combination
of a protein, polypeptide, or peptide having Zcytor16 binding
activity and a pharmaceutically acceptable carrier is said to be
administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. An agent is
physiologically significant if its presence results in a detectable
change in the physiology of a recipient patient. For example, an
agent used to treat inflammation is physiologically significant if
its presence alleviates the inflammatory response.
[0333] A pharmaceutical composition comprising Zcytor16 (or
Zcytor16 analog or fusion protein) can be furnished in liquid form,
in an aerosol, or in solid form. Liquid forms, are illustrated by
injectable solutions and oral suspensions. Exemplary solid forms
include capsules, tablets, and controlled-release forms. The latter
form is illustrated by miniosmotic pumps and implants (Bremer et
al., Pharm. Biotechnol. 10:239 (1997); Ranade, "Implants in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 95-123 (CRC Press 1995); Bremer et al., "Protein Delivery
with Infusion Pumps," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);
Yewey et al., "Delivery of Proteins from a Controlled Release
Injectable Implant," in Protein Delivery: Physical Systems, Sanders
and Hendren (eds.), pages 93-117 (Plenum Press 1997)).
[0334] Liposomes provide one means to deliver therapeutic
polypeptides to a subject intravenously, intraperitoneally,
intrathecally, intramuscularly, subcutaneously, or via oral
administration, inhalation, or intranasal administration. Liposomes
are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg
et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61
(1993), Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug
Delivery Using Liposomes as Carriers," in Drug Delivery Systems,
Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)).
Liposomes are similar in composition to cellular membranes and as a
result, liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0335] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents (Scherphof et al.,
Ann. N.Y. Acad. Sci. 446:368 (1985)). After intravenous
administration, small liposomes (0.1 to 1.0 .mu.m) are typically
taken up by cells of the reticuloendothelial system, located
principally in the liver and spleen, whereas liposomes larger than
3.0 .mu.m are deposited in the lung. This preferential uptake of
smaller liposomes by the cells of the reticuloendothelial system
has been used to deliver chemotherapeutic agents to macrophages and
to tumors of the liver.
[0336] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means (Claassen et al., Biochim. Biophys. Acta 802:428 (1984)). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system (Allen et al., Biochim. Biophys. Acta
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993)).
[0337] Liposomes can also be prepared to target particular cells or
organs by varying phospholipid composition or by inserting
receptors or ligands into the liposomes. For example, liposomes,
prepared with a high content of a nonionic surfactant, have been
used to target the liver (Hayakawa et al., Japanese Patent
04-244,018; Kato et al., Biol. Pharm. Bull 16:960 (1993)). These
formulations were prepared by mixing soybean phospatidylcholine,
.alpha.-tocopherol, and ethoxylated hydrogenated castor oil
(HCO-60) in methanol, concentrating the mixture under vacuum, and
then reconstituting the mixture with water. A liposomal formulation
of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived
sterylglucoside mixture (SG) and cholesterol (Ch) has also been
shown to target the liver (Shimizu et al., Biol. Pharm. Bull 20:881
(1997)).
[0338] Alternatively, various targeting ligands can be bound to the
surface of the liposome, such as antibodies, antibody fragments,
carbohydrates, vitamins, and transport proteins. For example,
liposomes can be modified with branched type galactosyllipid
derivatives to target asialoglycoprotein (galactose) receptors,
which are exclusively expressed on the surface of liver cells (Kato
and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:287 (1997);
Murahashi et al., Biol Pharm. Bull. 20:259 (1997)). Similarly, Wu
et al., Hepatology 27:772 (1998), have shown that labeling
liposomes with asialofetuin led to a shortened liposome plasma
half-life and greatly enhanced uptake of asialofetuin-labeled
liposome by hepatocytes. On the other hand, hepatic accumulation of
liposomes comprising branched type galactosyllipid derivatives can
be inhibited by preinjection of asialofetuin (Murahashi et al.,
Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serum
albumin liposomes provide another approach for targeting liposomes
to liver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681
(1997)). Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe a
hepatocyte-directed liposome vesicle delivery system, which has
specificity for hepatobiliary receptors associated with the
specialized metabolic cells of the liver.
[0339] In a more general approach to tissue targeting, target cells
are prelabeled with biotinylated antibodies specific for a ligand
expressed by the target cell (Harasym et al., Adv. Drug Deliv. Rev.
32:99 (1998)). After plasma elimination of free antibody,
streptavidin-conjugated liposomes are administered. In another
approach, targeting antibodies are directly attached to liposomes
(Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).
[0340] Polypeptides having Zcytor16 binding activity can be
encapsulated within liposomes using standard techniques of protein
microencapsulation (see, for example, Anderson et al., Infect.
Immun. 31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990),
and Cohen et al., Biochim. Biophys. Acta 1063:95 (1991), Alving et
al. "Preparation and Use of Liposomes in Immunological Studies," in
Liposome Technology, 2nd Edition, Vol. III, Gregoriadis (ed.), page
317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124
(1987)). As noted above, therapeutically useful liposomes may
contain a variety of components. For example, liposomes may
comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,
Biochim. Biophys. Acta 1150:9 (1993)).
[0341] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney
and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin.
Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
nanospheres can also provide carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0342] The present invention also contemplates chemically modified
polypeptides having binding Zcytor16 activity such as zcytor16
monomeric, homodimeric, heterodimeric or multimeric soluble
receptors, and Zcytor16 antagonists, for example anti-zcytor16
antibodies or binding polypeptides, which a polypeptide is linked
with a polymer, as discussed above.
[0343] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0344] As an illustration, pharmaceutical compositions may be
supplied as a kit comprising a container that comprises a
polypeptide with a Zcytor16 extracellular domain, e.g., zcytor16
monomeric, homodimeric, heterodimeric or multimeric soluble
receptors, or a Zcytor16 antagonist (e.g., an antibody or antibody
fragment that binds a Zcytor16 polypeptide). Therapeutic
polypeptides can be provided in the form of an injectable solution
for single or multiple doses, or as a sterile powder that will be
reconstituted before injection. Alternatively, such a kit can
include a dry-powder disperser, liquid aerosol generator, or
nebulizer for administration of a therapeutic polypeptide. Such a
kit may further comprise written information on indications and
usage of the pharmaceutical composition. Moreover, such information
may include a statement that the Zcytor16 composition is
contraindicated in patients with known hypersensitivity to
Zcytor16.
13. Therapeutic Uses of Zcytor16 Nucleotide Sequences
[0345] The present invention includes the use of Zcytor16
nucleotide sequences to provide Zcytor16 to a subject in need of
such treatment. In addition, a therapeutic expression vector can be
provided that inhibits Zcytor16 gene expression, such as an
anti-sense molecule, a ribozyme, or an external guide sequence
molecule.
[0346] There are numerous approaches to introduce a Zcytor16 gene
to a subject, including the use of recombinant host cells that
express Zcytor16, delivery of naked nucleic acid encoding Zcytor16,
use of a cationic lipid carrier with a nucleic acid molecule that
encodes Zcytor16, and the use of viruses that express Zcytor16,
such as recombinant retroviruses, recombinant adeno-associated
viruses, recombinant adenoviruses, and recombinant Herpes simplex
viruses (see, for example, Mulligan, Science 260:926 (1993),
Rosenberg et al., Science 242:1575 (1988), LaSalle et al., Science
259:988 (1993), Wolff et al., Science 247:1465 (1990), Breakfield
and Deluca, The New Biologist 3:203 (1991)). In an ex vivo
approach, for example, cells are isolated from a subject,
transfected with a vector that expresses a Zcytor16 gene, and then
transplanted into the subject.
[0347] In order to effect expression of a Zcytor16 gene, an
expression vector is constructed in which a nucleotide sequence
encoding a Zcytor16 gene is operably linked to a core promoter, and
optionally a regulatory element, to control gene transcription. The
general requirements of an expression vector are described
above.
[0348] Alternatively, a Zcytor16 gene can be delivered using
recombinant viral vectors, including for example, adenoviral
vectors (e.g., Kass-Eisler et al., Proc. Nat'l Acad. Sci. USA
90:11498 (1993), Kolls et al., Proc. Nat'l Acad. Sci. USA 91:215
(1994), Li et al., Hum. Gene Ther. 4:403 (1993), Vincent et al.,
Nat. Genet. 5:130 (1993), and Zabner et al., Cell 75:207 (1993)),
adenovirus-associated viral vectors (Flotte et al., Proc. Nat'l
Acad. Sci. USA 90:10613 (1993)), alphaviruses such as Semliki
Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857
(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,
Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat.
Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus
vectors (Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus
vectors (Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993),
Panicali and Paoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)),
pox viruses, such as canary pox virus or vaccinia virus
(Fisher-Hoch et al., Proc. Nat'l Acad. Sci. USA 86:317 (1989), and
Flexner et al., Ann. N.Y. Acad. Sci. 569:86 (1989)), and
retroviruses (e.g., Baba et al., J. Neurosurg 79:729 (1993), Ram et
al., Cancer Res. 53:83 (1993), Takamiya et al., J. Neurosci. Res
33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993), Vile and
Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S. Pat.
No. 5,399,346). Within various embodiments, either the viral vector
itself, or a viral particle which contains the viral vector may be
utilized in the methods and compositions described below.
[0349] As an illustration of one system, adenovirus, a
double-stranded DNA virus, is a well-characterized gene transfer
vector for delivery of a heterologous nucleic acid molecule (for a
review, see Becker et al., Meth. Cell Biol 43:161 (1994); Douglas
and Curiel, Science & Medicine 4:44 (1997)). The adenovirus
system offers several advantages including: (i) the ability to
accommodate relatively large DNA inserts, (ii) the ability to be
grown to high-titer, (iii) the ability to infect a broad range of
mammalian cell types, and (iv) the ability to be used with many
different promoters including ubiquitous, tissue specific, and
regulatable promoters. In addition, adenoviruses can be
administered by intravenous injection, because the viruses are
stable in the bloodstream.
[0350] 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 is deleted from the viral vector, and the virus will not
replicate unless the E1 gene is provided by the host cell. When
intravenously administered to intact animals, adenovirus primarily
targets the liver. Although an adenoviral delivery system with an
E1 gene deletion cannot replicate in the host cells, the host's
tissue will express and process an encoded heterologous protein.
Host cells will also secrete the heterologous protein if the
corresponding gene includes a secretory signal sequence. Secreted
proteins will enter the circulation from tissue that expresses the
heterologous gene (e.g., the highly vascularized liver).
[0351] Moreover, adenoviral vectors containing various deletions of
viral genes can be used to reduce or eliminate immune responses to
the vector. Such adenoviruses are E1-deleted, and in addition,
contain deletions of E2A or E4 (Lusky et al., J. Virol. 72:2022
(1998); Raper et al., Human Gene Therapy 9:671 (1998)). The
deletion of E2b has also been reported to reduce immune responses
(Amalfitano et al., J. Virol. 72:926 (1998)). 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 a review, see Yeh. and Perricaudet, FASEB J. 11:615
(1997)).
[0352] High titer stocks of recombinant viruses capable of
expressing a therapeutic gene can be obtained from infected
mammalian cells using standard methods. For example, recombinant
herpes simplex virus can be prepared in Vero cells, as described by
Brandt et al., J. Gen. Virol. 72:2043 (1991), Herold et al., J.
Gen. Virol 75:1211 (1994), Visalli and Brandt, Virology 185:419
(1991), Grau et al., Invest. Ophthalmol. Vis. Sci. 30:2474 (1989),
Brandt et al., J. Virol. Meth. 36:209 (1992), and by Brown and
MacLean (eds.), HSV Virus Protocols (Humana Press 1997).
[0353] Alternatively, an expression vector comprising a Zcytor16
gene can be introduced into a subject's cells by lipofection in
vivo using liposomes. Synthetic cationic lipids can be used to
prepare liposomes for in vivo transfection of a gene encoding a
marker (Felgner et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987);
Mackey et al., Proc. Nat'l Acad. Sci. USA 85:8027 (1988)). The use
of lipofection to introduce exogenous genes into specific organs in
vivo has certain practical advantages. Liposomes can be used to
direct transfection to particular cell types, which is particularly
advantageous in a tissue with cellular heterogeneity, such as the
pancreas, liver, kidney, and brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting. Targeted
peptides (e.g., hormones or neurotransmitters), proteins such as
antibodies, or non-peptide molecules can be coupled to liposomes
chemically.
[0354] Electroporation is another alternative mode of
administration. For example, Aihara and Miyazaki, Nature
Biotechnology 16:867 (1998), have demonstrated the use of in vivo
electroporation for gene transfer into muscle.
[0355] In an alternative approach to gene therapy, a therapeutic
gene may encode a Zcytor16 anti-sense RNA that inhibits the
expression of Zcytor16. Suitable sequences for anti-sense molecules
can be derived from the nucleotide sequences of Zcytor16 disclosed
herein.
[0356] Alternatively, an expression vector can be constructed in
which a regulatory element is operably linked to a nucleotide
sequence that encodes a ribozyme. Ribozymes can be designed to
express endonuclease activity that is directed to a certain target
sequence in a mRNA molecule (see, for example, Draper and Macejak,
U.S. Pat. No. 5,496,698, McSwiggen, U.S. Pat. No. 5,525,468,
Chowrira and McSwiggen, U.S. Pat. No. 5,631,359, and Robertson and
Goldberg, U.S. Pat. No. 5,225,337). In the context of the present
invention, ribozymes include nucleotide sequences that bind with
Zcytor16 mRNA.
[0357] In another approach, expression vectors can be constructed
in which a regulatory element directs the production of RNA
transcripts capable of promoting RNase P-mediated cleavage of mRNA
molecules that encode a Zcytor16 gene. According to this approach,
an external guide sequence can be constructed for directing the
endogenous ribozyme, RNase P, to a particular species of
intracellular mRNA, which is subsequently cleaved by the cellular
ribozyme (see, for example, Altman et al., U.S. Pat. No. 5,168,053,
Yuan et al., Science 263:1269 (1994), Pace et al., international
publication No. WO 96/18733, George et al., international
publication No. WO 96/21731, and Werner et al., international
publication No. WO 97/33991). For example, the external guide
sequence can comprise a ten to fifteen nucleotide sequence
complementary to Zcytor16 mRNA, and a 3'-NCCA nucleotide sequence,
wherein N is preferably a purine. The external guide sequence
transcripts bind to the targeted mRNA species by the formation of
base pairs between the mRNA and the complementary external guide
sequences, thus promoting cleavage of mRNA by RNase P at the
nucleotide located at the 5'-side of the base-paired region.
[0358] In general, the dosage of a composition comprising a
therapeutic vector having a Zcytor16 nucleotide sequence, such as a
recombinant virus, will vary depending upon such factors as the
subject's age, weight, height, sex, general medical condition and
previous medical history. Suitable routes of administration of
therapeutic vectors include intravenous injection, intraarterial
injection, intraperitoneal injection, intramuscular injection,
intratumoral injection, and injection into a cavity that contains a
tumor. As an illustration, Horton et al., Proc. Nat'l Acad. Sci.
USA 96:1553 (1999), demonstrated that intramuscular injection of
plasmid DNA encoding interferon-.alpha. produces potent antitumor
effects on primary and metastatic tumors in a murine model.
[0359] A composition comprising viral vectors, non-viral vectors,
or a combination of viral and non-viral vectors of the present
invention can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby vectors or viruses
are combined in a mixture with a pharmaceutically acceptable
carrier. As noted above, a composition, such as phosphate-buffered
saline is said to be a "pharmaceutically acceptable carrier" if its
administration can be tolerated by a recipient subject. Other
suitable carriers are well-known to those in the art (see, for
example, Remington's Pharmaceutical Sciences, 19th Ed. (Mack
Publishing Co. 1995), and Gilman's the Pharmacological Basis of
Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985)).
[0360] For purposes of therapy, a therapeutic gene expression
vector, or a recombinant virus comprising such a vector, and a
pharmaceutically acceptable carrier are administered to a subject
in a therapeutically effective amount. A combination of an
expression vector (or virus) and a pharmaceutically acceptable
carrier is said to be administered in a "therapeutically effective
amount" if the amount administered is physiologically significant.
An agent is physiologically significant if its presence results in
a detectable change in the physiology of a recipient subject. For
example, an agent used to treat inflammation is physiologically
significant if its presence alleviates the inflammatory
response.
[0361] When the subject treated with a therapeutic gene expression
vector or a recombinant virus is a human, then the therapy is
preferably somatic cell gene therapy. That is, the preferred
treatment of a human with a therapeutic gene expression vector or a
recombinant virus does not entail introducing into cells a nucleic
acid molecule that can form part of a human germ line and be passed
onto successive generations (i.e., human germ line gene
therapy).
14. Production of Transgenic Mice
[0362] Transgenic mice can be engineered to over-express the
Zcytor16 gene in all tissues or under the control of a
tissue-specific or tissue-preferred regulatory element. These
over-producers of Zcytor16 can be used to characterize the
phenotype that results from over-expression, and the transgenic
animals can serve as models for human disease caused by excess
Zcytor16. Transgenic mice that over-express Zcytor16 also provide
model bioreactors for production of Zcytor16, such as soluble
Zcytor16, in the milk or blood of larger animals. Methods for
producing transgenic mice are well-known to those of skill in the
art (see, for example, Jacob, "Expression and Knockout of
Interferons in Transgenic Mice," in Overexpression and Knockout of
Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (Academic
Press, Ltd. 1994), Monastersky and Robl (eds.), Strategies in
Transgenic Animal Science (ASM Press 1995), and Abbud and Nilson,
"Recombinant Protein Expression in Transgenic Mice," in Gene
Expression Systems: Using Nature for the Art of Expression,
Fernandez and Hoeffler (eds.), pages 367-397 (Academic Press, Inc.
1999)).
[0363] For example, a method for producing a transgenic mouse that
expresses a Zcytor16 gene can begin with adult, fertile males
(studs) (B6C3f1, 2-8 months of age (Taconic Farms, Germantown,
N.Y.)), vasectomized males (duds) (B6D2f1, 2-8 months, (Taconic
Farms)), prepubescent fertile females (donors) (B6C3f1, 4-5 weeks,
(Taconic Farms)) and adult fertile females (recipients) (B6D2f1,
2-4 months, (Taconic Farms)). The donors are acclimated for one
week and then injected with approximately 8 IU/mouse of Pregnant
Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, Mo.)
I.P., and 46-47 hours later, 8 IU/mouse of human Chorionic
Gonadotropin (hCG (Sigma)) I.P. to induce superovulation. Donors
are mated with studs subsequent to hormone injections. Ovulation
generally occurs within 13 hours of hCG injection. Copulation is
confirmed by the presence of a vaginal plug the morning following
mating.
[0364] Fertilized eggs are collected under a surgical scope. The
oviducts are collected and eggs are released into urinanalysis
slides containing hyaluronidase (Sigma). Eggs are washed once in
hyaluronidase, and twice in Whitten's W640 medium (described, for
example, by Menino and O'Claray, Biol. Reprod. 77:159 (1986), and
Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated
with 5% CO.sub.2, 5% O.sub.2, and 90% N.sub.2 at 37.degree. C. The
eggs are then stored in a 37.degree. C./5% CO.sub.2 incubator until
microinjection.
[0365] Ten to twenty micrograms of plasmid DNA containing a
Zcytor16 encoding sequence is linearized, gel-purified, and
resuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a
final concentration of 5-10 nanograms per microliter for
microinjection. For example, the Zcytor16 encoding sequences can
encode a polypeptide comprising the amino acid sequence of SEQ ID
NO:2.
[0366] Plasmid DNA is microinjected into harvested eggs contained
in a drop of W640 medium overlaid by warm, CO.sub.2-equilibrated
mineral oil. The DNA is drawn into an injection needle (pulled from
a 0.75 mm ID, 1 mm OD borosilicate glass capillary), and injected
into individual eggs. Each egg is penetrated with the injection
needle, into one or both of the haploid pronuclei.
[0367] Picoliters of DNA are injected into the pronuclei, and the
injection needle withdrawn without coming into contact with the
nucleoli. The procedure is repeated until all the eggs are
injected. Successfully microinjected eggs are transferred into an
organ tissue-culture dish with pre-gassed W640 medium for storage
overnight in a 37.degree. C./5% CO.sub.2 incubator.
[0368] The following day, two-cell embryos are transferred into
pseudopregnant recipients. The recipients are identified by the
presence of copulation plugs, after copulating with vasectomized
duds. Recipients are anesthetized and shaved on the dorsal left
side and transferred to a surgical microscope. A small incision is
made in the skin and through the muscle wall in the middle of the
abdominal area outlined by the ribcage, the saddle, and the hind
leg, midway between knee and spleen. The reproductive organs are
exteriorized onto a small surgical drape. The fat pad is stretched
out over the surgical drape, and a baby serrefine (Roboz,
Rockville, Md.) is attached to the fat pad and left hanging over
the back of the mouse, preventing the organs from sliding back
in.
[0369] With a fine transfer pipette containing mineral oil followed
by alternating W640 and air bubbles, 12-17 healthy two-cell embryos
from the previous day's injection are transferred into the
recipient. The swollen ampulla is located and holding the oviduct
between the ampulla and the bursa, a nick in the oviduct is made
with a 28 g needle close to the bursa, making sure not to tear the
ampulla or the bursa.
[0370] The pipette is transferred into the nick in the oviduct, and
the embryos are blown in, allowing the first air bubble to escape
the pipette. The fat pad is gently pushed into the peritoneum, and
the reproductive organs allowed to slide in. The peritoneal wall is
closed with one suture and the skin closed with a wound clip. The
mice recuperate on a 37.degree. C. slide warmer for a minimum of
four hours.
[0371] The recipients are returned to cages in pairs, and allowed
19-21 days gestation. After birth, 19-21 days postpartum is allowed
before weaning. The weanlings are sexed and placed into separate
sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off
the tail with clean scissors.
[0372] Genomic DNA is prepared from the tail snips using, for
example, a QIAGEN DNEASY kit following the manufacturer's
instructions. Genomic DNA is analyzed by PCR using primers designed
to amplify a Zcytor16 gene or a selectable marker gene that was
introduced in the same plasmid. After animals are confirmed to be
transgenic, they are back-crossed into an inbred strain by placing
a transgenic female with a wild-type male, or a transgenic male
with one or two wild-type female(s). As pups are born and weaned,
the sexes are separated, and their tails snipped for
genotyping.
[0373] To check for expression of a transgene in a live animal, a
partial hepatectomy is performed. A surgical prep is made of the
upper abdomen directly below the zyphoid process. Using sterile
technique, a small 1.5-2 cm incision is made below the sternum and
the left lateral lobe of the liver exteriorized. Using 4-0 silk, a
tie is made around the lower lobe securing it outside the body
cavity. An atraumatic clamp is used to hold the tie while a second
loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed
proximal to the first tie. A distal cut is made from the Dexon tie
and approximately 100 mg of the excised liver tissue is placed in a
sterile petri dish. The excised liver section is transferred to a
14 ml polypropylene round bottom tube and snap frozen in liquid
nitrogen and then stored on dry ice. The surgical site is closed
with suture and wound clips, and the animal's cage placed on a
37.degree. C. heating pad for 24 hours post operatively. The animal
is checked daily post operatively and the wound clips removed 7-10
days after surgery. The expression level of Zcytor16 mRNA is
examined for each transgenic mouse using an RNA solution
hybridization assay or polymerase chain reaction.
[0374] In addition to producing transgenic mice that over-express
Zcytor16, it is useful to engineer transgenic mice with either
abnormally low or no expression of the gene. Such transgenic mice
provide useful models for diseases associated with a lack of
Zcytor16. As discussed above, Zcytor16 gene expression can be
inhibited using anti-sense genes, ribozyme genes, or external guide
sequence genes. To produce transgenic mice that under-express the
Zcytor16 gene, such inhibitory sequences are targeted to Zcytor16
mRNA, including the fragments or the entire 5' UTR, coding region,
and 3' UTR, e.g., as shown in SEQ ID NO:37 and described herein.
Methods for producing transgenic mice that have abnormally low
expression of a particular gene are known to those in the art (see,
for example, Wu et al., "Gene Underexpression in Cultured Cells and
Animals by Antisense DNA and RNA Strategies," in Methods in Gene
Biotechnology, pages 205-224 (CRC Press 1997)).
[0375] An alternative approach to producing transgenic mice that
have little or no Zcytor16 gene expression is to generate mice
having at least one normal Zcytor16 allele replaced by a
nonfunctional Zcytor16 gene. One method of designing a
nonfunctional Zcytor16 gene is to insert another gene, such as a
selectable marker gene, within a nucleic acid molecule that encodes
Zcytor16. Standard methods for producing these so-called "knockout
mice" are known to those skilled in the art (see, for example,
Jacob, "Expression and Knockout of Interferons in Transgenic Mice,"
in Overexpression and Knockout of Cytokines in Transgenic Mice,
Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et
al., "New Strategies for Gene Knockout," in Methods in Gene
Biotechnology, pages 339-365 (CRC Press 1997)).
[0376] Polynucleotides and polypeptides of the present invention
will additionally find use as educational tools as a laboratory
practicum kits for courses related to genetics and molecular
biology, protein chemistry and antibody production and analysis.
Due to its unique polynucleotide and polypeptide sequence molecules
of zcytor16 can be used as standards or as "unknowns" for testing
purposes. For example, zcytor16 polynucleotides can be used as an
aid, such as, for example, to teach a student how to prepare
expression constructs for bacterial, viral, and/or mammalian
expression, including fusion constructs, wherein zcytor16 is the
gene to be expressed; for determining the restriction endonuclease
cleavage sites of the polynucleotides; determining mRNA and DNA
localization of zcytor16 polynucleotides in tissues (i.e., by
Northern and Southern blotting as well as polymerase chain
reaction); and for identifying related polynucleotides and
polypeptides by nucleic acid hybridization.
[0377] Zcytor16 polypeptides can be used educationally as an aid to
teach preparation of antibodies; identifying proteins by Western
blotting; protein purification; determining the weight of expressed
zcytor16 polypeptides as a ratio to total protein expressed;
identifying peptide cleavage sites; coupling amino and carboxyl
terminal tags; amino acid sequence analysis, as well as, but not
limited to monitoring biological activities of both the native and
tagged protein (i.e., receptor binding, signal transduction,
proliferation, and differentiation) in vitro and in vivo. Zcytor16
polypeptides can also be used to teach analytical skills such as
mass spectrometry, circular dichroism to determine conformation,
especially of the four alpha helices, x-ray crystallography to
determine the three-dimensional structure in atomic detail, nuclear
magnetic resonance spectroscopy to reveal the structure of proteins
in solution. For example, a kit containing the zcytor16 can be
given to the student to analyze. Since the amino acid sequence
would be known by the professor, the specific protein can be given
to the student as a test to determine the skills or develop the
skills of the student, the teacher would then know whether or not
the student has correctly analyzed the polypeptide. Since every
polypeptide is unique, the educational utility of zcytor16 would be
unique unto itself.
[0378] Moreover, since zcytor16 has a tissue-specific expression
and is a polypeptide with a class II cytokine receptor structure
and a distinct chromosomal localization, and expressin pattern,
activity can be measured using proliferation assays; luciferase and
binding assays described herein. Moreover, expression of zcytor16
polynucleotides and polypeptides in lymphoid and other tissues can
be analyzed in order to train students in the use of diagnostic and
tissue-specific identification and methods. Moreover zcytor16
polynucleotides can be used to train students on the use of
chromosomal detection and diagnostic methods, since it's locus is
known. Moreover, students can be specifically trained and educated
about human chromosome 1, and more specifically the locus 6q23-q24
wherein the zcytor16 gene is localized. Such assays are well known
in the art, and can be used in an educational setting to teach
students about cytokine receptor proteins and examine different
properties, such as cellular effects on cells, enzyme kinetics,
varying antibody binding affinities, tissue specificity, and the
like, between zcytor16 and other cytokine receptor polypeptides in
the art.
[0379] The antibodies which bind specifically to zcytor16 can be
used as a teaching aid to instruct students how to prepare affinity
chromatography columns to purify zcytor16, cloning and sequencing
the polynucleotide that encodes an antibody and thus as a practicum
for teaching a student how to design humanized antibodies.
Moreover, antibodies which bind specifically to zcytor16 can be
used as a teaching aid for use in detection e.g., of activated
CD91+ cells, cell sorting, or ovarian cancer tissue using
histological, and in situ methods amongst others known in the art.
The zcytor16 gene, polypeptide or antibody would then be packaged
by reagent companies and sold to universities and other educational
entities so that the students gain skill in art of molecular
biology. Because each gene and protein is unique, each gene and
protein creates unique challenges and learning experiences for
students in a lab practicum. Such educational kits containing the
zcytor16 gene, polypeptide or antibody are considered within the
scope of the present invention.
15. Therapeutic Uses of Antibodies or Polypeptides that Bind to
IL-TIF or have IL-TIF Antagonizing Activity
[0380] IL-TIF polynucleotides are expressed in T-cells, activated
T- and B-cells, and lymphoid tissue. The human IL-TIF nucleotide
sequence is represented in SEQ ID NO:14
[0381] Analysis of SEQ ID NO:14 reveals that there are two possible
initiation Methionine residues for a IL-TIF cytokine polypeptide
translated therefrom. The two deduced IL-TIF polypeptide amino acid
sequences are shown in SEQ ID NO:15 (a 179 amino acid polypeptide
having the initiating Met at nucleotide 21 in SEQ ID NO:14 a 167
amino acid polypeptide having the initiating Met at nucleotide 57
in SEQ ID NO:14). Although both of these sequences encode a IL-TIF
polypeptide, based on similarity of the IL-TIF sequence to IL-10
and other cytokines, and the presence of a strong signal sequence,
amino acid 34 (Ala) to 179 (IIe) of SEQ ID NO:15 encodes a fully
functional secreted IL-TIF cytokine polypeptide. N-terminal
sequence analysis shows that the mature start at residue 34 (Ala)
of SEQ ID NO:15.
[0382] In general, cytokines are predicted to have a four-alpha
helix structure, with the 1.sup.st and 4.sup.th helices being most
important in ligand-receptor interactions. The 1.sup.st and
4.sup.th helices are more highly conserved among members of the
family. Referring to the human IL-TIF amino acid sequence shown in
SEQ ID NO:15, alignment of human IL-TIF, human IL-10, human zcyto10
(WO US98/25228), and human Human MDA7 (Genbank Accession No.
Q13007) amino acid sequences suggests that IL-TIF helix A is
defined by amino acid residues 53 (Thr) to 65 (Leu) of SEQ ID
NO:15; helix B by amino acid residues 92 (Met) to 103 (Val) of SEQ
ID NO:15; helix C by amino acid residues 115 (Met) to 128 (Arg) of
SEQ ID NO:15; and helix D by amino acid residues 161 (Ile) to 174
(Leu) of SEQ ID NO:15. Structural analysis suggests that the A/B
loop is long, the B/C loop is short and the C/D loop is long. This
loop structure results in an up-up-down-down helical organization.
Four cysteine residues are conserved between IL-10 and IL-TIF
corresponding to amino acid residues 20, 40, 89 and 132 of SEQ ID
NO:15. Consistent cysteine placement is further confirmation of the
four-helical-bundle structure.
[0383] The corresponding polynucleotides encoding the IL-TIF
polypeptide regions, domains, motifs, residues and sequences
described herein are as shown in SEQ ID NO:1. Moreover, the
corresponding IL-TIF polypeptide regions, domains, motifs, residues
and sequences described herein are also as shown in SEQ ID
NO:15.
[0384] Four-helical bundle cytokines are also grouped by the length
of their component helices. "Long-helix" form cytokines generally
consist of between 24-30 residue helices and include IL-6, ciliary
neutrotrophic factor (CNTF), leukemia inhibitory factor (LIF) and
human growth hormone (hGH). "Short-helix" form cytokines generally
consist of between 18-21 residue helices and include IL-2, IL-4 and
GM-CSF. IL-TIF is believed to be a new member of the short-helix
form cytokine group. Studies using CNTF and IL-6 demonstrated that
a CNTF helix can be exchanged for the equivalent helix in IL-6,
conferring CTNF-binding properties to the chimera. Thus, it appears
that functional domains of four-helical cytokines determined on the
basis of structural homology, irrespective of sequence identity,
and can maintain functional integrity in a chimera (Kallen et al.,
J. Biol. Chem. 274:11859-11867, 1999). Using similar methods,
putative regions conferring receptor binding specificity in IL-TIF
comprise the regions of amino acid residues of 34 (Ala) to 179
(Ile) of SEQ ID NO:15 that include: residues 65-72, residues
97-103, and residues 133-152. These regions will be useful for
preparing chimeric molecules, particularly with other short-helix
form cytokines to determine and modulate receptor binding
specificity.
[0385] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a IL-TIF
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0386] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein. Hopp/Woods hydrophilicity
profiles can be used to determine regions that have the most
antigenic potential (Hopp et al., 1981, ibid. and Hopp, 1986,
ibid.). In IL-TIF these regions include: (1) amino acid number 41
(Arg) to amino acid number 46 (Asn) of SEQ ID NO:15; (2) amino acid
number 133 (His) to amino acid number 138 (Asp) of SEQ ID NO:15;
(3) amino acid number 146 (Gln) to amino acid number 151 (Thr) of
SEQ ID NO:15; (4) amino acid number 149 (Lys) to amino acid number
154 (Lys) of SEQ ID NO:15; and (5) amino acid number 157 (Glu) to
amino acid number 162 (Lys) of SEQ ID NO:15. Moreover, IL-TIF
antigenic epitopes as predicted by a Jameson-Wolf plot, e.g., using
DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.) serve as
preferred antigens, and are readily determined by one of skill in
the art. Such antigens include (1) amino acid number 40 (Cys) to
amino acid number 47 (Phe) of SEQ ID NO:15; (2) amino acid number
64 (Ser) or 67 (Asp) to amino acid number 71 (Asp) or 74 (Leu) of
SEQ ID NO:15; (3) amino acid number 106 (Pro) or 107 (Gln) to amino
acid number 112 (Gln) or 115 (Met) of SEQ ID NO:15; (4) amino acid
number 125 (Leu) to amino acid number 130 (Ser) or 131 (Thr) of SEQ
ID NO:15; (5) amino acid number 135 (Glu) to amino acid number 138
(Asp) or 140 (His) of SEQ ID NO:15; and (6) amino acid number 146
(Gln) or 156 (Gly) to amino acid number 159 (Gly) of SEQ ID
NO:15.
[0387] Antigenic epitope-bearing peptides and polypeptides
preferably contain at least four to ten amino acids, at least ten
to fifteen amino acids, or about 15 to about 30 amino acids of 34
(Ala) to 179 (Ile) of SEQ ID NO:15. Such epitope-bearing peptides
and polypeptides can be produced by fragmenting a IL-TIF
polypeptide, or by chemical peptide synthesis, as described herein.
Moreover, epitopes can be selected by phage display of random
peptide libraries (see, for example, Lane and Stephen, Curr. Opin.
Immunol. 5:268 (1993); and Cortese et al., Curr. Opin. Biotechnol.
7:616 (1996)). Standard methods for identifying epitopes and
producing antibodies from small peptides that comprise an epitope
are described, for example, by Mole, "Epitope Mapping," in Methods
in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The
Humana Press, Inc. 1992); Price, "Production and Characterization
of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production Engineering and Clinical Application, Ritter and Ladyman
(eds.), pages 60-84 (Cambridge University Press 1995), and Coligan
et al. (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5
and pages 9.4.1-9.4.11 (John Wiley & Sons 1997).
[0388] IL-TIF polypeptides can also be used to prepare antibodies
that bind to IL-TIF epitopes, peptides or polypeptides. The IL-TIF
polypeptide or a fragment thereof serves as an antigen (immunogen)
to inoculate an animal and elicit an immune response. Such
antibodies can be used to block the biological action of
pro-inflammatory IL-TIF and are useful as anti-inflammatory
therapeutics in a variety of diseases as described herein. 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 IL-TIF polypeptide (e.g., from 34 (Ala) to 179
(Ile) of SEQ ID NO:15). Polypeptides comprising a larger portion of
a IL-TIF polypeptide, i.e., from 30 to 100 residues up to the
entire length of the amino acid sequence of SEQ ID NO:15 are
included. Antigens or immunogenic epitopes can also include
attached tags, adjuvants and carriers, as described herein.
Suitable antigens include the IL-TIF polypeptide encoded by 34
(Ala) to 179 (Ile) of SEQ ID NO:15 from amino acid number 34 to
amino acid number 179, or a contiguous 9 to 144, or 30 to 144 amino
acid fragment thereof. Other suitable antigens include polypeptides
comprising isolated helices and fragments of the
four-helical-bundle structure, as described herein. Preferred
peptides to use as antigens are hydrophilic peptides such as those
predicted by one of skill in the art from a hydrophobicity plot, as
described herein. Moreover, IL-TIF antigenic epitopes as predicted
by a Jameson-Wolf plot, e.g., using DNASTAR Protean program
(DNASTAR, Inc., Madison, Wis.) serve as preferred antigens, and are
readily determined by one of skill in the art, and described
herein.
[0389] Antibodies from an immune response generated by inoculation
of an animal with these antigens (or immunogens) can be isolated
and purified as described herein. Methods for preparing and
isolating polyclonal and monoclonal antibodies are well known in
the art. See, for example, Current Protocols in Immunology,
Cooligan, et al. (eds.), National Institutes of Health, John Wiley
and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989;
and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:
Techniques and Applications, CRC Press, Inc., Boca Raton, Fla.,
1982.
[0390] 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 IL-TIF polypeptide or a
fragment thereof. The immunogenicity of a IL-TIF 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 IL-TIF 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.
[0391] As used herein, the term "antibodies" includes polyclonal
antibodies, affinity-purified polyclonal antibodies, monoclonal
antibodies, and antigen-binding fragments, such as F(ab').sub.2 and
Fab proteolytic fragments. Genetically engineered intact antibodies
or fragments, such as chimeric antibodies, Fv fragments, single
chain antibodies and the like, as well as synthetic antigen-binding
peptides and polypeptides, are also included. Non-human antibodies
may be humanized by grafting non-human CDRs onto human framework
and constant regions, or by incorporating the entire non-human
variable domains (optionally "cloaking" them with a human-like
surface by replacement of exposed residues, wherein the result is a
"veneered" antibody). In some instances, humanized antibodies may
retain non-human residues within the human variable region
framework domains to enhance proper binding characteristics.
Through humanizing antibodies, biological half-life may be
increased, and the potential for adverse immune reactions upon
administration to humans is reduced. Moreover, human antibodies can
be produced in transgenic, non-human animals that have been
engineered to contain human immunoglobulin genes as disclosed in
WIPO Publication WO 98/24893. It is preferred that the endogenous
immunoglobulin genes in these animals be inactivated or eliminated,
such as by homologous recombination.
[0392] 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-IL-TIF antibodies
herein bind to a IL-TIF polypeptide, peptide or epitope with an
affinity at least 10-fold greater than the binding affinity to
control (non-IL-TIF) polypeptide. It is preferred that the
antibodies exhibit a binding affinity (K.sub.a) of 10.sup.6
M.sup.-1 or greater, preferably 10.sup.7 M.sup.-1 or greater, more
preferably 10.sup.8 M.sup.-1 or greater, and most preferably
10.sup.9 M.sup.-1 or greater. The binding affinity of an antibody
can be readily determined by one of ordinary skill in the art, for
example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci.
51: 660-672, 1949).
[0393] Whether anti-IL-TIF antibodies do not significantly
cross-react with related polypeptide molecules is shown, for
example, by the antibody detecting IL-TIF polypeptide but not known
related polypeptides using a standard Western blot analysis
(Ausubel et al., ibid.). Examples of known related polypeptides are
those disclosed in the prior art, such as known orthologs, and
paralogs, and similar known members of a protein family. Screening
can also be done using non-human IL-TIF, and IL-TIF mutant
polypeptides. Moreover, antibodies can be "screened against" known
related polypeptides, to isolate a population that specifically
binds to the IL-TIF polypeptides. For example, antibodies raised to
IL-TIF are adsorbed to related polypeptides adhered to insoluble
matrix; antibodies specific to IL-TIF 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-IL-TIF antibodies can be detected by a
number of methods in the art, and disclosed below.
[0394] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which bind to IL-TIF 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
IL-TIF protein or polypeptide.
[0395] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to IL-TIF protein or peptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled IL-TIF protein or peptide). Genes encoding
polypeptides having potential IL-TIF polypeptide binding domains
can be obtained by screening random peptide libraries displayed on
phage (phage display) or on bacteria, such as E. coli. Nucleotide
sequences encoding the polypeptides can be obtained in a number of
ways, such as through random mutagenesis and random polynucleotide
synthesis. These random peptide display libraries can be used to
screen for peptides which interact with a known target which can be
a protein or polypeptide, such as a ligand or receptor, a
biological or synthetic macromolecule, or organic or inorganic
substances. Techniques for creating and screening such random
peptide display libraries are known in the art (Ladner et al., U.S.
Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner
et al., U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No.
5,571,698) and random peptide display libraries and kits for
screening such libraries are available commercially, for instance
from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego,
Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia
LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the IL-TIF sequences disclosed
herein to identify proteins which bind to IL-TIF. These "binding
polypeptides" which interact with IL-TIF 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 IL-TIF
polypeptides; for detecting or quantitating soluble IL-TIF
polypeptides as marker of underlying pathology or disease. These
binding polypeptides can also act as IL-TIF "antagonists" to block
IL-TIF binding and signal transduction in vitro and in vivo. These
anti-IL-TIF binding polypeptides would be useful for inhibiting
IL-TIF activity or protein-binding. Such anti-IL-TIF binding
polypeptides can be used to block the biological action of
pro-inflammatory IL-TIF and are useful as anti-inflammatory
therapeutics in a variety of diseases as described herein.
[0396] Antibodies to IL-TIF may be used for tagging cells that
express IL-TIF; for isolating IL-TIF by affinity purification; for
diagnostic assays for determining circulating levels of IL-TIF
polypeptides; for detecting or quantitating soluble IL-TIF as a
marker of underlying pathology or disease; in analytical methods
employing FACS; for screening expression libraries; for generating
anti-idiotypic antibodies; and as neutralizing antibodies or as
antagonists to block IL-TIF 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 IL-TIF or fragments thereof may be used in vitro to
detect denatured IL-TIF or fragments thereof in assays, for
example, Western Blots or other assays known in the art.
[0397] Antibodies or polypeptides herein can also be directly or
indirectly conjugated to drugs, toxins, radionuclides and the like,
and these conjugates used for in vivo diagnostic or therapeutic
applications. For instance, polypeptides or antibodies of the
present invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary molecule
(receptor or antigen, respectively, for instance). More
specifically, anti-IL-TIF antibodies, or bioactive fragments or
portions thereof, can be coupled to detectable or cytotoxic
molecules and delivered to a mammal having cells, tissues or organs
that express the anti-complementary molecule.
[0398] Suitable detectable molecules may be directly or indirectly
attached to the polypeptide or antibody, and include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like. Suitable
cytotoxic molecules may be directly or indirectly attached to the
polypeptide or antibody, and include bacterial or plant toxins (for
instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and
the like), as well as therapeutic radionuclides, such as
iodine-131, rhenium-188 or yttrium-90 (either directly attached to
the polypeptide or antibody, or indirectly attached through means
of a chelating moiety, for instance). Polypeptides or antibodies
may also be conjugated to cytotoxic drugs, such as adriamycin. For
indirect attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule can be conjugated with a member of
a complementary/anticomplementary pair, where the other member is
bound to the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/anticomplementary
pair.
[0399] In another embodiment, polypeptide-toxin fusion proteins or
antibody-toxin fusion proteins can be used for targeted cell or
tissue inhibition or ablation (for instance, to treat cancer cells
or tissues). Alternatively, if the polypeptide has multiple
functional domains (i.e., an activation domain or a receptor
binding domain, plus a targeting domain), a fusion protein
including only the targeting domain may be suitable for directing a
detectable molecule, a cytotoxic molecule or a complementary
molecule to a cell or tissue type of interest. In instances where
the domain only fusion protein includes a complementary molecule,
the anti-complementary molecule can be conjugated to a detectable
or cytotoxic molecule. Such domain-complementary molecule fusion
proteins thus represent a generic targeting vehicle for
cell/tissue-specific delivery of generic
anti-complementary-detectable/cytotoxic molecule conjugates. Such
cytokine toxin fusion proteins can be used for in vivo killing of
target tissues.
[0400] In another embodiment, IL-TIF cytokine fusion proteins or
antibody-cytokine fusion proteins can be used for in vivo killing
of target tissues (for example, leukemia, lymphoma, lung cancer,
colon cancer, melanoma, pancreatic cancer, ovanian cancer, blood
and bone marrow cancers, or other cancers wherein IL-TIF receptors
are expressed) (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 IL-TIF polypeptides or
anti-IL-TIF antibodies target an undesirable cell or tissue (i.e.,
a tumor or a leukemia), and the fused cytokine mediated improved
target cell lysis by effector cells. Suitable cytokines for this
purpose include interleukin 2 and granulocyte-macrophage
colony-stimulating factor (GM-CSF), for instance.
[0401] In yet another embodiment, if the IL-TIF polypeptide or
anti-IL-TIF antibody targets vascular cells or tissues, such
polypeptide or antibody may be conjugated with a radionuclide, and
particularly with a beta-emitting radionuclide, to reduce
restenosis. Such therapeutic approaches pose less danger to
clinicians who administer the radioactive therapy. For instance,
iridium-192 impregnated ribbons placed into stented vessels of
patients until the required radiation dose was delivered showed
decreased tissue growth in the vessel and greater luminal diameter
than the control group, which received placebo ribbons. Further,
revascularisation and stent thrombosis were significantly lower in
the treatment group. Similar results are predicted with targeting
of a bioactive conjugate containing a radionuclide, as described
herein.
[0402] The bioactive polypeptide or antibody conjugates described
herein can be delivered intravenously, intraarterially or
intraductally, or may be introduced locally at the intended site of
action.
[0403] Moreover, Inflammation is a protective response by an
organism to fend off an invading agent. Inflammation is a cascading
event that involves many cellular and humoral mediators. On one
hand, suppression of inflammatory responses can leave a host
immunocompromised; however, if left unchecked, inflammation can
lead to serious complications including chronic inflammatory
diseases (e.g., rheumatoid arthritis, multiple sclerosis,
inflammatory bowel disease and the like), septic shock and multiple
organ failure. Importantly, these diverse disease states share
common inflammatory mediators. The collective diseases that are
characterized by inflammation have a large impact on human
morbidity and mortality. Therefore it is clear that
anti-inflammatory antibodies and binding polypeptides, such as
anti-IL-TIF antibodies and binding polypeptides described herein,
could have crucial therapeutic potential for a vast number of human
and animal diseases, from asthma and allergy to autoimmunity and
septic shock. As such, use of anti-inflammatory anti IL-TIF
antibodies and binding polypeptides described herein can be used
therapeutically as IL-TIF antagonists described herein,
particularly in diseases such as arthritis, endotoxemia,
inflammatory bowel disease, psoriasis, related disease and the
like.
[0404] Within one aspect the present invention provides an isolated
polypeptide, comprising at least 15 contiguous amino acid residues
of an amino acid sequence of SEQ ID NO:2 selected from the group
consisting of: (a) amino acid residues 28 to 127; (b) amino acid
residues 132 to 231; (c) amino acid residues 28 to 231; (d) amino
acid residues 23 to 230; (e) amino acid residues 23 to 231; (f)
amino acid residues 22 to 230; (g) amino acid residues 22 to 231;
and (h) amino acid residues 1 to 231. In one embodiment, the
isolated polypeptide disclosed above comprises an amino acid
sequence selected from the group consisting of: (a) amino acid
residues 28 to 127; (b) amino acid residues 132 to 231; (c) amino
acid residues 28 to 231; (d) amino acid residues 23 to 230; (e)
amino acid residues 23 to 231; (f) amino acid residues 22 to 230;
(g) amino acid residues 22 to 231; and (h) amino acid residues 1 to
231. In another embodiment, the isolated polypeptide disclosed
above consists of an amino acid sequence selected from the group
consisting of: (a) amino acid residues 28 to 127; (b) amino acid
residues 132 to 231; (c) amino acid residues 28 to 231; (d) amino
acid residues 23 to 230; (e) amino acid residues 23 to 231; (f)
amino acid residues 22 to 230; (g) amino acid residues 22 to 231;
and (h) amino acid residues 1 to 231.
[0405] Within a second aspect the present invention provides an
isolated polypeptide, comprising an amino acid sequence that is at
least 70% identical to a reference amino acid sequence of SEQ ID
NO:2 selected from the group consisting of: (a) amino acid residues
28 to 127; (b) amino acid residues 132 to 231; (c) amino acid
residues 28 to 231; (d) amino acid residues 23 to 230; (e) amino
acid residues 23 to 231; (f) amino acid residues 22 to 230; (g)
amino acid residues 22 to 231; and (h) amino acid residues 1 to
231. In one embodiment, the isolated polypeptide disclosed above
has an amino acid sequence that is at least 80% identical to the
reference amino acid sequence. In another embodiment, the isolated
polypeptide disclosed above has an amino acid sequence that is at
least 90% identical to the reference amino acid sequence. In
another embodiment, the isolated polypeptide disclosed above
comprises either amino acid residues 22 to 231 of SEQ ID NO:2 or
amino acid residues 28 to 231 of SEQ ID NO:2.
[0406] Within a third aspect the present invention provides an
isolated nucleic acid molecule, wherein the nucleic acid molecule
is either (a) a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:3, or (b) a nucleic acid molecule that
remains hybridized following stringent wash conditions to a nucleic
acid molecule consisting of the nucleotide sequence of nucleotides
64 to 693, or 82 to 693 of SEQ ID NO:1, or the complement of the
nucleotide sequence of nucleotides 64 to 693, or 82 to 693 of SEQ
ID NO:1; or (c) SEQ ID NO:1 or SEQ ID NO:37, or the complement of
the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:37. In one
embodiment, the isolated nucleic acid molecule is as disclosed
above wherein any difference between the amino acid sequence
encoded by the nucleic acid molecule and the corresponding amino
acid sequence of SEQ ID NO:2 is due to a conservative amino acid
substitution. In another embodiment, the isolated nucleic acid
molecule disclosed above comprises the nucleotide sequence of
nucleotides 1 to 693 of SEQ ID NO:1.
[0407] Within another aspect the present invention provides a
vector, comprising the isolated nucleic acid molecule as disclosed
above.
[0408] Within another aspect the present invention provides an
expression vector, comprising the isolated nucleic acid molecule as
disclosed above, a transcription promoter, and a transcription
terminator, wherein the promoter is operably linked with the
nucleic acid molecule, and wherein the nucleic acid molecule is
operably linked with the transcription terminator.
[0409] Within another aspect the present invention provides a
recombinant host cell comprising the expression vector as disclosed
above, wherein the host cell is selected from the group consisting
of bacterium, yeast cell, fungal cell, insect cell, mammalian cell,
and plant cell.
[0410] Within another aspect the present invention provides a
method of producing Zcytor16 protein, the method comprising
culturing recombinant host cells that comprise the expression
vector as disclosed above, and that produce the Zcytor16 protein.
In one embodiment, the method disclosed above, further comprises
isolating the Zcytor16 protein from the cultured recombinant host
cells.
[0411] Within another aspect the present invention provides an
antibody or antibody fragment that specifically binds with the
polypeptide as disclosed above. In one embodiment, the antibody
disclosed above is selected from the group consisting of: (a)
polyclonal antibody, (b) murine monoclonal antibody, (c) humanized
antibody derived from (b), and (d) human monoclonal antibody.
[0412] Within another aspect the present invention provides an
anti-idiotype antibody that specifically binds with the antibody as
disclosed above.
[0413] Within another aspect the present invention provides a
fusion protein, comprising the polypeptide as disclosed above. In
one embodiment, the fusion protein disclosed above further
comprises an immunoglobulin moiety.
[0414] Within another aspect the present invention provides an
isolated polynucleotide that encodes a soluble cytokine 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:2 from amino acid 22 to 231 or 28 to 231, and wherein the
soluble cytokine receptor polypeptide encoded by the polynucleotide
sequence binds IL-TIF or antagonizes IL-TIF activity.
[0415] Within another aspect the present invention provides an
isolated polynucleotide as disclosed above, wherein the soluble
cytokine receptor polypeptide encoded by the polynucleotide forms a
homodimeric, heterodimeric or multimeric receptor complex. In one
embodiment, the isolated polynucleotide is as disclosed above,
wherein the soluble cytokine receptor polypeptide encoded by the
polynucleotide forms a heterodimeric or multimeric receptor complex
further comprising a soluble Class I or Class II cytokine receptor.
In another embodiment, the isolated polynucleotide is as disclosed
above, wherein the soluble cytokine receptor polypeptide encoded by
the polynucleotide forms a heterodimeric or multimeric receptor
complex further comprising a soluble CRF2-4 receptor polypeptide
(SEQ ID NO:35), a soluble IL-10 receptor polypeptide (SEQ ID
NO:36), or soluble zcytor11 receptor polypeptide (SEQ ID
NO:34).
[0416] Within another aspect the present invention provides an
isolated polynucleotide that encodes a soluble cytokine receptor
polypeptide comprising a sequence of amino acid residues as shown
in SEQ ID NO:2 from amino acid 22 to 231 or 28 to 231, wherein the
soluble cytokine receptor polypeptide encoded by the polynucleotide
forms a homodimeric, heterodimeric or multimeric receptor complex.
In one embodiment, the isolated polynucleotide is as disclosed
above, wherein the soluble cytokine receptor polypeptide encoded by
the polynucleotide further comprises a soluble Class I or Class II
cytokine receptor. In another embodiment, the isolated
polynucleotide is as disclosed above, wherein the soluble cytokine
receptor polypeptide encoded by the polynucleotide forms a
heterodimeric or multimeric receptor complex further comprising a
soluble CRF2-4 receptor polypeptide (SEQ ID NO:35), a soluble IL-10
receptor polypeptide (SEQ ID NO:36), or soluble zcytor11 receptor
polypeptide (SEQ ID NO:34). In another embodiment, the isolated
polynucleotide is as disclosed above, wherein the soluble cytokine
receptor polypeptide further encodes an intracellular domain. In
another embodiment, the isolated polynucleotide is as disclosed
above, wherein the soluble cytokine receptor polypeptide further
comprises an affinity tag.
[0417] Within another 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 cytokine receptor polypeptide having an amino
acid sequence as shown in SEQ ID NO:2 from amino acid 22 to 231 or
28 to 231; and a transcription terminator; and (b) a second
transcription promoter; a second DNA segment encoding a soluble
Class I or Class II 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 comprising 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
polypeptide comprising a soluble CRF2-4 receptor polypeptide (SEQ
ID NO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), or
soluble zcytor11 receptor polypeptide (SEQ ID NO:34).
[0418] Within another 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.
[0419] Within another aspect the present invention provides a
cultured cell comprising an expression vector 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.
[0420] Within another aspect the present invention provides a
cultured cell into which has been introduced an expression vector
as disclosed above, wherein the cell expresses a heterodimeric or
multimeric soluble receptor polypeptide encoded by the DNA
segments. In one embodiment is provided a cell as disclosed above,
wherein the cell secretes a soluble cytokine receptor polypeptide
heterodimer or multimeric complex. In another embodiment is
provided a cell as disclosed above, wherein the cell secretes a
soluble cytokine receptor polypeptide heterodimer or multimeric
complex that binds IL-TIF or antagonizes IL-TIF activity.
[0421] 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:2 from amino acid 22 to 231 or 28 to 231; and at
least one other DNA segment encoding a soluble Class I or Class II
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 encoding a fusion protein is as disclosed above, wherein
at least one other DNA segment encodes a polypeptide comprising a
soluble CRF2-4 receptor polypeptide (SEQ ID NO:35), a soluble IL-10
receptor polypeptide (SEQ ID NO:36), or soluble zcytor11 receptor
polypeptide (SEQ ID NO:34).
[0422] 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.
[0423] 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.
[0424] 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.
[0425] Within another aspect the present invention provides an
isolated soluble cytokine 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:2 from amino acid 22
to 231 or 28 to 231, and wherein the soluble cytokine receptor
polypeptide binds IL-TIF or antagonizes IL-TIF activity.
[0426] Within another aspect the present invention provides an
isolated polypeptide as disclosed above, wherein the soluble
cytokine receptor polypeptide forms a homodimeric, heterodimeric or
multimeric receptor complex. In one embodiment, the isolated
polypeptide is as disclosed above, wherein the soluble cytokine
receptor polypeptide forms a heterodimeric or multimeric receptor
complex further comprising a soluble Class I or Class II cytokine
receptor. In another embodiment, the isolated polypeptide is as
disclosed above, wherein the soluble cytokine receptor polypeptide
forms a heterodimeric or multimeric receptor complex further
comprising a soluble CRF2-4 receptor polypeptide (SEQ ID NO:35), a
soluble IL-10 receptor polypeptide (SEQ ID NO:36), or soluble
zcytor11 receptor polypeptide (SEQ ID NO:34).
[0427] Within another aspect the present invention provides an
isolated soluble cytokine receptor polypeptide comprising a
sequence of amino acid residues as shown in SEQ ID NO:2 from amino
acid 22 to 231 or 28 to 231, wherein the soluble cytokine receptor
polypeptide forms a homodimeric, heterodimeric or multimeric
receptor complex. In one embodiment, the isolated soluble cytokine
receptor polypeptide is as disclosed above, wherein the soluble
cytokine receptor polypeptide forms a heterodimeric or multimeric
receptor complex further comprising a soluble Class I or Class II
cytokine receptor. In another embodiment, the isolated soluble
cytokine receptor polypeptide is as disclosed above, wherein the
soluble cytokine receptor polypeptide forms a heterodimeric or
multimeric receptor complex comprising a soluble CRF2-4 receptor
polypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide
(SEQ ID NO:36), or soluble zcytor11 receptor polypeptide (SEQ ID
NO:34). In another embodiment, the isolated soluble cytokine
receptor polypeptide is as disclosed above, wherein the soluble
cytokine receptor polypeptide further comprises an affinity tag,
chemical moiety, toxin, or label.
[0428] Within another aspect the present invention provides an
isolated heterodimeric or multimeric soluble receptor complex
comprising soluble receptor subunits, wherein at least one of the
soluble receptor subunits comprises a soluble cytokine receptor
polypeptide comprising a sequence of amino acid residues as shown
in SEQ ID NO:2 from amino acid 22 to 231 or 28 to 231. In one
embodiment, the isolated heterodimeric or multimeric soluble
receptor complex disclosed above, further comprises a soluble Class
I or Class II cytokine receptor polypeptide. In another embodiment,
the isolated heterodimeric or multimeric soluble receptor complex
disclosed above, further comprises a soluble CRF2-4 receptor
polypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide
(SEQ ID NO:36), or soluble zcytor11 receptor polypeptide (SEQ ID
NO:34).
[0429] Within another aspect the present invention provides a
method of producing a soluble cytokine 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.
[0430] Within another aspect the present invention provides a
method of producing an antibody to soluble cytokine receptor
polypeptide comprising: inoculating an animal with a soluble
cytokine receptor polypeptide selected from the group consisting
of: (a) a polypeptide comprising a monomeric or homodimeric soluble
cytokine receptor comprising a polypeptide as shown in SEQ ID NO:2
from amino acid 22 to 231 or 28 to 231; (b) a polypeptide of (a)
further comprising a soluble cytokine receptor heterodimeric or
multimeric receptor complex comprising a soluble Class I or Class
II cytokine receptor polypeptide; (c) a polypeptide of (a) further
comprising a soluble cytokine receptor heterodimeric or multimeric
receptor complex comprising a soluble CRF2-4 receptor polypeptide
(SEQ ID NO:35); (d) a polypeptide of (a) further comprising a
soluble cytokine receptor heterodimeric or multimeric receptor
complex comprising a soluble IL-10 receptor polypeptide (SEQ ID
NO:36); and wherein the polypeptide elicits an immune response in
the animal to produce the antibody; and isolating the antibody from
the animal.
[0431] 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 polypeptide as shown in SEQ ID NO:2
from amino acid 22 to 231 or 28 to 231. In one embodiment, the
antibody disclosed above is a monoclonal antibody.
[0432] Within another aspect the present invention provides an
antibody which specifically binds to a homodimeric, heterodimeric
or multimeric receptor complex as disclosed above.
[0433] Within another aspect the present invention provides a
method for inhibiting IL-TIF-induced proliferation or
differentiation of hematopoietic cells and hematopoietic cell
progenitors comprising culturing bone marrow or peripheral blood
cells with a composition comprising an amount of soluble cytokine
receptor polypeptide as shown in SEQ ID NO:2 from amino acid 22 to
231 or 28 to 231, sufficient to reduce proliferation or
differentiation of the hematopoietic cells in the bone marrow or
peripheral blood cells as compared to bone marrow or peripheral
blood cells cultured in the absence of soluble cytokine 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 macrophages or T cells.
[0434] Within another aspect the present invention provides a
method of reducing IL-TIF-induced or IL-9 induced inflammation
comprising administering to a mammal with inflammation an amount of
a composition of a polypeptide as shown in SEQ ID NO:2 from amino
acid 22 to 231 or 28 to 231 sufficient to reduce inflammation.
[0435] Within another aspect the present invention provides a
method of suppressing an inflammatory response in a mammal with
inflammation comprising: (1) determining a level of serum amyloid A
protein; (2) administering a composition comprising a soluble
zcytor16 cytokine receptor polypeptide as disclosed above in an
acceptable pharmaceutical vehicle; (3) determining a post
administration level of serum amyloid A protein; (4) comparing the
level of serum amyloid A protein in step (1) to the level of serum
amyloid A protein in step (3), wherein a lack of increase or a
decrease in serum amyloid A protein level is indicative of
suppressing an inflammatory response.
[0436] Within another aspect the present invention provides a
method for detecting a genetic abnormality in a patient,
comprising: obtaining a genetic sample from a patient; producing a
first reaction product by incubating the genetic sample with a
polynucleotide comprising at least 14 contiguous nucleotides of SEQ
ID NO:1 or the complement of SEQ ID NO:1 or of SEQ ID NO:37 or the
complement of SEQ ID NO:37, under conditions wherein said
polynucleotide will hybridize to complementary polynucleotide
sequence; visualizing the first reaction product; and comparing
said first reaction product to a control reaction product from a
wild type patient, wherein a difference between said first reaction
product and said control reaction product is indicative of a
genetic abnormality in the patient.
[0437] Within another aspect the present invention provides a
method for detecting a cancer in a patient, comprising: obtaining a
tissue or biological sample from a patient; incubating the tissue
or biological sample with an antibody as disclosed above under
conditions wherein the antibody binds to its complementary
polypeptide in the tissue or biological sample; visualizing the
antibody bound in the tissue or biological sample; and comparing
levels of antibody bound in the tissue or biological sample from
the patient to a normal control tissue or biological sample,
wherein an increase in the level of antibody bound to the patient
tissue or biological sample relative to the normal control tissue
or biological sample is indicative of a cancer in the patient.
[0438] Within another aspect the present invention provides a
method for detecting a cancer in a patient, comprising: obtaining a
tissue or biological sample from a patient; labeling a
polynucleotide comprising at least 14 contiguous nucleotides of SEQ
ID NO:1 or the complement of SEQ ID NO:1 or of SEQ ID NO:37 or the
complement of SEQ ID NO:37; incubating the tissue or biological
sample with under conditions wherein the polynucleotide will
hybridize to complementary polynucleotide sequence; visualizing the
labeled polynucleotide in the tissue or biological sample; and
comparing the level of labeled polynucleotide hybridization in the
tissue or biological sample from the patient to a normal control
tissue or biological sample, wherein an increase in the labeled
polynucleotide hybridization to the patient tissue or biological
sample relative to the normal control tissue or biological sample
is indicative of a cancer in the patient.
[0439] Within another aspect the present invention provides a
method of treating a mammal afflicted with an inflammatory disease
in which IL-TIF or serum amyloid A plays a role, comprising:
administering an antagonist of IL-TIF or serum amyloid A to the
mammal such that the inflammation is reduced, wherein the
antagonist is selected from the group consisting of: a polypeptide
or cytokine binding domain fragment of SEQ ID NO:2; a soluble
receptor comprising a polypeptide or cytokine binding domain
fragment of SEQ ID NO:34; an antibody that specifically binds a
polypeptide or cytokine binding domain fragment of SEQ ID NO:2; and
an antibody or binding polypeptide that specifically binds a
polypeptide or polypeptide fragment of IL-TIF (SEQ ID NO:15). In
one embodiment is provided the method described above, wherein the
disease is a chronic inflammatory disease. In another embodiment is
provided the method described above, wherein the disease is a
chronic inflammatory disease selected from the group consisting of:
inflammatory bowel disease; ulcerative colitis; Crohn's disease;
arthritis; and psoriasis. In another embodiment is provided the
method described above, wherein the disease is an acute
inflammatory disease. In another embodiment is provided the method
described above, wherein the disease is an acute inflammatory
disease selected from the group consisting of: endotoxemia;
septicemia; toxic shock syndrome; and infectious disease. In
another embodiment is provided the method described above, wherein
the antagonist soluble receptor comprising a polypeptide or
cytokine binding domain fragment of zcytor11 (SEQ ID NO:34) further
comprises a polypeptide or cytokine binding domain fragment of
CRF2-4 (SEQ ID NO:35).
[0440] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Cloning of zcytor16 and Construction of Mammalian Expression
Vectors That Express zcytor16 Soluble Receptors: zcytor16CEE
zcytor16CFLG zcytor16CHIS and zcytor16-Fc4
[0441] A. Cloning of Zcytor16 Extracellular domain
[0442] Scanning of a translated human genomic database resulted in
identification of a class II cytokine receptor named zcytor16. The
sequence for zcytor16 was subsequently identified a clone from an
in-house derived shallow tonsil library. The insert in the tonsil
library clone was sequenced, and shown to encode the zcytor16
extracellular domain. The polynucleotide sequence of the zcytor16
clone is shown in SEQ ID NO:1 and polypeptide sequence shown in SEQ
ID NO:2.
B. Mammalian Expression Construction of Soluble zcytor16 Receptor
zcytor16-Fc4 Construction of mammalian expression vectors that
express zcytor16 soluble receptor zcytor16sR/Fc4
[0443] An expression vector was prepared to express the soluble
zcytor16 polypeptide (zcytor16sR, i.e., from residue 22 (Thr) to
residue 231 (Pro) of SEQ ID NO:2; SEQ ID NO:13) fused to a
C-terminal Fc4 tag (SEQ ID NO:5).
[0444] A PCR generated zcytor16 DNA fragment of about 630 bp was
created using oligo ZC29,181 (SEQ ID NO:6) and oligo ZC29,182 (SEQ
ID NO:7) as PCR primers to add BamHI and Bgl2 restriction sites at
5' and 3' ends respectively, of the zcytor16 DNA encoding the
soluble receptor. A plasmid containing the zcytor16 cDNA (SEQ ID
NO:1) (Example 1A) was used as a template. PCR amplification of the
zcytor16 fragment was performed as follows: One cycle at 94.degree.
C. for 1 minute; 25 cycles at 94.degree. C. for 30 seconds,
68.degree. C. for 90 seconds, followed by an additional 68.degree.
C. incubation for 4 minutes, and hold at 10.degree. C. The reaction
was purified by chloroform/phenol extraction and isopropanol
precipitation, and digested with BamHI and Bgl2 (Boehringer
Mannheim, Indianapolis, Ind.). A band of approximately 630 bp, as
visualized by 1% agarose gel electrophoresis, was excised and the
DNA was purified using a QiaexII.TM. purification kit (Qiagen,
Valencia, Calif.) according to the manufacturer's instruction.
[0445] The Fc4/pzmp20 plasmid is a mammalian expression vector
containing an expression cassette having the CMV promoter, human
tPA leader peptide, multiple restriction sites for insertion of
coding sequences, a Fc4 tag, 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, an
enhancer and an origin of replication, as well as a DHFR gene, and
SV40 terminator. The zcytor16sR/Fc4/pzmp20 expression vector uses
the human tPA leader peptide (SEQ ID NO:8 and SEQ ID NO:9) and
attaches the Fc4 tag (SEQ ID NO:5) to the C-terminus of the
extracellular portion of the zcytor16 polypeptide sequence. Fc4 is
the Fc region derived from human IgG, which contains a mutation so
that it no longer binds the Fc receptor
[0446] About 30 ng of the restriction digested zcytor16sR insert
and about 10 ng of the digested vector (which had been cut with
Bgl2) were ligated at 11.degree. C. overnight. One microliter of
ligation reaction was electroporated into DH10B competent cells
(Gibco BRL, Rockville, 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, which was 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. Mega prep kit (Qiagen) according to manufacturer's
instruction.
[0447] Similar methods are used to prepare non-zcytor16 subunits of
heterodimeric and multimeric receptors, such as CRF2-4 and IL-1 OR
tagged with Fc4.
C. Construction of zcytor16 Mammalian Expression Vector Containing
zcytor16CEE zcytor16CFLG and zcytor16CHIS
[0448] An expression vector is prepared for the expression of the
soluble, extracellular domain of the zcytor16 polypeptide (e.g.,
amino acids 22-231 of SEQ ID NO:2; SEQ ID NO:13), pC4zcytor16CEE,
wherein the construct is designed to express a zcytor16 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:10).
[0449] A zcytor16 DNA fragment comprising the zcytor16
extracellular cytokine binding domain (e.g., SEQ ID NO:13) is
created using PCR, and purified. The excised DNA is subcloned into
a plasmid expression vector that has a signal peptide, e.g., the
native zcytor16 signal peptide, tPA leader, and attaches a Glu-Glu
tag (SEQ ID NO:10) to the C-terminus of the zcytor16
polypeptide-encoding polynucleotide sequence. Such an expression
vector mammalian expression vector contains an expression cassette
having a mammalian promoter, multiple restriction sites for
insertion of coding sequences, a stop codon and a mammalian
terminator. The plasmid can also have an E. coli origin of
replication, a mammalian selectable marker expression unit having
an SV40 promoter, enhancer and origin of replication, a DHFR gene
and the SV40 terminator.
[0450] Restriction digested zcytor16 insert and previously digested
vector are ligated using standard molecular biological techniques,
and electroporated into competent cells such as DH10B competent
cells (GIBCO BRL, Gaithersburg, Md.) according to manufacturer's
direction and plated onto LB plates containing 50 mg/ml ampicillin,
and incubated overnight. Colonies are screened by restriction
analysis of DNA prepared from individual colonies. The insert
sequence of positive clones is verified by sequence analysis. A
large-scale plasmid preparation is done using a QIAGEN.RTM. Maxi
prep kit (Qiagen) according to manufacturer's instructions.
[0451] The same process is used to prepare the zcytor16 soluble
homodimeric, heterodimeric or multimeric receptors (including
non-zcytor16 soluble receptor subunits, such as, soluble CRF2-4 or
IL-10R) with a C-terminal HIS tag, composed of 6 His residues in a
row (SEQ ID NO:12); and a C-terminal FLAG (SEQ ID NO:11) tag,
zcytor16CFLAG. 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:10).
Example 2
Transfection And Expression of Soluble Receptor Polypeptides
[0452] The day before the transfection, BHK 570 cells (ATCC No.
CRL-10314; ATCC, Manasas, Va.) were plated in a 10-cm plate with
50% confluence in normal BHK DMEM (Gibco/BRL High Glucose) media.
The day of the transfection, the cells were washed once with Serum
Free (SF) DMEM, followed by transfection with the
zcytor16sR/Fc4/pzmp20 expression plasmids. Sixteen micrograms of
zcytor16sR-Fc4 DNA construct (Example 1B) were diluted into a total
final volume of 640 .mu.l SF DMEM. A diluted LipofectAMINE.TM.
(Gibco BRL, Gaithersburg, Md.) mixture (35 .mu.l LipofectAMINE.TM.
in 605 .mu.L SF media) was added to the DNA mix, and incubated for
30 minutes at room temperature. Five milliliters of SF media was
added to the DNA/LipofectAMINE.TM. mixture, which was then added to
BHK cells. The cells were incubated at 37.degree. C./5% CO.sub.2
for 5 hours, after which 6.4 ml of BHK media with 10% FBS was
added. The cells were incubated overnight at 37.degree. C./5%
CO.sub.2.
[0453] Approximately 24 hours post-transfection, the BHK cells were
split into selection media with 1 .mu.M methotrexate (MTX). The
cells were repeatedly split in this manner until stable
zcytor16sR-Fc4/BHK cell lines were identified. To detect the
expression level of the zcytor16 soluble receptor fusion proteins,
the transfected BHK cells were washed with PBS and incubated in SF
media for 72 hours. The SF condition media was collected and 20
.mu.l of the sample was run on 10% SDS-PAGE gel under reduced
conditions. The protein bands were transferred to nitrocellulose
filter by Western blot, and the fusion proteins were detected using
goat-anti-human IgG/HRP conjugate (Jackson ImmunoResearch
Laboratories, Inc, West Grove, Pa.). An expression vector
containing a different soluble receptor fused to the Fc4 was used
as a control. The expression level of the stable zcytor16sR-Fc4/BHK
cells was approximately 2 mg/L.
[0454] For protein purification, the transfected BHK cells were
transferred into T-162 flasks. Once the cells reached about 80%
confluence, they were washed with PBS and incubated in 100 ml SF
media for 72 hours, and then the condition media was collected for
protein purification (Example 11).
Example 3
Expression of zcytor16 Soluble Receptor in E. coli
[0455] A. Construction of Expression Vector pCZR225 that Expresses
huzcytor16/MBP-6H Fusion Polypeptide
[0456] An expression plasmid containing a polynucleotide encoding a
zcytor16 soluble receptor fused C-terminally to maltose binding
protein (MBP) is constructed via homologous recombination. The
fusion polypeptide contains an N-terminal approximately 388 amino
acid MBP portion fused to the zcytor16 soluble receptor (e.g., SEQ
ID NO:13). A fragment of zcytor16 cDNA (SEQ ID NO:1) is isolated
using PCR as described herein. Two primers are used in the
production of the zcytor16 fragment in a standard PCR reaction: (1)
one containing about 40 bp of the vector flanking sequence and
about 25 bp corresponding to the amino terminus of the zcytor16,
and (2) another containing about 40 bp of the 3' end corresponding
to the flanking vector sequence and about 25 bp corresponding to
the carboxyl terminus of the zcytor16. Two .mu.l of the 100 .mu.l
PCR reaction is run on a 1.0% agarose gel with 1.times.TBE buffer
for analysis, and the expected approximately fragment is seen. The
remaining PCR reaction is combined with the second PCR tube and
precipitated with 400 .mu.l of absolute ethanol. The precipitated
DNA used for recombining into an appropriately restriction digested
recipient vector pTAP98 to produce the construct encoding the
MBP-zcytor16 fusion, as described below.
[0457] Plasmid pTAP98 is 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 is constructed using yeast homologous
recombination. 100 ng of EcoR1 cut pMAL-c2 is recombined with 1
.mu.g Pvu1 cut pRS316, 1 .mu.g linker, and 1 .mu.g Sca1/EcoR1 cut
pRS316 are combined in a PCR reaction. PCR products are
concentrated via 100% ethanol precipitation.
[0458] Competent yeast cells (S. cerevisiae) are combined with
about 10 .mu.l of a mixture containing approximately 1 .mu.g of the
zcytor16 receptor PCR product above, and 100 ng of digested pTAP98
vector, and electroporated using standard methods and plated onto
URA-D plates and incubated at 30.degree. C.
[0459] After about 48 hours, the Ura+ yeast transformants from a
single plate are picked, DNA isolated, and transformed into
electrocompetent E. coli cells (e.g., MC1061, Casadaban et. al. J.
Mol. Biol. 138, 179-207), and plated on MM/CA+AMP 100 mg/L plates
(Pryor and Leiting, Protein Expression and Purification 10:309-319,
1997). using standard procedures. Cells are grown in MM/CA with 100
.mu.g/ml Ampicillin for two hours, shaking, at 37.degree. C. 1 ml
of the culture is induced with 1 mM IPTG. 2-4 hours later the 250
.mu.l of each culture is 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
are vortexed for one minute and heated to 65.degree. C. for 10
minutes. 20 .mu.l are loaded per lane on a 4%-12% PAGE gel (NOVEX).
Gels are run in 1XMES buffer. The positive clones are designated
pCZR225 and subjected to sequence analysis.
[0460] One microliter of sequencing DNA is used to transform strain
BL21. The cells are electropulsed at 2.0 kV, 25 .mu.F and 400 ohms.
Following electroporation, 0.6 ml MM/CA with 100 mg/L Ampicillin.
Cells are grown in MM/CA and induced with ITPG as described above.
The positive clones are used to grow up for protein purification of
the huzcytor16/MBP-6H fusion protein using standard techniques.
Example 4
Zcytor16 Soluble Receptor Polyclonal Antibodies
[0461] Polyclonal antibodies are prepared by immunizing female New
Zealand white rabbits with the purified huzcytor16/MBP-6H
polypeptide (Example 3), or the purified recombinant zcytor16CEE
soluble receptor (Example 1; Example 11). The rabbits are each
given an initial intraperitoneal (IP) injection of about 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 are bled and the serum is collected. The rabbits are then
boosted and bled every three weeks.
[0462] The zcytor16-specific polyclonal antibodies are affinity
purified from the rabbit serum using an CNBr-SEPHAROSE 4B protein
column (Pharmacia LKB) that is prepared using about 10 mg of the
appropriate purified zcytor16 polypeptide per gram CNBr-SEPHAROSE,
followed by 20.times. dialysis in PBS overnight. Zcytor16-specific
antibodies are 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-zcytor16 affinity
purified antibodies is determined using standard methods.
Example 5
Zcytor16 Receptor Monoclonal Antibodies
[0463] Zcytor16 receptor Monoclonal antibodies are prepared by
immunizing male BalbC mice (Harlan Sprague Dawley, Indianapolis,
Ind.) with the purified recombinant zcytor16 proteins described
herein. The mice are 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 are bled and the serum is collected,
and antibody titer assessed.
[0464] Splenocytes are harvested from 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 are
identified by ELISA using purified recombinant zcytor16 soluble
receptor protein (Example 6C) as an antibody target and by FACS
using Baf3 cells expressing the zcytor16 sequence (Example 8) as an
antibody target. The resulting hybridomas positive by both methods
are cloned three times by limiting dilution.
Example 6
Assessing Zcytor16 Receptor Heterodimerization Using ORIGEN
Assay
[0465] Soluble zcytor16 receptor (Example 11), or gp130 (Hibi, M.
et al., Cell 63:1149-1157, 1990) are biotinylated by reaction with
a five-fold molar excess of sulfo-NHS-LC-Biotin (Pierce, Inc.,
Rockford, Ill.) according to the manufacturer's protocol. Soluble
zcytor16 receptor and another soluble receptor subunit, for
example, soluble IL-10R (sIL-10R) or CRF2-4 receptor (CRF2-4),
soluble zcytor11 receptor (U.S. Pat. No. 5,965,704) or soluble
zcytor7 receptor (U.S. Pat. No. 5,945,511) are labeled with a five
fold molar excess of Ru--BPY--NHS (Igen, Inc., Gaithersburg, Md.)
according to manufacturer's protocol. The biotinylated and
Ru--BPY--NHS-labeled forms of the soluble zcytor16 receptor can be
respectively designated Bio-zcytor16 receptor and Ru-zcytor16; the
biotinylated and Ru--BPY--NHS-labeled forms of the other soluble
receptor subunit can be similarly designated. Assays can be carried
out using conditioned media from cells expressing a ligand, such as
IL-TIF, that binds zcytor16 heterodimeric receptors, or using
purified IL-TIF.
[0466] For initial receptor binding characterization a panel of
cytokines or conditioned medium are tested to determine whether
they can mediate homodimerization of zcytor16 receptor and if they
can mediate the heterodimerization of zcytor16 receptor with the
soluble receptor subunits described above. To do this, 50 .mu.l of
conditioned media or TBS-B containing purified cytokine, is
combined with 50 .mu.l of TBS-B (20 mM Tris, 150 mM NaCl, 1 mg/ml
BSA, pH 7.2) containing e.g., 400 ng/ml of Ru-zcytor16 receptor and
Bio-zcytor16, or 400 ng/ml of Ru-zcytor16 receptor and e.g.,
Bio-gp130, or 400 ng/ml of e.g., Ru-CRF2-4 and Bio-zcytor16.
Following incubation for one hour at room temperature, 30 .mu.g of
streptavidin coated, 2.8 mm magnetic beads (Dynal, Inc., Oslo,
Norway) are added and the reaction incubated an additional hour at
room temperature. 200 .mu.l ORIGEN assay buffer (Igen, Inc.,
Gaithersburg, Md.) is then added and the extent of receptor
association measured using an M8 ORIGEN analyzer (Igen, Inc.).
Example 7
Construct for Generating a zcytor16 Receptor Heterodimer
[0467] A vector expressing a secreted human zcytor16 heterodimer is
constructed. In this construct, the extracellular cytokine-binding
domain of zcytor16 (e.g., SEQ ID NO:13) is fused to the heavy chain
of IgG gamma 1 (IgG.gamma.1) while the extracellular portion of the
heteromeric cytokine receptor subunit (e.g., an CRF2-4, IL-9,
IL-10, zcytor7, zcytor11, IL-4 receptor component) is fused to a
human kappa light chain (human .kappa. light chain).
A. Construction of IgG Gamma 1 and Human .kappa. Light Chain Fusion
Vectors
[0468] The heavy chain of IgG.gamma.1 can be cloned into the
Zem229R mammalian expression vector (ATCC deposit No. 69447) such
that any desired cytokine receptor extracellular domain having a 5'
EcoRI and 3' NheI site can be cloned in resulting in an N-terminal
extracellular domain-C-terminal IgG.gamma.1 fusion. The IgG.gamma.1
fragment used in this construct is made by using PCR to isolate the
IgG.gamma.1 sequence from a Clontech hFetal Liver cDNA library as a
template. PCR products are purified using methods described herein
and digested with MluI and EcoRI (Boerhinger-Mannheim), ethanol
precipitated and ligated with oligos which comprise an desired
restriction site linker, into Zem229R previously digested with and
EcoRI using standard molecular biology techniques disclosed
herein.
[0469] The human .kappa. light chain can be cloned in the Zem228R
mammalian expression vector (ATCC deposit No. 69446) such that any
desired cytokine receptor extracellular domain having a 5' EcoRI
site and a 3' KpnI site can be cloned in resulting in a N-terminal
cytokine extracellular domain-C-terminal human .kappa. light chain
fusion. As a KpnI site is located within the human .kappa. light
chain sequence, a special primer is designed to clone the 3' end of
the desired extracellular domain of a cytokine receptor into this
KpnI site: The primer is designed so that the resulting PCR product
contains the desired cytokine receptor extracellular domain with a
segment of the human .kappa. light chain up to the KpnI site. This
primer preferably comprises a portion of at least 10 nucleotides of
the 3' end of the desired cytokine receptor extracellular domain
fused in frame 5' to fragment cleaved at the KpnI site. The human
.kappa. light chain fragment used in this construct is made by
using PCR to isolate the human .kappa. light chain sequence from
the same Clontech human Fetal Liver cDNA library used above. PCR
products are purified using methods described herein and digested
with MluI and EcoRI (Boerhinger-Mannheim), ethanol precipitated and
ligated with the MluI/EcoRI linker described above, into Zem228R
previously digested with and EcoRI using standard molecular biology
techniques disclosed herein.
B. Insertion of zcytor16 Receptor or Heterodimeric Subunit
Extracellular Domains into Fusion Vector Constructs
[0470] Using the construction vectors above, a construct having
zcytor16 fused to IgG.gamma.1 is made. This construction is done by
PCRing the extracellular cytokine-binding domain of zcytor16
receptor (SEQ ID NO:13) from a tonsil cDNA library (Clontech) or
plasmid (Example 1A) using standard methods and oligos that provide
EcoRI and NheI restriction sites. The resulting PCR product is
digested with EcoRI and NheI, gel purified, as described herein,
and ligated into a previously EcoRI and NheI digested and
band-purified Zem229R/IgG.gamma.1 described above. The resulting
vector is sequenced to confirm that the zcytor16/IgG gamma 1 fusion
is correct.
[0471] A separate construct having a heterodimeric cytokine
receptor subunit extracellular domain fused to .kappa. light is
also constructed as above. The CRF2-4/human .kappa. light chain
construction is performed as above by PCRing from, e.g., a
lymphocyte cDNA library (Clontech) using standard methods, and
oligos that provide EcoRI and KpnI restriction sites. The resulting
PCR product is digested with EcoRI and KpnI and then ligating this
product into a previously EcoRI and KpnI digested and band-purified
Zem228R/human .kappa. light chain vector described above. The
resulting vector is sequenced to confirm that the cytokine receptor
subunit/human .kappa. light chain fusion is correct.
D. Co-expression of the zcytor16 and Heterodimeric Cytokine
Receptor Subunit Extracellular Domain
[0472] Approximately 15 .mu.g of each of vectors above, are
co-transfected into mammalian cells, e.g., BHK-570 cells (ATCC No.
CRL-10314) using LipofectaminePlus.TM. reagent (Gibco/BRL), as per
manufacturer's instructions. The transfected cells are selected for
10 days in DMEM+5% FBS (Gibco/BRL) containing 1 .mu.M of
methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5 mg/ml G418
(Gibco/BRL) for 10 days. The resulting pool of transfectants is
selected again in 10 .mu.m of MTX and 0.5 mg/ml G418 for about 10
days.
[0473] The resulting pool of doubly selected cells is used to
generate protein. Three Factories (Nunc, Denmark) of this pool are
used to generate 10 L of serum free conditioned medium. This
conditioned media is passed over a 1 ml protein-A column and eluted
in about 10, 750 microliter fractions. The fractions having the
highest protein concentration are pooled and dialyzed (10 kD MW
cutoff) against PBS. Finally the dialyzed material is submitted for
amino acid analysis (AAA) using routine methods.
Example 8
Determination of Receptor Subunits That Heterodimerize or
Multimerize With zcytor16 Receptor Using a Proliferation Assay
[0474] Using standard methods described herein, cells expressing a
BaF3/zcytor16-MPL chimera (wherein the extracellular domain of the
zcytor16 (e.g., SEQ ID NO:13) is fused in frame to the
intracellular signaling domain of the mpl receptor) are tested for
proliferative response in the presence of IL-TIF. Such cells serve
as a bioassay cell line to measure ligand binding of monomeric or
homodimeric zcytor16 receptors. In addition, BaF3/zcytor16-MPL
chimera cells transfected with an additional heterodimeric cytokine
receptor subunit can be assessed for proliferative response in the
presence of IL-TIF. In the presence of IL-TIF, if the
BaF3/zcytor16-MPL cells signal, this would suggest that zcytor16
receptor can homodimerize to signal. Transfection of the
BaF3/MPL-zcytor16 cell line with and additional MPL-class II
cytokine receptor fusion that signals in the presence of the IL-TIF
ligand, such as CRF2-4, determines which heterodimeric cytokine
receptor subunits are required for zcytor16 receptor signaling. Use
of MPL-receptor fusions for this purpose alleviates the requirement
for the presence of an intracellular signaling domain for the
zcytor16 receptor.
[0475] Each independent receptor complex cell line is then assayed
in the presence of IL-TIF and proliferation measured using routine
methods (e.g., Alamar Blue assay). The BaF3/MPL-zcytor16 bioassay
cell line serves as a control for the monomeric or homodimeric
receptor activity, and is thus used as a baseline to compare
signaling by the various receptor complex combinations. The
untransfected bioassay cell line serves as a control for the
background activity, and is thus used as a baseline to compare
signaling by the various receptor complex combinations. A
BaF3/MPL-zcytor16 without ligand (IL-TIF) is also used as a
control. The IL-TIF in the presence of the correct receptor
complex, is expected to increase proliferation of the
BaF3/zcytor16-MPL receptor cell line approximately 5 fold over
background or greater in the presence of IL-TIF. Cells expressing
the components of zcytor16 heterodimeric and multimeric receptors
should proliferate in the presence of IL-TIF.
Example 9
Reconstitution of zcytor16 Receptor In Vitro
[0476] To identify components involved in the zcytor16-signaling
complex, receptor reconstitution studies are performed as follows.
BHK 570 cells (ATCC No. CRL-10314) transfected, using standard
methods described herein, with a luciferase reporter mammalian
expression vector plasmid serve as a bioassay cell line to measure
signal transduction response from a transfected zcytor16 receptor
complex to the luciferase reporter in the presence of IL-TIF. BHK
cells do not endogenously express the zcytor16 receptor. An
exemplary luciferase reporter mammalian expression vector is the
KZ134 plasmid which was constructed with complementary
oligonucleotides that contain STAT transcription factor binding
elements from 4 genes. A modified c-fos Sis inducible element
(m67SIE, or hSIE) (Sadowski, H. et al., Science 261:1739-1744,
1993), the p21 SIE1 from the p21 WAF1 gene (Chin, Y. et al.,
Science 272:719-722, 1996), the mammary gland response element of
the .beta.-casein gene (Schmitt-Ney, M. et al., Mol. Cell. Biol.
11:3745-3755, 1991), and a STAT inducible element of the Fcg RI
gene, (Seidel, H. et al., Proc. Natl. Acad. Sci. 92:3041-3045,
1995). These oligonucleotides contain Asp718-XhoI compatible ends
and 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 is used to stably transfect BHK, or BaF3 cells, using
standard transfection and selection methods, to make a BHK/KZ134 or
BaF3/KZ134 cell line respectively.
[0477] The bioassay cell line is transfected with zcytor16-mpl
fusion receptor alone, or co-transfected along with one of a
variety of other known receptor subunits. Receptor complexes
include but are not limited to zcytor16-mpl receptor only, various
combinations of zcytor16-mpl receptor with one or more of the
CRF2-4, IL-9, IL-10, zcytor11, zcytor7 class II cytokine receptor
subunits, or IL-4 receptor components, or the IL-2 receptor
components (IL-2R.alpha., IL-2R.beta., IL-2R.gamma.); zcytor16-mpl
receptor with one or more of the IL-4/IL-13 receptor family
receptor components (IL-4R.alpha., IL-13R.alpha., IL-13R.alpha.'),
as well as other Interleukin receptors (e.g., IL-15 R.alpha.,
IL-7R.alpha., IL-9R.alpha., IL-21R (zalpha11; WIPO publication WO
00/17235; Parrish-Novak, J et al., Nature 408:57-63, 2000)). Each
independent receptor complex cell line is then assayed in the
presence of cytokine-conditioned media or purified cytokines and
luciferase activity measured using routine methods. The
untransfected bioassay cell line serves as a control for the
background luciferase activity, and is thus used as a baseline to
compare signaling by the various receptor complex combinations. The
conditioned medium or cytokine that binds the zcytor16 receptor in
the presence of the correct receptor complex, is expected to give a
luciferase readout of approximately 5 fold over background or
greater.
[0478] As an alternative, a similar assay can be performed wherein
Baf3/zcytor16-mpl cell lines are co-transfected as described above
and proliferation measured (Example 8).
Example 10
COS Cell Transfection and Secretion Trap
[0479] COS cell transfections were performed as follows: A mixture
of 0.5 .mu.g DNA and 5 .mu.l lipofectamine (Gibco BRL) in 92 ul
serum free DMEM media (55 mg sodium pyruvate, 146 mg L-glutamine, 5
mg transferrin, 2.5 mg insulin, 1 g selenium and 5 mg fetuin in 500
ml DMEM) was incubated at room temperature for 30 minutes and then
400 .mu.l serum free DMEM media added. A 500 .mu.l mixture was
added onto COS cells plated on 12-well tissue culture plate at
1.5.times.10.sup.5 COS cells/well and previously incubated for 5
hours at 37.degree. C. An additional 500 .mu.l 20% FBS DMEM media
(100 ml FBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 ml
DMEM) was added and the plates were incubated overnight.
[0480] The secretion trap was performed as follows: Media was
rinsed off cells with PBS and 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). Cells were
permeated with 0.1% Triton-X in PBS for 15 minutes and washed again
with TNT. C ells were blocked for 1 hour with TNB (0.1M Tris-HCl,
0.15M NaCl and 0.5% Blocking Reagent (NEN Renaissance TSA-Direct
Kit; NEN) in H.sub.2O Cells were again washed with TNT. Cells were
then incubated for 1 hour with 1-3 .mu.g/ml zcytor16 soluble
receptor Fc4 fusion protein (zcytor16sR-Fc4) (Example 11) in TNB.
Cells were washed with TNT, and then incubated for another hour
with 1:200 diluted goat-anti-human Ig-HRP (Fc specific; Jackson
ImmunoResearch Laboratories, Inc.) in TNB. Cells were again washed
with TNT. Antibodies positively binding to the zcytor16sR-Fc4 were
detected with fluorescein tyramide reagent diluted 1:50 in dilution
buffer (NEN kit) and incubated for 4-6 minutes. Cells were again
washed with TNT. Cells were preserved with Vectashield Mounting
Media (Vector Labs) diluted 1:5 in TNT. Cells were visualized using
FITC filter on fluorescent microscope.
[0481] Since zcytor16 is a Class II cytokine receptor, the binding
of zcytor16sR/Fc4 fusion protein with known or orphan Class II
cytokines was tested. The pZP7 expression vectors containing cDNAs
of cytokines (including human IL-TIF, interferon alpha, interferon
beta, interferon gamma, IL-10, amongst others) were transfected
into COS cells, and the binding of zcytor16sR/Fc4 to transfected
COS cells were carried out using the secretion trap assay described
above. Human IL-TIF showed positive binding. Based on these data,
human IL-TIF and zcytor16 is a potential ligand-receptor pair.
Example 11
Purification of zcytor16-Fc4 Polypeptide From Transfected BHK 570
Cells
[0482] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
zcytor16 polypeptide containing C-terminal fusion to human Fc4
(zcytor16-Fc4; Example 1). About 16,500 ml of conditioned media
from BHK 570 cells transfected with zcytor16-Fc4 (Example 2) was
filtered through a 0.2 um 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 Poros protein A50 column (20 ml bed
volume, Applied Biosystems) was packed and washed with 400 ml PBS
(Gibco/BRL) The supplemented conditioned media was passed over the
column with a flow rate of 15 ml/minute, followed by washing with
800 ml PBS (BRL/Gibco). Zcytor16-Fc4 was eluted from the column
with 0.1 M Glycine pH 3.0 and 5 ml fractions were collected
directly into 0.5 ml 2M Tris pH 7.8, to adjust the final pH to 7.4
in the fractions.
[0483] Column performance was characterized through western
blotting of reducing SDS-PAGE gels of the starting media and column
pass through. Western blotting used anti-human IgG HRP (Amersham)
antibody, which showed an immunoreactive protein at 60,000 Da in
the starting media, with nothing in the pass through, suggesting
complete capture. The protein A50 eluted fractions were
characterized by reducing SDS PAGE gel. This gel showed an
intensely Coomassie stained band at 60,000 Da in fractions 3 to 11.
Fractions 3 to 11 were pooled.
[0484] Protein A 50 elution pool was concentrated from 44 ml to 4
ml using a 30,000 Da Ultrafree Biomax centrifugal concentrator (15
ml volume, Millipore). A Sephacryl S-300 gel filtration column (175
ml bed volume; Pharmacia) was washed with 350 ml PBS (BRL/Gibco).
The concentrated pool was injected over the column with a flow rate
of 1.5 ml/min, followed by washing with 225 ml PBS (BRL/Gibco).
Eluted peaks were collected into 2 ml fractions.
[0485] Eluted fractions were characterized by reducing and
non-reducing silver stained (Geno Technology) SDS PAGE gels.
Reducing silver stained SDS PAGE gels showed an intensely stained
band at 60,000 Da in fractions 14-31, while non-reducing silver
stained SDS PAGE gels showed an intensely stained band at 160,000
Da in fractions 14-31. Fractions 1-13 showed many bands of various
sizes. Fractions 14-31 were pooled, concentrated to 22 ml using
30,000 Da Ultrafree Biomax centrifugal concentrator (15 ml volume,
Millipore). This concentrate was filtered through a 0.2 .mu.m
Acrodisc sterilizing filter (Pall Corporation).
[0486] The protein concentration of the concentrated 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 1.50 mg/ml.
Example 12
Human zcytor16 Tissue Distribution in Tissue Panels Using Northern
Blot and PCR
[0487] A. Human zcytor16 Tissue Distribution using Northern Blot
and Dot Blot
[0488] Northern blot analysis was performed using Human Multiple
Tissue Northern Blots I, II, III (Clontech) and an in house
generated U-937 northern blot. U-937 is a human monoblastic
promonocytic cell line. The cDNA probe was generated using oligos
ZC25,963 (SEQ ID NO:16) and ZC28,354 (SEQ ID NO:17). The PCR
conditions were as follows: 940 for 1 minute; 30 cycles of
94.degree., 15 seconds; 60.degree., 30 seconds; 72.degree., 30
seconds and a final extension for 5 minutes at 72.degree.. The 364
bp product was gel purified by gel electrophoresis on a 1% TBE gel
and the band was excised with a razor blade. The cDNA was extracted
from the agarose using the QIAquick Gel Extraction Kit (Qiagen). 94
ng of this fragment was radioactively labeled with .sup.32P-dCTP
using Rediprime II (Amersham), a random prime labeling system,
according to the manufacturer's specifications. Unincorporated
radioactivity was removed using a Nuc-Trap column (Stratagene)
according to manufacturer's instructions. Blots were prehybridized
at 650 for 3 hours in ExpressHyb (Clontech) solution. Blots were
hybridized overnight at 650 in Expresshyb solution containing
1.0.times.10.sup.6 cpm/ml of labeled probe, 0.1 mg/ml of salmon
sperm DNA and 0.5 .mu.g/ml of human cot-1 DNA. Blots were washed in
2.times.SSC, 0.1% SDS at room temperature with several solution
changes then washed in 0.1.times.SSC. 0.1% SDS at 550 for 30
minutes twice. Transcripts of approximately 1.6 kb and 3.0 kb size
were detected in spleen and placenta, but not other tissues
examined. The same sized transcripts plus an additional approximate
1.2 kb transcript was detected in U-937 cell line.
B. Tissue Distribution in tissue cDNA panels using PCR
[0489] A panel of cDNAs from human tissues was screened for
zcytor16 expression using PCR. The panel was made in-house and
contained 94 marathon cDNA and cDNA samples from various normal and
cancerous human tissues and cell lines are shown in Table 5, below.
The cDNAs came from in-house libraries or marathon cDNAs from
in-house RNA preps, Clontech RNA, or Invitrogen RNA. The marathon
cDNAs were made using the marathon-Ready.TM. kit (Clontech, Palo
Alto, Calif.) and QC tested with clathrin primers ZC21195 (SEQ ID
NO:18) and ZC21196 (SEQ ID NO:19) and then diluted based on the
intensity of the clathrin band. To assure quality of the panel
samples, three tests for quality control (QC) were run: (1) To
assess the RNA quality used for the libraries, the in-house cDNAs
were tested for average insert size by PCR with vector oligos that
were specific for the vector sequences for an individual cDNA
library; (2) Standardization of the concentration of the cDNA in
panel samples was achieved using standard PCR methods to amplify
full length alpha tubulin or G3PDH cDNA using a 5' vector oligo
ZC14,063 (SEQ ID NO:20) and 3' alpha tubulin specific oligo primer
ZC17,574 (SEQ ID NO:21) or 3' G3PDH specific oligo primer ZC17,600
(SEQ ID NO:22); and (3) a sample was sent to sequencing to check
for possible ribosomal or mitochondrial DNA contamination. The
panel was set up in a 96-well format that included a human genomic
DNA (Clontech, Palo Alto, Calif.) positive control sample. Each
well contained approximately 0.2-100 pg/.mu.l of cDNA. The PCR
reactions were set up using oligos ZC25,963 (SEQ ID NO:16) and
ZC27,659 (SEQ ID NO:23), Advantage 2 DNA Polymerase Mix (Clontech)
and Rediload dye (Research Genetics, Inc., Huntsville, Ala.). The
amplification was carried out as follow: 1 cycle at 94.degree. C.
for 2 minutes, 30 cycles of 94.degree. C. for 20 seconds,
58.degree. C. for 30 seconds and 72.degree. C. for 1 minute,
followed by 1 cycle at 72.degree. C. for 5 minutes. About 10 .mu.l
of the PCR reaction product was subjected to standard Agarose gel
electrophoresis using a 2% agarose gel. The correct predicted DNA
fragment size was not observed in any tissue or cell line.
Subsequent experiments showing expression of zcytor16 indicated
that the negative results from this panel were likely due to the
primers used.
TABLE-US-00005 TABLE 5 Tissue/Cell line #samples Tissue/Cell line
#samples Adrenal gland 1 Bone marrow 3 Bladder 1 Fetal brain 3 Bone
Marrow 1 Islet 2 Brain 1 Prostate 3 Cervix 1 RPMI #1788 (ATCC #
CCL-156) 2 Colon 1 Testis 4 Fetal brain 1 Thyroid 2 Fetal heart 1
WI38 (ATCC # CCL-75 2 Fetal kidney 1 ARIP (ATCC # CRL-1674 - rat) 1
Fetal liver 1 HaCat - human keratinocytes 1 Fetal lung 1 HPV (ATCC
# CRL-2221) 1 Fetal muscle 1 Adrenal gland 1 Fetal skin 1 Prostate
SM 2 Heart 2 CD3+ selected PBMC's 1 Ionomycin + PMA stimulated K562
(ATCC # CCL-243) 1 HPVS (ATCC # CRL-2221) - 1 selected Kidney 1
Heart 1 Liver 1 Pituitary 1 Lung 1 Placenta 2 Lymph node 1 Salivary
gland 1 Melanoma 1 HL60 (ATCC # CCL-240) 3 Pancreas 1 Platelet 1
Pituitary 1 HBL-100 1 Placenta 1 Renal mesangial 1 Prostate 1
T-cell 1 Rectum 1 Neutrophil 1 Salivary Gland 1 MPC 1 Skeletal
muscle 1 Hut-102 (ATCC # TIB-162) 1 Small intestine 1 Endothelial 1
Spinal cord 1 HepG2 (ATCC # HB-8065) 1 Spleen 1 Fibroblast 1
Stomach 1 E. Histo 1 Testis 2 Thymus 1 Thyroid 1 Trachea 1 Uterus 1
Esophagus tumor 1 Gastric tumor 1 Kidney tumor 1 Liver tumor 1 Lung
tumor 1 Ovarian tumor 1 Rectal tumor 1 Uterus tumor 1
[0490] An additional panel of cDNAs from human tissues was screened
for zcytor16 expression using PCR. This panel was made in-house and
contained 77 marathon cDNA and cDNA samples from various normal and
cancerous human tissues and cell lines are shown in Table 6, below.
Aside from the PCR reaction, the assay was carried out as per
above. The PCR reactions were set up using oligos ZC25,963 (SEQ ID
NO:30) and ZC25,964 (SEQ ID NO:31), Advantage 2 DNA Polymerase Mix
(Clontech) and Rediload dye (Research Genetics, Inc., Huntsville,
Ala.). The amplification was carried out as follow: 1 cycle at
94.degree. C. for 1 minute, 38 cycles of 94.degree. C. for 10
seconds, 60.degree. C. for 30 seconds and 72.degree. C. for 30
seconds, followed by 1 cycle at 72.degree. C. for 5 minutes. The
correct predicted DNA fragment size was observed in bone marrow,
fetal heart, fetal kidney, fetal muscle, fetal skin, heart, mammary
gland, placenta, salivary gland, skeletal muscle, small intestine,
spinal cord, spleen, kidney, fetal brain, esophageal tumor, uterine
tumor, stomach tumor, ovarian tumor, rectal tumor, lung tumor and
RPMI-1788 (a B-lymphocyte cell line). Zcytor16 expression was not
observed in the other tissues and cell lines tested in this panel.
The expression pattern of zcytor16 shows expression in certain
tissue-specific tumors especially, e.g., ovarian cancer, stomach
cancer, uterine cancer, rectal cancer, lung cancer and esophageal
cancer, where zcytor16 is not expressed in normal tissue, but is
expressed in the tumor tissue. One of skill in the art would
recognize that the polynucleotides, polypeptides, antibodies, and
binding partners of the present invention can be used as a
diagnostic to detect cancer, or cancer tissue in a biopsy, tissue,
or histologic sample, particularly e.g., ovarian cancer, stomach
cancer, uterine cancer, rectal cancer, lung cancer and esophageal
cancer tissue. Such diagnostic uses for the molecules of the
present invention are known in the art and described herein.
[0491] In addition, because the expression pattern of zcytor16, one
of IL-TIF's receptors, shows expression in certain specific tissues
as well as tissue-specific tumors, binding partners including the
natural liganed, IL-TIF, can also be used as a diagnostic to detect
specific tissues (normal or abnormal), cancer, or cancer tissue in
a biopsy, tissue, or histologic sample, where IL-TIF receptors are
expressed, and particularly e.g., ovarian cancer, stomach cancer,
uterine cancer, rectal cancer, lung cancer and esophageal cancer
tissue. IL-TIF can also be used to target other tissues wherein its
receptors, e.g., zcytor16 and zcytor11 are expressed. Moreover,
such binding partners could be conjugated to chemotherapeutic
agents, toxic moieties and the like to target therapy to the site
of a tumor or diseased tissue. Such diagnostic and targeted therapy
uses are known in the art and described herein.
[0492] A commercial 1st strand cDNA panel (Human Blood Fractions
MTC Panel, Clontech, Palo Alto, Calif.) was also assayed as above.
The panel contained the following samples: mononuclear cells,
activated mononuclear cells, resting CD4+ cells, activated CD4+
cells, resting CD8+ cells, activated CD8+ cells, resting CD 14+
cells, resting CD 19+ cells and activated CD19+ cells. Activated
CD4+ cells and activated CD19+ cells showed zcytor16 expression,
whereas the other cells tested, including resting CD4+ cells and
resting CD 19+ cells, did not.
TABLE-US-00006 TABLE 6 Tissue #samples Tissue #samples adrenal
gland 1 bladder 1 bone marrow 3 brain 2 cervix 1 colon 1 fetal
brain 3 fetal heart 2 fetal kidney 1 fetal liver 2 fetal lung 1
fetal skin 1 heart 2 fetal muscle 1 kidney 2 liver 1 lung 1 lymph
node 1 mammary 1 melanoma 1 gland ovary 1 pancreas 1 pituitary 2
placenta 3 prostate 3 rectum 1 salivary gland 2 skeletal muscle 1
small intestine 1 spinal cord 2 spleen 1 uterus 1 stomach 1
adipocyte library 1 testis 5 islet 1 thymus 1 prostate SMC 1
thyroid 2 RPMI 1788 1 trachea 1 WI38 1 esophageal 1 lung tumor 1
tumor liver tumor 1 ovarian tumor 1 rectal tumor 1 stomach tumor 1
uterine tumor 2 CD3+ library 1 HaCAT library 1 HPV library 1 HPVS
library 1 MG63 library 1 K562 1
C. Tissue Distribution in Human Tissue and Cell Line RNA Panels
Using RT-PCR
[0493] A panel of RNAs from human cell lines was screened for
zcytor16 expression using RT-PCR. The panels were made in house and
contained 84 RNAs from various normal and cancerous human tissues
and cell lines as shown in Tables 7-10 below. The RNAs were made
from in house or purchased tissues and cell lines using the RNAeasy
Midi or Mini Kit (Qiagen, Valencia, Calif.). The panel was set up
in a 96-well format with 100 ngs of RNA per sample. The RT-PCR
reactions were set up using oligos ZC25,963 (SEQ ID NO:30) and
ZC25,964 (SEQ ID NO:31), Rediload dye and SUPERSCRIPT One Step
RT-PCR System (Life Technologies, Gaithersburg, Md.). The
amplification was carried out as follows: one cycle at 550 for 30
minutes followed by 40 cycles of 94.degree., 15 seconds;
59.degree., 30 seconds; 72.degree., 30 seconds; then ended with a
final extension at 720 for 5 minutes. 8 to 10 .mu.ls of the PCR
reaction product was subjected to standard Agarose gel
electrophoresis using a 4% agarose gel. The correct predicted cDNA
fragment size of 184 bps was observed in cell lines U-937, HL-60,
ARPE-19, HaCat#1, HaCat#2, HaCat#3, and HaCat#4; bladder, cancerous
breast, normal breast adjacent to a cancer, bronchus, colon,
ulcerative colitis colon, duodenum, endometrium, esophagus,
gastro-esophageal, heart left ventricle, heart ventricle, ileum,
kidney, lung, lymph node, lymphoma, mammary adenoma, mammary gland,
cancerous ovary, pancreas, parotid and skin, spleen lymphoma and
small bowel. Zcytor16 expression was not observed in the other
tissues and cell lines tested in this panel.
[0494] Zcytor16 is detectably expressed by PCR in normal tissues:
such as, the digestive system, e.g., esophagus, gastro-esophageal,
pancreas, duodenum, lleum, colon, small bowel; the female
reproductive system, e.g., mammary gland, endometrium, breast
(adjacent to cancerous tissues); and others systems, e.g., lymph
nodes, skin, parotid, bladder, bronchus, heart ventricles, and
kidney. Moreover, Zcytor16 is detectably expressed by PCR in
several human tumors: such as tumors associated with female
reproductive tissues e.g., mammary adenoma, ovary cancer, uterine
cancer, other breast cancers; and other tissues such as lymphoma,
stomach tumor, and lung tumor. The expression of zcytor16 is found
in normal tissues of female reproductive organs, and in some tumors
associated with these organs. As such, zcytor16 can serve as a
marker for these tumors wherein the zcytor16 may be over-expressed.
Several cancers positive for zcytor16 are associated with
ectodermal/epithelial origin (mammary adenoma, and other breast
cancers). Hence, zcytor16 can serve as a marker for epithelial
tissue, such as epithelial tissues in the digestive system and
female reproductive organs (e.g., endometrial tissue, columnar
epithelium), as well as cancers involving epithelial tissues.
Moreover, in a preferred embodiment, zcytor16 can serve as a marker
for certain tissue-specific tumors especially, e.g., ovarian
cancer, stomach cancer, uterine cancer, rectal cancer, lung cancer
and esophageal cancer, where zcytor16 is not expressed in normal
tissue, but is expressed in the tumor tissue. Use of
polynucleotides, polypeptides, and antibodies of the present
invention for diagnostic purposes are known in the art, and
disclosed herein.
TABLE-US-00007 TABLE 7 Tissue #samples Tissue #samples adrenal
gland 6 duodenum 1 bladder 3 endometrium 5 brain 2 cancerous
endometrium 1 brain meningioma 1 gastric cancer 1 breast 1
esophagus 7 cancerous breast 4 gastro-esophageal 1 normal breast
adjacent 5 heart aorta 1 to cancer bronchus 3 heart left ventricle
4 colon 15 heart right ventricle 2 cancerous colon 1 heart
ventricle 1 normal colon adjacent 1 ileum 3 to cancer ulcerative
colitis colon 1 kidney 15 cancerous kidney 1
TABLE-US-00008 TABLE 8 Tissue/Cell Line #samples Tissue/Cell Line
#samples 293 1 HBL-100 1 C32 1 Hs-294T 1 HaCat#1 1 Molt4 1 HaCat#2
1 RPMI 1 HaCat#3 1 U-937 1 HaCat#4 1 A-375 1 WI-38 1 HCT-15 1 WI-38
+ 2 um ionomycin #1 1 HT-29 1 WI-38 + 2 um ionomycin #2 1 MRC-5 1
WI-38 + 5 um ionomycin #1 1 RPT-1 1 WI-38 + 5 um ionomycin #2 1
RPT-2 1 Caco-2, 1 WM-115 1 Caco-2, differentiated 1 A-431 1 DLD-1 1
WERI-Rb-1 1 HRE 1 HEL-92.1.7 1 HRCE 1 HuH-7 1 MCF7 1 MV-4-11 1 PC-3
1 U-138 1 TF-1 1 CCRF-CEM 1 5637 1 Y-79 1 143B 1 A-549 1 ME-180 1
EL-4 1 prostate epithelia 1 HeLa 229 1 U-2 OS 1 HUT 78 1 T-47D 1
NCI-H69 1 Mg-63 1 SaOS2 1 Raji 1 USMC 1 U-373 MG 1 UASMC 2 A-172 1
AoSMC 1 CRL-1964 1 UtSMC 1 CRL-1964 + butryic acid 1 HepG2 1 HUVEC
1 HepG2-IL6 1 SK-Hep-1 1 NHEK#1 1 SK-Lu-1 1 NHEK#2 1 Sk-MEL-2 1
NHEK#3 1 K562 1 NHEK#4 1 BeWo 1 ARPE-19 1 FHS74.Int 1 G-361 1 HL-60
1 HISM 1 Malme 3M 1 3AsubE 1 FHC 1 INT407 1 HREC 1
TABLE-US-00009 TABLE 9 Tissue #samples Tissue #samples liver 10
lung 13 lymph node 1 cancerous lung 2 lymphoma 4 normal lung
adjacent 1 to cancer mammary adenoma 1 muscle 3 mammary gland 3
neuroblastoma 1 melinorioma 1 omentum 2 osteogenic sarcoma 2 ovary
6 pancreas 4 cancerous ovary 2 skin 5 parotid 7 sarcoma 2 salivary
gland 4
TABLE-US-00010 TABLE 10 Tissue #samples Tissue #samples small bowel
10 uterus 11 spleen 3 uterine cancer 1 spleen lymphoma 1 thyroid 9
stomach 13 stomach cancer 1
Example 13
Construction of Expression Vector Expressing Full-Length
zcytor11
[0495] The entire zcytor11 receptor (commonly owned U.S. Pat. No.
5,965,704) was isolated by digestion with EcoRI and XhoI from
plasmid pZP7P, containing full-length zcytor11 receptor cDNA (SEQ
ID NO:24) and a puromycin resistance gene. The digest was run on a
1% low melting point agarose (Boerhinger Mannheim) gel and the
approximately 1.5 kb zcytor11 cDNA was isolated using Qiaquick.TM.
gel extraction kit (Qiagen) as per manufacturer's instructions. The
purified zcytor11 cDNA was inserted into an expression vector as
described below.
[0496] Recipient expression vector pZP7Z was digested with EcoRI
(BRL) and XhoI (Boehringer Mannheim) as per manufacturer's
instructions, and gel purified as described above. This vector
fragment was combined with the EcoRI and XhoI cleaved zcytor11
fragment isolated above in a ligation reaction. The ligation was
run using T4 Ligase (BRL) at 12.degree. C. overnight. A sample of
the ligation was electroporated in to DH10B electroMAX.TM.
electrocompetent E. coli cells (25 .mu.F, 200.OMEGA., 1.8V).
Transformants were plated on LB+Ampicillin plates, and single
colonies were picked into 2 ml LB+Ampicillin and grown overnight.
Plasmid DNA was isolated using Wizard Minipreps (Promega), and each
was digested with EcoRI and XhoI to confirm the presence of insert.
The insert was approximately 1.5 kb, and was full-length. Digestion
with SpeI and PstI was used to confirm the identity of the
vector.
Example 14
Construction of BaF3 Cells Expressing the CRF2-4 Receptor
(BaF3/CRF2-4 Cells) and BaF3 Cells Expressing the CRF2-4 Receptor
with the zcytor11 Receptor (BaF3/CRF2-4/zcytor11 Cells)
[0497] BaF3 cells expressing the full-length CFR2-4 receptor were
constructed, using 30 .mu.g of a CFR2-4 expression vector,
described below. The BaF3 cells expressing the CFR2-4 receptor were
designated as BaF3/CFR2-4. These cells were used as a control, and
were further transfected with full-length zcytor11 receptor (U.S.
Pat. No. 5,965,704) and used to construct a screen for IL-TIF
activity as described below.
A. Construction of BaF3 Cells Expressing the CRF2-4 Receptor
[0498] The full-length cDNA sequence of CRF2-4 (Genbank Accession
No. Z17227) was isolated from a Daudi cell line cDNA library, and
then cloned into an expression vector pZP7P, as described in
Example 6.
[0499] BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell
line derived from murine bone marrow (Palacios and Steinmetz, Cell
41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:
4133-4135, 1986), was maintained in complete media (RPMI medium
(JRH Bioscience Inc., Lenexa, Kans.) supplemented with 10%
heat-inactivated fetal calf serum, 2 ng/ml murine IL-3 (mIL-3) (R
& D, Minneapolis, Minn.), 2 mM L-glutaMax-1.TM. (Gibco BRL), 1
mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics (GIBCO BRL)).
Prior to electroporation, CRF2-4/pZP7P was prepared and purified
using a Qiagen Maxi Prep kit (Qiagen) as per manufacturer's
instructions. For electroporation, BaF3 cells were washed once in
serum-free RPMI media and then resuspended in serum-free RPMI media
at a cell density of 10.sup.7 cells/ml. One ml of resuspended BaF3
cells was mixed with 30 .mu.g of the CRF2-4/pZP7P plasmid DNA and
transferred to separate disposable electroporation chambers (GIBCO
BRL). Following a 15-minute incubation at room temperature the
cells were given two serial shocks (800 lFad/300 V.; 1180 lFad/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 2 .mu.g/ml puromycin in a T-162 flask to isolate the
puromycin-resistant pool. Pools of the transfected BaF3 cells,
hereinafter called BaF3/CRF2-4 cells, were assayed for signaling
capability as described below. Moreover these cells were further
transfected with zcytor11 receptor as described below.
B. Construction of BaF3 Cells Expressing CRF2-4 and zcytor11
Receptors
[0500] BaF3/CRF2-4 cells expressing the full-length zcytor11
receptor were constructed as per Example 5A above, using 30 .mu.g
of the zcytor11 expression vector, described in Example 6 above.
Following recovery, transfectants were selected using 200 g/ml
zeocin and 2 .mu.g/ml puromycin. The BaF3/CRF2-4 cells expressing
the zcytor11 receptor were designated as BaF3/CRF2-4/zcytor11
cells. These cells were used to screen for IL-TIF activity as well
as zcytor16 antagonist activity described IN Example 15.
Example 15
Screening for IL-TIF Antagonist Activity Using BaF3/CRF2-4/zcytor11
Cells Using an Alamar Blue Proliferation Assay
[0501] A. Screening for IL-TIF Activity Using BaF3/CRF2-4/zcytor11
cells using an Alamar Blue Proliferation Assay
[0502] Purified IL-TIF-CEE (Example 19) was used to test for the
presence of proliferation activity as described below. Purified
zcytor16-Fc4 (Example 11) was used to antagonize the proliferative
response of the IL-TIF in this assay as described below.
[0503] BaF3/CRF2-4/zcytor11 cells were spun down and washed in the
complete media, described in Example 7A above, but without mIL-3
(hereinafter referred to as "m/L-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.
[0504] Proliferation of the BaF3/CRF2-4/zcytor11 cells was assessed
using IL-TIF-CEE protein diluted with mIL-3 free media to 50, 10,
2, 1, 0.5, 0.25, 0.13, 0.06 ng/ml concentrations. 100 .mu.l of the
diluted protein was added to the BaF3/CRF2-4/zcytor11 cells. The
total assay volume is 200 .mu.l. The assay plates were incubated at
37.degree. C., 5% CO.sub.2 for 3 days at which time Alamar Blue
(Accumed, Chicago, Ill.) was added at 20 .mu.l/well. Plates were
again incubated at 37.degree. C., 5% CO.sub.2 for 24 hours. Alamar
Blue gives a fluourometric readout based on number of live cells,
and is thus a direct measurement of cell proliferation in
comparison to a negative control. Plates were again incubated at
37.degree. C., 5% CO.sub.2 for 24 hours. Plates were read on the
Fmax.TM. plate reader (Molecular Devices Sunnyvale, Calif.) using
the SoftMax.TM. Pro program, at wavelengths 544 (Excitation) and
590 (Emmission). Results confirmed the dose-dependent proliferative
response of the BaF3/CRF2-4/zcytor11 cells to IL-TIF-CEE. The
response, as measured, was approximately 15-fold over background at
the high end of 50 ng/ml down to a 2-fold induction at the low end
of 0.06 ng/ml. The BaF3 wild type cells, and BaF3/CRF2-4 cells did
not proliferate in response to IL-TIF-CEE, showing that IL-TIF is
specific for the CRF2-4/zcytor11heterodimeric receptor.
[0505] In order to determine if zcytor16 is capable of antagonizing
IL-TIF activity, the assay described above was repeated using
purified soluble zcytor16/Fc4. When IL-TIF was combined with
zcytor16 at 10 g/ml, the response to IL-TIF at all concentrations
was brought down to background. That the presence of soluble
zcytor16 ablated the proliferative effects of IL-TIF demonstrates
that it is a potent antagonist of the IL-TIF ligand.
Example 16
IL-TIF Activation of a Reporter Mini-Gene in MES 13 Cells and
Inhibition of Activity by zcytor16-Fc4
[0506] MES 13 cells (ATCC No. CRL-1927) were plated at 10,000
cells/well in 96-well tissue culture clusters (Costar) in DMEM
growth medium (Life Technologies) supplemented with pyruvate and
10% serum (HyClone). Next day, the medium was switched to serum
free DMEM medium by substituting 0.1% BSA (Fraction V; Sigma) for
serum. This medium also contained the adenoviral construct KZ136
(below) that encodes a luciferase reporter mini-gene driven by SRE
and STAT elements, at a 1000:1 multiplicity of infection (m.o.i.),
i.e. 1000 adenoviral particles per cell. After allowing 24 h for
the incorporation of the adenoviral construct in the cells, the
media were changed and replaced with serum-free media. Human
recombinant IL-TIF with or without a recombinant zcytor16-Fc4
fusion was added at the indicated final concentration in the well
(as described in Table 11, below). Dilutions of both the IL-TIF and
zcytor16-Fc4 were performed in serum-free medium. 0.1% BSA was
added for a basal assay control. 4 h later, cells were lysed and
luciferase activity, denoting activation of the reporter gene, was
determined in the lysate using an Luciferase Assay System assay kit
(Promega) and a Labsystems Luminoskan luminometer (Labsystems,
Helsinki, Finland). Activity was expressed as luciferase units (LU)
in the lysate. Results are shown in Table 11, below.
TABLE-US-00011 TABLE 11 Level of IL-TIF (ng/ml) LU w/o zcytoR16 LU
w/ 10 .mu.g/ml zcytoR16 0 (basal BSA control) 103 .+-. 2 104 .+-. 2
0.03 105 .+-. 3 104 .+-. 4 0.3 108 .+-. 4 99 .+-. 6 3 134 .+-. 8 98
.+-. 15 30 188 .+-. 16 110 .+-. 3 300 258 .+-. 21 112 .+-. 30
[0507] These results demonstrate two things: First, that MES 13
cells respond to human recombinant IL-TIF and therefore possess
endogenous functional receptors for the cytokine. Second, that the
zcytoR16-Fc4 receptor fusion acts as an antagonist that effectively
blocks the response to IL-TIF, even at the highest dose that this
cytokine was used. Therefore, zcyto16 is an effective antagonist of
IL-TIF on cells (MES 13) that are intrinsically capable of
responding to IL-TIF, i.e. cells that do not require exogenous
expression of additional receptor components to respond to the
cytokine.
[0508] The construction of the adenoviral KZ136 vector was as
follows. The original KZ136 vector is disclosed in Poulsen, L K et
al. J. Biol. Chem. 273:6228-6232, 1998. The CMV promoter/enhancer
and SV40 pA sequences were removed from pACCMV.pLpA (T. C. Becker
et al., Meth. Immunology 43:161-189, 1994.) and replaced with a
linker containing Asp718/KpnI and HindIII sites (oligos ZC13252
(SEQ ID NO:26) and ZC13453 (SEQ ID NO:27)). The STAT/SRE driven
luciferase reporter cassette was exised from vector KZ136 (Poulsen,
L K et al., supra.).) as a Asp718/KpnI-HindIII fragment and
inserted into the adapted pAC vector. Recombinant KZ136 Adenovirus
was produced by transfection with JM17 Adenovirus into 293 cells as
described in T. C. Becker et al. supra.). Plaque purified virus was
amplified and used to infect cultured cells at 5-50 pfu/cell 12-48
hours before assay. Luciferase reporter assays were performed as
described in 96 well microplates as per Poulsen, L K et al.,
supra.).
B. IL-TIF Reporter Gene Assay on HCT-15 and HT-29 Cell Lines
[0509] HCT-15 (human colon adenocarcinoma, ATCC #CCL-225) and HT-29
(human epithelial colorectal adenocarcinoma, ATCC #HTB-38) cells
were plated at 3000 cells/100 ul/well in RPMI (Life Technologies)
supplemented with 10% fetal bovine serum, 1% Glutamax, and 1% Na
pyruvate into 96 well opaque plates (Costar). These cells were
incubated 24 hours in a 37.degree. C., 5% CO2 incubator.
[0510] Cells were then infected with KZ136 adenovirus
(STAT/SRE/Lucif) described above in 50 .mu.l/well DMEM/F12 media
(Life Technologies) supplemented with 1% ITS and 2% HEPES (Serum
Free Media) using an M.O.I. of 5000. Cells were incubated for 24
hours.
[0511] After removal of the adenovirus, cell samples were diluted
in serum free media with serial concentrations of IL-TIF in the
presence or absence of Zcytor16-Fc4 fusion soluble receptor, and
added to a 96-well plate in a volume of 100 .mu.l/well, and
incubated for 4 hours. Serum-free medium alone was used as a
background control. Cells were then lysed and exposed to luciferase
substrate using reagents and protocols from Promega as per
manufacturer's instructions. Plates were read on the Berthold
MicroLumat Plus LB96V2R (Perkin-Elmer Life Sciences) using a 40
.mu.l injection and 5 second integration protocol.
[0512] HCT-15 cells showed a 2.5 fold induction over media alone to
the IL-TIF at 10 ng/ml and a 2.2 fold induction at 5 ng/ml. This
IL-TIF response was blocked to background levels when Zcytor16-Fc4
fusion soluble receptor was added in at 1 .mu.g/ml.
[0513] HT-29 cells showed a nice dose response to IL-TIF starting
with a 6.2 fold induction at 10 ng/ml titering down to a 2 fold
induction at 0.08 ng/ml. This IL-TIF response was completely
blocked down to background levels when 1 .mu.g/ml Zcytor16-Fc4
fusion soluble receptor was added in.
Example 17
Construct for Generating CEE-Tagged IL-TIF
[0514] Oligonucleotides were designed to generate a PCR fragment
containing the Kozak sequence and the coding region for IL-TIF,
without its stop codon. These oligonucleotides were designed with a
KpnI site at the 5' end and a BamHI site at the 3' end to
facilitate cloning into pHZ200-CEE, our standard vector for
mammalian expression of C-terminal Glu-Glu tagged (SEQ ID NO:10)
proteins. The pHZ200 vector contains an MT-1 promoter.
[0515] PCR reactions were carried out using Turbo Pfu polymerase
(Stratagene) to amplify a IL-TIF cDNA fragment. About 20 ng human
IL-TIF polynucleotide template (SEQ ID NO:14), and oligonucleotides
ZC28590 (SEQ ID NO:28) and ZC28580 (SEQ ID NO:29) were used in the
PCR reaction. PCR reaction conditions were as follows: 95.degree.
C. for 5 minutes; 30 cycles of 95.degree. C. for 60 seconds,
55.degree. C. for 60 seconds, and 72.degree. C. for 60 seconds; and
72.degree. C. for 10 minutes; followed by a 4.degree. C. hold. PCR
products were separated by agarose gel electrophoresis and purified
using a QiaQuick.TM. (Qiagen) gel extraction kit. The isolated,
approximately 600 bp, DNA fragment was digested with KpnI and BamHI
(Boerhinger-Mannheim), gel purified as above and ligated into
pHZ200-CEE that was previously digested with KpnI and BamHI.
[0516] About one microliter of the ligation reaction was
electroporated into DH10B ElectroMax.TM. competent cells (GIBCO
BRL, Gaithersburg, Md.) according to manufacturer's direction and
plated onto LB plates containing 100 .mu.g/ml ampicillin, and
incubated overnight. Colonies were picked and screened by PCR using
oligonucleotides ZC28,590 (SEQ ID NO:28) and ZC28,580 (SEQ ID
NO:29), with PCR conditions as described above. Clones containing
inserts were then sequenced to confirm error-free IL-TIF inserts.
Maxipreps of the correct pHZ200-IL-TIF-CEE construct, as verified
by sequence analysis, were performed.
Example 18
Transfection And Expression Of IL-TIF Soluble Receptor
Polypeptides
[0517] BHK 570 cells (ATCC No. CRL-10314), were plated at about
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 an expression
plasmid containing IL-TIF-CEE described above (Example 17), using
Lipofectin.TM. (Gibco BRL), in serum free (SF) DMEM according to
manufacturer's instructions.
[0518] 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.
[0519] Approximately 10-12 days post-transfection, colonies were
mechanically picked to 12-well plates in one ml of 5% FCS/DMEM with
5 .mu.M MTX, then grown to confluence. Positive expressing clonal
colonies Conditioned media samples were then tested for expression
levels via SDS-PAGE and Western analysis. A high-expressing clone
was picked and expanded for ample generation of conditioned media
for purification of the IL-TIF-CEE expressed by the cells (Example
19).
Example 19
Purification of IL-TIF-CEE from BHK 570 Cells
[0520] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying IL-TIF
polypeptide containing C-terminal GluGlu (EE) tags (SEQ ID NO:10).
Conditioned media from BHK cells expressing IL-TIF-CEE (Example 18)
was concentrated with an Amicon S10Y3 spiral cartridge on a ProFlux
A30. A Protease inhibitor solution was added to the concentrated
conditioned media 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
conditioned media were determined via SDS-PAGE and Western blot
analysis with the anti-EE HRP conjugated antibody.
[0521] About 100 ml column of anti-EE G-Sepharose (prepared as
described below) was poured in a Waters AP-5, 5 cm.times.10 cm
glass column. The column was flow packed and equilibrated on a
BioCad Sprint (PerSeptive BioSystems, Framingham, Mass.) with
phosphate buffered saline (PBS) pH 7.4. The concentrated
conditioned media was thawed, 0.2 micron sterile filtered, pH
adjusted to 7.4, then loaded on the column overnight with about 1
ml/minute flow rate. The column was washed with 10 column volumes
(CVs) of phosphate buffered saline (PBS, pH 7.4), then plug eluted
with 200 ml of PBS (pH 6.0) containing 0.5 mg/ml EE peptide
(Anaspec, San Jose, Calif.) at 5 ml/minute. The EE peptide used has
the sequence EYMPME (SEQ ID NO:10). The column was 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.
[0522] To separate IL-TIF-CEE 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 was 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.
[0523] To separate IL-TIF-CEE polypeptide from free EE peptide and
any contaminating co-purifying proteins, the pooled concentrated
fractions were subjected to size exclusion chromatography on a
1.5.times.90 cm Sephadex S200 (Pharmacia, Piscataway, N.J.) column
equilibrated and loaded in PBS at a flow rate of 1.0 ml/min using a
BioCad Sprint. 1.5 ml fractions were collected across the entire
chromatography and the absorbance at 280 and 215 nM were monitored.
The peak fractions were characterized via SDS-PAGE Silver staining,
and only the most pure fractions were pooled. This material
represented purified IL-TIF-CEE polypeptide.
[0524] This purified material was finally subjected to a 4 ml
ActiClean Etox (Sterogene) column to remove any remaining
endotoxins. The sample was passed over the PBS equilibrated gravity
column four times then the column was washed with a single 3 ml
volume of PBS, which was pooled with the "cleaned" sample. The
material was then 0.2 micron sterile filtered and stored at
-80.degree. C. until it was aliquoted.
[0525] On Western blotted, Coomassie Blue and Silver stained
SDS-PAGE gels, the IL-TIF-CEE polypeptide was one major band. The
protein concentration of the purified material was performed by BCA
analysis (Pierce, Rockford, Ill.) and the protein was aliquoted,
and stored at -80.degree. C. according to standard procedures.
[0526] To prepare anti-EE Sepharose, a 100 ml bed volume of protein
G-Sepharose (Pharmacia, Piscataway, N.J.) was washed 3 times with
100 ml of PBS containing 0.02% sodium azide using a 500 ml Nalgene
0.45 micron filter unit. The gel was washed with 6.0 volumes of 200
mM triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.), and an
equal volume of EE antibody solution containing 900 mg of antibody
was added. After an overnight incubation at 4.degree. C., unbound
antibody was removed by washing the resin with 5 volumes of 200 mM
TEA as described above. The resin was resuspended in 2 volumes of
TEA, transferred to a suitable container, and
dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in
TEA, was added to a final concentration of 36 mg/ml of protein
G-Sepharose gel. The gel was rocked at room temperature for 45 min
and the liquid was removed using the filter unit as described
above. Nonspecific sites on the gel were then blocked by incubating
for 10 min. at room temperature with 5 volumes of 20 mM
ethanolamine in 200 mM TEA. The gel was then washed with 5 volumes
of PBS containing 0.02% sodium azide and stored in this solution at
4.degree. C.
Example 20
Human zcytor11 Tissue Distribution in Tissue Panels Using Northern
Blot and PCR
[0527] A. Human zcytor11 Tissue Distribution in Tissue Panels Using
PCR
[0528] A panel of cDNAs from human tissues was screened for
zcytor11 expression using PCR. The panel was made in-house and
contained 94 marathon cDNA and cDNA samples from various normal and
cancerous human tissues and cell lines are shown in Table 6 above.
Aside from the PCR reaction, the method used was as shown in
Example 12. The PCR reactions were set up using oligos ZC14,666
(SEQ ID NO: 32) and ZC14,742 (SEQ ID NO:33), Advantage 2 cDNA
polymerase mix (Clontech, Palo Alto, Calif.), and Rediload dye
(Research Genetics, Inc., Huntsville, Ala.). The amplification was
carried out as follows: 1 cycle at 94.degree. C. for 2 minutes, 40
cycles of 94.degree. C. for 15 seconds, 51.degree. C. for 30
seconds and 72.degree. C. for 30 seconds, followed by 1 cycle at
72.degree. C. for 7 minutes. The correct predicted DNA fragment
size was observed in bladder, brain, cervix, colon, fetal brain,
fetal heart, fetal kidney, fetal liver, fetal lung, fetal skin,
heart, kidney, liver, lung, melanoma, ovary, pancreas, placenta,
prostate, rectum, salivary gland, small intestine, testis, thymus,
trachea, spinal cord, thyroid, lung tumor, ovarian tumor, rectal
tumor, and stomach tumor. Zcytor11 expression was not observed in
the other tissues and cell lines tested in this panel.
[0529] A commercial 1st strand cDNA panel (Human Blood Fractions
MTC Panel, Clontech, Palo Alto, Calif.) was also assayed as above.
The panel contained the following samples: mononuclear cells,
activated mononuclear cells, resting CD4+ cells, activated CD4+
cells, resting CD8+ cells, activated CD8+ cells, resting CD14+
cells, resting CD19+ cells and activated CD19+ cells. The following
samples showed positive expression of zcytor11: mononuclear cells,
resting CD8+ and resting CD 19+ cells.
B. Tissue Distribution of Zcytor11 in Human Cell Line and Tissue
Panels Using RT-PCR
[0530] A panel of RNAs from human cell lines was screened for
zcytor11 expression using RT-PCR. The panels were made in house and
contained 84 RNAs from various normal and cancerous human tissues
and cell lines as shown in Tables 7-10 above. The RNAs were made
from in house or purchased tissues and cell lines using the RNAeasy
Midi or Mini Kit (Qiagen, Valencia, Calif.). The panel was set up
in a 96-well format with 100 ngs of RNA per sample. The RT-PCR
reactions were set up using oligos ZC14,666 (SEQ ID NO:32) and
ZC14,742 (SEQ ID NO:33), Rediload dye and SUPERSCRIPT One Step
RT-PCR System (Life Technologies, Gaithersburg, Md.). The
amplification was carried out as follows: one cycle at 500 for 30
minutes followed by 45 cycles of 94.degree., 15 seconds;
52.degree., 30 seconds; 72.degree., 30 seconds; then ended with a
final extension at 720 for 7 minutes. 8 to 10 uls of the PCR
reaction product was subjected to standard Agarose gel
electrophoresis using a 4% agarose gel. The correct predicted cDNA
fragment size was observed in adrenal gland, bladder, breast,
bronchus, normal colon, colon cancer, duodenum, endometrium,
esophagus, gastic cancer, gastro-esophageal cancer, heart
ventricle, ileum, normal kidney, kidney cancer, liver, lung, lymph
node, pancreas, parotid, skin, small bowel, stomach, thyroid, and
uterus. Cell lines showing expression of zcytor11 were A-431,
differentiated CaCO2, DLD-1, HBL-100, HCT-15, HepG2, HepG2+IL6,
HuH7, and NHEK #1-4. Zcytor11 expression was not observed in the
other tissues and cell lines tested in this panel.
[0531] In addition, because the expression pattern of zcytor11, one
of IL-TIF's receptors, shows expression in certain specific
tissues, binding partners including the natural ligand, IL-TIF, can
also be used as a diagnostic to detect specific tissues (normal or
abnormal), cancer, or cancer tissue in a biopsy, tissue, or
histologic sample, particularly in tissues where IL-TIF receptors
are expressed. IL-TIF can also be used to target other tissues
wherein its receptors, e.g., zcytor16 and zcytor11 are expressed.
Moreover, such binding partners could be conjugated to
chemotherapeutic agents, toxic moieties and the like to target
therapy to the site of a tumor or diseased tissue. Such diagnostic
and targeted therapy uses are known in the art and described
herein.
[0532] The expression patterns of zcytor11 (above) and zcytor16
(Example 12, and Example 21) indicated target tissues and cell
types for the action of IL-TIF, and hence IL-TIF antagonsists, such
as zcytor16. The zcytor11 expression generally overlapped with
zcytor16 expression in three physiologic systems: digestive system,
female reproductive system, and immune system. Moreover, the
expression pattern of the receptor (zcytor11) indicated that an
IL-TIF antagonist such as zcytor16 would have therapeutic
application for human disease in two areas: inflammation (e.g.,
IBD, Chron's disease, pancreatitis) and cancer (e.g., ovary,
colon). That is, the polynucleotides, polypeptides and antibodies
of the present invention can be used to antagonize the
inflammatory, and other cytokine-induced effects of IL-TIF
interaction with the cells expressing the zcytor11 receptor.
[0533] Moreover, the expression of zcytor11 appeared to be
downregulated or absent in an ulcerative colitis tissue, HepG2
liver cell line induced by IL-6, activated CD8+ T-cells and CD
19+B-cells. However, zcytor16 appeared to be upregulated in
activated CD19+B-cells (Example 12), while zcytor11 is
downregulated in activated CD19+ cells, as compared to the resting
CD19+ cells (above). The expression of zcytor11 and zcytor16 has a
reciprocal correlation in this case. These RT-PCR experiments
demonstrate that CD19+ peripheral blood cells, B lymphocytes,
express receptors for IL-TIF, namely zcytoR11 and zcytoR16.
Furthermore B cells display regulated expression of zcytoR11 and
zcytoR16. B-lymphocytes activated with mitogens decrease expression
of zcytoR11 and increase expression of zcytoR16. This represents a
classical feedback inhibition that would serve to dampen the
activity of IL-TIF on B cells and other cells as well. Soluble
zcytoR16 would act as an antagonist to neutralize the effects of
IL-TIF on B cells. This would be beneficial in diseases where B
cells are the key players: Autoimmune diseases including systemic
lupus erythmatosus (SLE), myasthenia gravis, immune complex
disease, and B-cell cancers that are exacerbated by IL-TIF. Also
autoimmune diseases where B cells contribute to the disease
pathology would be targets for zcytoR16 therapy: Multiple
sclerosis, inflammatory bowel disease (IBD) and rheumatoid
arthritis are examples. ZcytoR16 therapy would be beneficial to
dampen or inhibit B cells producing IgE in atopic diseases
including asthma, allergy and atopic dermatitis where the
production of IgE contributes to the pathogenesis of disease.
[0534] B cell malignancies may exhibit a loss of the "feedback
inhibition" described above. Administration of zcytoR16 would
restore control of IL-TIF signaling and inhibit B cell tumor
growth. The administration of zcytoR16 following surgical resection
or chemotherapy may be useful to treat minimal residual disease in
patients with B cell malignancies. The loss of regulation may lead
to sustain or increased expression of zcytoR11. Thus creating a
target for therapeutic monoclonal antibodies targeting
zcytoR11.
Example 21
Identification of Cells Expressing zcytor16 Using In Situ
Hybridization
[0535] Specific human tissues were isolated and screened for
zcytor16 expression by in situ hybridization. Various human tissues
prepared, sectioned and subjected to in situ hybridization included
cartilage, colon, appendix, intestine, fetal liver, lung, lymph
node, lymphoma, ovary, pancreas, placenta, prostate, skin, spleen,
and thymus. The tissues were fixed in 10% buffered formalin and
blocked in paraffin using standard techniques. Tissues were
sectioned at 4 to 8 microns. Tissues were prepared using a standard
protocol ("Development of non-isotopic in situ hybridization" at
The Laboratory of Experimental Pathology (LEP), NIEHS, Research
Triangle Park, N.C.; web address
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 7 minutes. This step was followed
by acetylation and re-hydration of the tissues.
[0536] One in situ probe was designed against the human zcytor16
sequence (nucleotide 1-693 of SEQ ID NO:1), and isolated from a
plasmid containing SEQ ID NO:1 using standard methods. T3 RNA
polymerase was used to generate an antisense probe. The probe was
labeled with digoxigenin (Boehringer) using an In Vitro
transcription System (Promega, Madison, Wis.) as per manufacturer's
instruction.
[0537] In situ hybridization was performed with a
digoxigenin-labeled zcytor16 probe (above). The probe was added to
the slides at a concentration of 1 to 5 pmol/ml for 12 to 16 hours
at 62.5.degree. C. Slides were subsequently washed in 2.times.SSC
and 0.1.times.SSC at 55.degree. C. The signals were amplified using
tyramide signal amplification (TSA) (TSA, in situ indirect kit;
NEN) and visualized with Vector Red substrate kit (Vector Lab) as
per manufacturer's instructions. The slides were then
counter-stained with hematoxylin (Vector Laboratories, Burlingame,
Calif.).
[0538] Signals were observed in several tissues tested: The lymph
node, plasma cells and other mononuclear cells in peripheral
tissues were strongly positive. Most cells in the lymphatic nodule
were negative. In lymphoma samples, positive signals were seen in
the mitotic and multinuclear cells. In spleen, positive signals
were seen in scattered mononuclear cells at the periphery of
follicles were positive. In thymus, positive signals were seen in
scattered mononuclear cells in both cortex and medulla were
positive. In fetal liver, a strong signal was observed in a mixed
population of mononuclear cells in sinusoid spaces. A subset of
hepatocytes might also have been positive. In the inflamed
appendix, mononuclear cells in peyer's patch and infiltration sites
were positive. In intestine, some plasma cells and ganglia nerve
cells were positive. In normal lung, zcytor16 was expressed in
alveolar epithelium and mononuclear cells in interstitial tissue
and circulation. In the lung carcinoma tissue, a strong signal was
observed in mostly plasma cells and some other mononuclear cells in
peripheral of lymphatic aggregates. In ovary carcinoma, epithelium
cells were strongly positive. Some interstitial cells, most likely
the mononuclear cells, were also positive. There was no signal
observed in the normal ovary. In both normal and pancreatitis
pancreas samples, acinar cells and some mononuclear cells in the
mesentery were positive. In the early term (8 weeks) placenta,
signal was observed in trophoblasts. In skin, some mononuclear
cells in the inflamed infiltrates in the superficial dermis were
positive. Keratinocytes were also weakly positive. In prostate
carcinoma, scatted mononuclear cells in interstitial tissues were
positive. In articular cartilage, chondrocytes were positive. Other
tissues tested including normal ovary and a colon adenocarcinoma
were negative.
[0539] In summary, the in situ data was consistent with expression
data described above for the zcytor16. Zcytor16 expression was
observed predominately in mononuclear cells, and a subset of
epithelium was also positive. These results confirmed the presence
of zcytor16 expression in immune cells and point toward a role in
inflammation, autoimmune disease, or other immune function, for
example, in binding pro-inflammatory cytokines, including but not
limited to IL-TIF. Moreover, detection of zcytor16 expression can
be used for example as an marker for mononuclear cells in
histologic samples.
[0540] Zcytor16 is expressed in mononuclear cells, including normal
tissues (lymph nodes, spleen, thymus, pancreas and fetal liver,
lung), and abnormal tissues (inflamed appendix, lung carcinoma,
ovary carcinoma, pancreatitis, inflamed skin, and prostate
carcinoma). It is notable that plasma cells in the lymph node,
intestine, and lung carcinoma are positive for zcytor16. Plasma
cells are immunologically activated lymphocytes responsible for
antibody synthesis. In addition, IL-TIF, is expressed in activated
T cells. In addition, the expression of zcytor16 is detected only
in activated (but not in resting) CD4+ and CD19+ cells (Example
12). Thus, zcytor16 can be used as a marker for or as a target in
isolating certain lymphocytes, such as mononuclear leucocytes and
limited type of activated leucocytes, such as activated CD4+ and CD
19+.
[0541] Furthermore, the presence of zcytor16 expression in
activated immune cells such as activated CD4+ and CD19+ cells
showed that zcytor16 may be involved in the body's immune defensive
reactions against foreign invaders: such as microorganisms and cell
debris, and could play a role in immune responses during
inflammation and cancer formation.
[0542] Moreover, as discussed herein, epithelium form several
tissues was positive for zcytor16 expression, such as hepatocytes
(endoderm-derived epithelia), lung alveolar epithelium
(endoderm-derived epithelia), and ovary carcinoma epithelium
(mesoderm-derived epithelium). The epithelium expression of
zcytor16 could be altered in inflammatory responses and/or
cancerous states in liver and lung. Thus, Zcyto16 could be used as
marker to monitor changes in these tissues as a result of
inflammation or cancer. Moreover, analysis of zcytor16 in situ
expression showed that normal ovary epithelium is negative for
zcytor16 expression, while it is strongly positive in ovary
carcinoma epithelium providing further evidence that zcytor16
polynucleotides, polypeptides and antibodies can be used as a
diagnostic marker and/or therapeutic target for the diagnosis and
treatment of ovarian cancers, and ovary carcinoma, as described
herein.
[0543] Zcytor16 was also detected in other tissues, such as acinar
cells in pancreas (normal and pancreatitis tissues), trophoblasts
in placenta (ectoderm-derived), chondrocytes in cartilage
(mesoderm-derived), and ganglia cells in intestine
(ectoderm-derived). As such, zcytor16 may be involved in
differentiation and/or normal functions of corresponding cells in
these organs. As such, potential utilities of zcytor16 include
maintenance of normal metabolism and pregnancy, bone
formation/homeostasis, and physiological function of intestine, and
the like.
Example 22
In Vivo Affects of IL-TIF Polypeptide
[0544] Mice (female, C57B1, 8 weeks old; Charles River Labs,
Kingston, N.Y.) were divided into three groups. An adenovirus
expressing an IL-TIF polypeptide (SEQ ID NO:15) was previously made
using standard methods. On day 0, parental or IL-TIF adenovirus was
administered to the first (n=8) and second (n=8) groups,
respectively, via the tail vein, with each mouse receiving a dose
of .about.1.times.10.sup.11 particles in .about.0.1 ml volume. The
third group (n=8) received no treatment. On days 12, mice were
weighed and blood was drawn from the mice. Samples were analyzed
for complete blood count (CBC) and serum chemistry. Statistically
significant elevations in neutrophil and platelet counts were
detected in the blood samples from the IL-TIF adenovirus
administered group relative to the parental adenovirus treated
group. Also, lymphocyte and red blood cell counts were
significantly reduced from the IL-TIF adenovirus administered group
relative to the parental adenovirus treated group. In addition, the
IL-TIF adenovirus treated mice decreased in body weight, while
parental adenovirus treated mice gained weight. Also the serum
zcyto18 level was increased and the glucose level decreased at day
3. In summary, zcyto18 adeno-mice displayed acute phase response
that can also be initiated by other pro-inflammatory cytokines such
as TNF-alpha, IL-1beta, and gp130 cytokines. The acute phase
response is the set of immediate inflammatory responses initiated
by pattern recognition molecules. The acute phase proteins provide
enhanced protection against microorganisms and modify inflammatory
responses by effects on cell trafficking and mediator release. For
example, SAA has potent leukocyte activating function including
induction of chemotaxis, enhancement of leukocyte adhesion to
endothelial cells, and increased phagocytosis. Understanding the
factors that initiate and alter the magnitude and duration of the
acute phase response represents an important step in the
development of new therapies for infectious and inflammatory
diseases.
[0545] The results suggested that IL-TIF affects hematopoiesis,
i.e., blood cell formation in vivo. As such, IL-TIF could have
biological activities effecting different blood stem cells, thus
resulting increase or decrease of certain differentiated blood
cells in a specific lineage. For instance, IL-TIF appears to reduce
lymphocytes, which is likely due to inhibition of the committed
progenitor cells that give rise to lymphoid cells. IL-TIF also
decreases red blood cells, supporting the notion that IL-TIF could
play a role in anemia, infection, inflammation, and/or immune
diseases by influencing blood cells involved in these process.
Antagonists against IL-TIF, such as antibodies or its soluble
receptor zcytor16, could be used as therapeutic reagents in these
diseases.
[0546] Moreover, these experiments using IL-TIF adenovirus in mice
suggest that IL-TIF over-expression increases the level of
neutrophils and platelets in vivo. It is conceivable that there are
other factors (such as cytokines and modifier genes) involved in
the responses to IL-TIF in the whole animal system. Nevertheless,
these data strongly support the involvement of IL-TIF in
hematopoiesis. Thus, IL-TIF and its receptors are suitable
reagents/targets for the diagnosis and treatment in variety of
disorders, such as inflammation, immune disorders, infection,
anemia, hematopoietic and other cancers, and the like.
Example 24
Chromosomal Assignment and Placement of Zcytor16
[0547] Zcytor16 was mapped to chromosome 6 by polymerase chain
reaction (PCR) using the commercially available version of the
Stanford G3 Human/Hamster Radiation Hybrid (RH) Mapping Panel
(Research Genetics, Inc., Huntsville, Ala.) in conjunction with
publicly available WWW servers (e.g, Stanford Human Genome Center,
Stanford University, CA server, http://shgc-www.stanford.edu/; and
National Center for Biotechnology Information, National Library of
Medicine, Bethesda, Md.,
http://www.ncbi.nlm.nih.gov/genemap99/).
[0548] The PCR reactions were carried out using Zcytor16 specific
sense and antisense primers ZC27,713 (SEQ ID NO:40), and ZC27,714
(SEQ ID NO:41). These yield a 226-bp amplicon in the 3' UTR. For
the PCR reactions, HotStarTaq DNA polymerase and buffer (Qiagen
Inc., Valencia, Calif.) were used. The PCR cycler conditions were
as follows: an initial 1 cycle 15 min denaturation at 95.degree.
C., followed by 35 cycles of a 1 min denaturation at 95.degree. C.,
1 min annealing at 56.degree. C. and 1 min and 15 s extension at
72.degree. C., and a final 1 cycle extension of 7 min at 72.degree.
C. The reactions were separated by electrophoresis on a 2% agarose
gel (EM Science, Gibbstown, N.J.) and visualized with ethidium
bromide staining. The data vector obtained for the Stanford G3 RH
mapping panel was: 01000 00000 10000 00000 00000 00010 00000 00000
00000 00000 10000 00010 00010 10000 00010 10000 000.
[0549] The radiation hybrid (RH) mapping linked Zcytor16 to
chromosome 6 on the Stanford G3 RH mapping panel. The RH results
placed Zcytor16 0 cR.sub.10000 from the Stanford G3 framework
marker SHGC-9657 (LOD=12.38, 1 cR.sub.10000=.about.29 kb).
SHGC-9657 (D6S1835) is a STS genomic, sequence tagged site for the
human interferon-gamma receptor (IFN.gamma.R1). IFN.gamma.R1 has
been mapped to 6q24.1-q24.2 by in situ hybridization (Papanicolaou
et al., Cytogenet. Cell Genet. 76:181-182, 1997) and is in the
D6S442 (158.5 cM)--D6S1581 (165.0 cM) interval on the NCBI GeneMap
'99. Due to the close proximity of Zcytor16 to IFN.gamma.R1, it can
be assumed that Zcytor16 is also located in the 6q23-q24
chromosomal region.
Example 25
IL-TIF-Expressing Transgenic Mice
A. Generation of Transgenic Mice Expressing Mouse IL-TIF
[0550] DNA fragments from a transgenic vector containing 5' and 3'
flanking sequences of the lymphoid specific E.mu.LCK promoter,
mouse IL-TIF (SEQ ID NO:42; polypeptide shown in SEQ ID NO:43), the
rat insulin II intron, IL-TIF cDNA and the human growth hormone
poly A sequence were prepared using standard methods, and used for
microinjection into fertilized B6C3f1 (Taconic, Germantown, N.Y.)
murine oocytes, using a standard microinjection protocol. See,
Hogan, B. et al., Manipulating the Mouse Embryo. A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 1994.
[0551] Twenty-five mice transgenic for mouse IL-TIF with the
lymphoid-specific E.mu.LCK promoter were identified among 154 pups.
Eleven of the transgenic pups died within hours of birth, 9
transgenic pups with a shiny appearance were necropsied the day of
birth, and 2 grew to adulthood. Expression levels were low in one
adult animal. Tissues from the necropsied pups were prepared and
histologically examined as described below.
[0552] The shiny appearance of the neonate pups appeared to be
associated with a stiffening of the skin, as if they were drying
out, resulting in a reduction of proper nursing. Their movements
became stiffened in general.
B. Genotypic and Expression Analysis from Transgenic Mice
[0553] From the mouse IL-TIF transgenic line driven by the E.mu.Lck
promoter, described above, newborn pups were observed for
abnormalities on day one (day of birth) and sacrificed for tissue
collection. All pups were given a unique ear tag number, and those
designated as having a shiny skin phenotype at the time of
sacrifice were noted. Of the twelve pups, six were observed to have
the shiny skin phenotype, with two designated as "severe"
phenotypes. Severe phenotypes were defined as small pups with
little mobility whose skin was especially shiny and very dry. Skin
was collected from the left lateral side of each pup, and frozen in
Tissue-Tek embedding medium.
[0554] Genotyping confirmed that shiny skin was a good indicator of
transgenic status, although no expression data was collected.
Frozen skin blocks were sectioned to 7 microns on a cryostat and
stained to look for the presence of CD3, CD4, CD8, mouse
macrophages, B-cells, CD80, and MHC class II. The staining protocol
involved binding of commercially available antibodies to the
tissue, detection with a peroxidase labeled secondary antibody, and
DAB chromogen reaction to visualize staining.
[0555] Transgenic animals were found to be higher in MHC class II
and CD80, which stain for antigen-presenting cells and dendritic
cells respectively. The macrophage marker also detected more cells
in the severe and non-severe transgenics than in the wild type
animals, although the distribution of these cells was very
localized in the high dermis. Animals classified as severe
phenotypes had the most robust staining with all three of these
markers, showing a dramatic increase in cell intensity and number
when compared to the wild type. This variability may be due to a
difference in expression level of IL-TIF in these transgenic
founder pups. The MHC class II positive cells were located in the
lower dermis arranged in loose open clusters, while the CD80
positive cells were predominantly below the dermis either in or
just above the muscle/fat layer. These two cell populations do not
appear to overlap. All other markers were of equivalent staining in
all animals. Toluidine blue staining for mast cells revealed slight
to no difference between wild type and transgenic animals.
C. Microscopic Evaluation of Tissues from Transgenic Mice: IL-TIF
TG with EuLck Promoter has a Neonatal Lethal-Histology
[0556] On the day of birth, pups from litters containing IL-TIF
transgenics were humanely euthanized and the whole body immersion
fixed in 10% buffered formalin. Six transgenic and two
non-transgenic pups were submitted for further workup. Four of the
six transgenics were noted to have shiny skin at the time of
euthanasia. The fixed tissues were trimmed into 5 sections
(longitudinal section of the head and cross sections of the upper
and lower thorax and upper and lower abdomen). The tissues were
embedded in paraffin, routinely processed, sectioned at 5 um (Jung
2065 Supercut microtome, Leica Microsystems, Wetzlar, Germany) and
stained with H&E. The stained tissues were evaluated under a
light microscope (Nikon Eclipse E600, Nikon Inc., Melville, N.Y.)
by a board (ACVP) certified veterinary pathologist.
[0557] On microscopic examination, the epidermis of two of the
transgenic pups was observed to be thicker than the epidermis of
the other six mice including the controls. No other abnormalities
were noted in the skin and other tissues of any of the mice.
Representative areas of skin from corresponding regions of the
thorax and abdomen were imaged with the 40.times. objective lens
and with a CoolSnap digital camera (Roper Scientific, Inc., San
Diego, Calif.) that was attached to the microscope. The thickness
of the epidermis was then determined using histomorphometry
software (Scion Image for Windows (NIH Image), Scion Corp.,
Frederick, Md., v. B4.0.2). The results were as follows:
TABLE-US-00012 Average thoracic skin Average abdominal skin
Genotype/phenotype thickness (.mu.m) thickness (.mu.m)
Non-transgenic/normal 5.2 5.4 Transgenic/non-shiny 5.0 6.7
Transgenic/shiny 8.2 7.4 Transgenic/all 7.1 7.1
[0558] There were insufficient numbers of mice to determine
statistical significance; however, the transgenics, especially
those with shiny skin, tended to have a thicker epidermis than the
non-shiny transgenics and non-transgenic controls. The shiny
transgenics may have a higher expression level of IL-TIF than the
non-shiny transgenics; however, expression levels were not
determined for these mice.
Example 26
In Vivo Affects of IL-TIF Polypeptide
[0559] A. Mice Infected with IL-TIF Adenovirus Show Induction of
SAA
[0560] Mice (female, C57B1, 8 weeks old; Charles River Labs,
Kingston, N.Y.) were divided into three groups. An adenovirus
expressing an IL-TIF polypeptide (SEQ ID NO:15) was previously made
using standard methods. On day 0, parental or IL-TIF adenovirus was
administered to the first (n=8) and second (n=8) groups,
respectively, via the tail vein, with each mouse receiving a dose
of .about.1.times.10.sup.11particles in .about.0.1 ml volume. The
third group (n=8) received no treatment. On day 12, mice were
weighed and blood was drawn from the mice. On day 20 of the study,
mice were sacrificed, body weight was recorded, and blood and
tissues were collected for analysis.
[0561] All blood samples were analyzed for complete blood count
(CBC) and serum chemistry. At both day 12 and 20, statistically
significant elevations in neutrophil and platelet counts were
detected in the blood samples from the IL-TIF adenovirus
administered group relative to the parental adenovirus treated
group. Also, lymphocyte counts were significantly reduced from the
IL-TIF adenovirus administered group relative to the parental
adenovirus treated group at day 12, but at day 20 the opposite
effect was observed. In addition, the IL-TIF adenovirus treated
mice decreased in body weight, while parental adenovirus treated
mice gained weight. Glucose was significantly reduced at both time
points in the serum samples from the IL-TIF adenovirus administered
group relative to the parental adenovirus treated group. Serum
albumin was also significantly reduced at both time points. Blood
urea nitrogen levels were significantly reduced at day 20. Serum
globulin levels were significantly increased the IL-TIF adenovirus
administered group relative to the parental adenovirus treated
group at both time points. Microscopically, one observed
histomorphological change attributed to IL-TIF was tubular
regeneration in the kidney. While not uncommon in mice, there was
an increased incidence and severity in this group of animals.
Nephropathy is characterized as multifocal areas of basophilia of
cortical tubular epithelial cells.
[0562] An additional experiment, identical in design to the one
described above, was carried out in order to verify results and
collect additional samples. In this study, body weight was recorded
every three days, blood was collected from the mice 3 days
following adenovirus injection, and mice were sacrificed for blood
and tissue collection on day 10 (n=4 per group) and day 20 (n=4 per
group). Elevated neutrophil and platelet counts were again detected
in blood samples from the IL-TIF adenovirus administered group
relative to the parental adenovirus treated group. This effect was
evident for neutrophils by day 3, but platelet count was not
significantly different until day 10. Also, lymphocyte counts were
significantly reduced from the IL-TIF adenovirus administered group
relative to the parental adenovirus treated group at 3 and 10, but
they were not elevated on day 20 as in the previous study. Again,
mice given IL-TIF adenovirus lost weight over the course of the
study, while control virus treated and untreated mice gained
weight. Serum chemistry parameters were consistent with the
previous study. Histological findings of tubular regeneration in
the kidney associated with IL-TIF adenovirus treatment were also
confirmed in this study. This was consistent with the additional
finding of moderate proteinurea in mice given IL-TIF adenovirus
(day 20).
[0563] The results suggested that IL-TIF affects hematopoiesis,
i.e., blood cell formation in vivo. As such, IL-TIF could have
biological activities effecting different blood stem cells, thus
resulting in an increase or decrease of certain differentiated
blood cells in a specific lineage. For instance, IL-TIF appears to
reduce lymphocytes, which is likely due to inhibition of the
committed progenitor cells that give rise to lymphoid cells,
supporting the notion that IL-TIF could play a role in anemia,
infection, inflammation, and/or immune diseases by influencing
blood cells involved in these processes. Antagonists against
IL-TIF, such as antibodies or its soluble receptor zcytor16, could
be used as therapeutic reagents in these diseases.
[0564] Moreover, these experiments using IL-TIF adenovirus in mice
suggest that IL-TIF over-expression increases the level of
neutrophils and platelets in vivo. It is conceivable that there are
other factors (such as cytokines and modifier genes) involved in
the responses to IL-TIF in the whole animal system. Nevertheless,
these data strongly support the involvement of IL-TIF in
hematopoiesis. Thus, IL-TIF, anti-IL-TIF antibodies, and its
receptors, such as zcytor16 and soluble zcytor11/CRF2-4, are
suitable reagents/targets for the diagnosis and treatment in
variety of disorders, such as inflammation, immune disorders,
infection, anemia, hematopoietic and other cancers, and the
like.
[0565] Association of IL-TIF expression with weight loss,
appearance of acute phase protein SAA, and metabolic perturbations
evidenced by decreased serum glucose, albumin and urea nitrogen
suggest that IL-TIF is a cytokine which acts early in certain
inflammatory responses. Mice given IL-TIF adenovirus may represent
a state of chronic inflammation, such as that observed in IBD,
ulcerative colitis, arthritis, psoriasis, asthma, and the like.
Certain detrimental inflammatory processes might be inhibited by
use of an antagonist to IL-TIF, such as anti-IL-TIF antibodies, and
its receptors, such as zcytor16 and soluble zcytor11/CRF2-4, and
the like.
B. IL-TIF is a Pro-Inflammatory Cytokine: Serum Level of SAA in
Adeno-IL-TIF Mice:
[0566] An ELISA was performed to determine the level of SAA in
IL-TIF-Adeno mice, using a Mouse SAA Immunoassay Kit and protocol
(Biosource International, California, USA). Diluted standards and
unknowns were plated along with HRP-anti-mouse SAA into assay
plates pre-coated with anti-mouse SAA antibody. Plates were
incubated for one hour at 37 degrees C. and then washed according
to kit instructions. Plates were developed for 15 minutes at room
temperature using TMB and stopped with 2M H.sub.2S0.sub.4, The
absorbance at 450 nm was read using a Spectromax 190 (Molecular
Devices, California, USA). The resulting data was analyzed using
Softmax Pro (Molecular Devices, California, USA) and Excel
(Microsoft Corp., Washington, USA).
[0567] Mice infected with IL-TIF-Adenovirus had highly elevated
levels of mSAA, over 10-fold, relative to the Parental-Adenovirus
control.
C. Flow Cytometry Analysis of IL-TIF-Adenovirus Infected Mice
[0568] To analyze the effects of IL-TIF expression in vivo by
adenovirus, we isolated peripheral blood, spleen, and bone marrow
from IL-TIF-adenovirus infected C57BL/6 mice, at day 10 and day 20
after infection. Approximately 100 .mu.l of blood was collected in
heparinized tubes, then depleted of red blood cells by hypotonic
lysis (cells were lysed in 4.5 ml dH.sub.2O for .about.5 seconds
before adding 1.5 ml 3.6% NaCl). Spleens were crushed between two
frosted glass slides, and the cells released were passed over a
Nytex membrane (cell strainer) and pelleted. Bone marrow was
obtained by crushing one femur in a mortar and pestle and passing
the cells over a cell strainer (Falcon). Cells were resuspended in
FACS wash buffer (WB=HBSS/1% BSA/10 mM hepes), counted in trypan
blue, and 1.times.10.sup.6 viable cells of each type were aliquoted
into 5 ml polystyrene tubes. Cells were washed and pelleted, then
incubated for 20 min on ice with cocktails of fluorescently-labeled
(FITC, PE, and CyChrome) monoclonal antibodies (PharMingen, San
Diego, Calif.) recognizing various cell surface markers used to
identify particular immune cell subsets. These markers include the
following (listed in the groups of 3 we tested). For blood
staining: CD3, Gr1, and B220; for spleen staining: CD62L, CD44, and
CD3; CD21, CD23, and B220; IgD, IgM, and B220; CD11b, Gr1, and CD8;
for bone marrow staining: CD11b, Gr1, CD3; IgD, IgM, and B220.
Cells were washed with 1.5 ml WB and pelleted, then resuspended in
0.4 ml of WB and analyzed on a FACScan using CellQuest software
(Becton Dickinson, Mountain View, Calif.).
[0569] We found that the fraction of neutrophils in the blood of
IL-TIF-adeno-treated mice was elevated 4-13 fold at Day 10 and
2-3-fold at Day 20. At Day 10, this difference resulted in a
concomitant decrease in the fraction of lymphocytes and monocytes
in the blood. In the bone marrow, we found that the total number of
B cells decreased .about.1.5-fold while the percentage of mature
recirculating B cells increased and the total number of immature B
cells dropped slightly at Day 10. At Day 20, many of these
differences were not apparent, though we did find a slight increase
in the fraction of mature recirculating B cells. In the spleen, the
total number of B cells decreased slightly (1.5-2-fold) on both
days tested, while on Day 20, the fraction of marginal zone B cells
(CD21+CD23-B220+) increased by 2-fold and the number of follicular
B cells (CD21+CD23+B220+) dropped 2-fold. Marginal zone B cells are
considered to be the first line of defense against pathogens, as
they are more sensitive to B cell mitogens (e.g. LPS) than the more
common follicular B cells, and when they encounter their cognate
antigen they differentiate very quickly into antibody-secreting
cells. It is possible that IL-TIF either enhances the conversion of
follicular to marginal zone B cells, or that it selectively
depletes the less mature follicular cells. The changes in B cell
numbers found in the bone marrow may reflect an enhanced
differentiation of pre/pro and/or immature B cells, or an increased
influx of recirculating B cells from the blood/spleen, and perhaps
a coincident increase in export of immature B cells to the
periphery. The actual number of mature BM B cells does not
increase, so IL-TIF may not enhance their proliferation.
Alternatively, IL-TIF may block differentiation of immature B cells
and thereby increase the relative representation of mature B
cells.
D. Zcytor16/Fc4 Neutralizes IL-TIF Activity In Vivo: SAA ELISA
Showing SAA Expression Induced By IL-TIF is Inhibited by
Zcytor16-Fc4 Injection:
[0570] To assess whether zcytor16 could inhibit the SAA induction
by IL-TIF mice (female, C3H/HEJ, 8 weeks old; Jackson Labs, Bar
Harbor, Me.) were divided into five groups of three animals each
and treated by IP injection of proteins as shown in Table 12
below:
TABLE-US-00013 TABLE 12 Group # IL-TIF Zcytor16 Group 1: -- --
Group 2: -- 100 .mu.g Group 3: 3 .mu.g -- Group 4: 3 .mu.g 20 .mu.g
Group 5: 3 .mu.g 100 .mu.g
[0571] The zcytor16 injections preceded the IL-TIF injection by 15
minutes. Both protein injections were given by the intraperitoneal
route. A blood sample was taken from each mouse prior to treatment,
then at 2 and 6 hours after treatment. Serum was prepared from each
of the samples for measurement of SAA and IL-TIF.
[0572] An ELISA was performed as described previously to determine
the level of SAA in mice treated with IL-TIF and a soluble receptor
for IL-TIF, zcytor16-Fc4 described herein. Mice treated with 3
.mu.g IL-TIF in conjunction with zcytor16-Fc4 at concentrations
between 20-100 ug showed a reduction in the level of SAA induced by
IL-TIF alone to background levels, demonstrating that zcytor16
inhibited the SAA induction activity of IL-TIF in vivo.
Example 27
Expression of IL-TIF in Inflammatory Bowel Disease mouse model
[0573] Inflammatory Bowel disease (IBD) is a multifactoral disease,
divided into two types, ulcerative colitis (UC) and Crohn's Disease
(CD). The etiology of these diseases is currently not known and
clinical manifestations differ. UC is restricted to the colon, and
symptoms include bloody diarrhea, weight loss and abdominal pain.
Macroscopic features of UC include punctuated ulcers and a
shortened colon. In contrast, Crohn's Disease can also affect other
parts of the bowel. Symptoms include diarrhea (which is less often
bloody than seen in UC), a low-grade fever and pain. Macroscopic
features include fibrotic and stenotic bowel with strictures, deep
ulcers, fissures and fistulas.
[0574] Several animal models are available that mimic these human
diseases. Three commonly used models of colitis for new drug
screening are the 2,4,6-trinitrobenzene sulphonic acid (TNBS)
induced rat model, the mouse T-cell transfer model, and the dextran
sodium sulfate, or DSS-induced mouse model. The DSS model was
derived from a model by Dr. S. Murthy, using a disease activity
index scoring system (S. N. S. Murthy, Treatment of Dextran Sulfate
Sodium-Induced Murine Colitis by Intracolonic Cyclosporin,
Digestive Diseases and Sciences, Vol. 38, No. 9 (September 1993),
pp. 1722-1734).
[0575] In the present study, an acute colitis resulted when mice
were fed DSS in their drinking water for 6 days. The animals
exhibited weight loss and bloody diarrhea, mimicking the condition
of UC patients. The mechanism of the DSS injury is not well
characterized, but it is thought that it induces a nonspecific
inflammatory immune response and mimics environmental effects on
the bowel. It is possible that H.sub.2S is produced, which could be
toxic to cells. In addition, changes in luminal bacterial flora
occur. Activated monocytes, macrophages and mast cells have been
demonstrated in the colon. Mediators for all three animal models
include inflammatory prostaglandins, leukotriene metabolites and
cytokines.
A. Method
[0576] Colitis was induced by DSS ingestion in Swiss Webster female
mice from Charles River Laboratories. The mice were 10 and 11 weeks
old at the start of the study. Mice were given 4% DSS in the
drinking water for a period of 6 days (treated mice), or were given
only normal drinking water (control mice). A Disease Activity Index
clinical score (DAI) was used, which comprises a combination of
measurements including stool quality, occult blood and weight loss.
DAI was obtained daily for each mouse beginning one day after DSS
treatment. After 6 days, DSS was removed from the drinking water of
the treated mice. All mice were monitored by DAI clinical score
until sacrifice at either 2, 7 or 10 days from the start of the
study. On each of days 2 and 7, four DSS-treated mice and one
control mouse were sacrificed. On day 10, four DSS-treated mice and
two control mice were sacrificed. For all animals after sacrifice,
the colon length was measured. Colon sections were fixed in 10%
neutral buffered formalin for histologic analysis or frozen for
mRNA extraction.
B. Histologic Scoring and Disease Activity Index (DAI) Scoring
[0577] Histologic index scores were obtained following the method
in reference 1. Generally, the colon sections were scored blinded
by a pathologist for crypt scores, hyperplastic epithelium, crypt
distortion and inflammation.
[0578] Daily, each mouse was graded as to a clinical score based on
weight loss, stool consistence and intestinal bleeding. Higher
scores were assigned for increasing amounts of weight loss,
diarrhea and bleeding. The daily score for each mouse was the mean
grade obtained from the three results/observations.
C. Results
[0579] The colon lengths for DSS-treated mice were somewhat shorter
on days 7 and 10 than non-treated controls, but the results may not
have been significant (not checked by a statistical application).
The clinical DAI scores reflected a rise in disease symptoms in the
DSS-treated mice similar to that seen in past studies using this
model. Occult blood was greatest on approximately days 4 and 5,
while loose stools were more prevalent on days 6 and 7.
Histopathology results show that disease scores were different from
the controls on all sacrifice days, especially days 7 (peak) and
10. The histopathology screening scores were: controls=0.5, day 2
DSS-treated mice=8.8, day 7 DSS-treated mice=21, day 10 DSS-treated
mice=18. Clinical and histopathology scores show that the
DSS-treated mice had significant colon disease relative to the
non-treated controls. The frozen tissue samples were used later for
mRNA determinations as described below.
D. Tissue Expression of IL-TIF RNA in Murine IBD Colon Samples
Using RT-PCR:
[0580] To determine the relative expression of mouse IL-TIF RNA
(SEQ ID NO:42) in an inflammatory bowel disease model, the distal
colons of DSS-treated mice were collected and snap frozen in liquid
nitrogen. In this experiment mice were treated with DSS and samples
were taken on days 2, 7 and 10 post-treatment. Samples from normal
untreated mice were collected as well. RNA was then isolated from
the samples using the standard RNeasy Midiprep.TM. Kit (Qiagen,
Valencia, Calif.) as per manufacturer's instructions.
[0581] The RT-PCR reactions used the `Superscript One-Step RT-PCR
System with Platinum Taq.` (Life Technologies, Gaithersburg, Md.)
Each 25 .mu.l reaction consisted of the following: 12.5 .mu.l of
2.times. Reaction Buffer, 0.5 ul (20 pmol/.mu.l) ZC39,289 (SEQ ID
NO:44), 0.5 .mu.l (20 pmol/ul) ZC39,290 (SEQ ID NO:45), 0.4 .mu.l
RT/Taq polymerase mix, 10 ul RNase-free water, 1.0 .mu.l total RNA
(100 ng/.mu.l). The amplification was carried out as follows: one
cycle at 500 for 30 minutes followed by 35 cycles of 94.degree., 30
seconds; 58.degree., 30 seconds; 72.degree., 60 seconds; then ended
with a final extension at 72.degree. for 7 minutes. 8 to 10 .mu.l
of the PCR reaction product was subjected to standard agarose gel
electrophoresis using a 2% agarose gel. The correct predicted cDNA
fragment size was observed as follows: There was a faint band in
both day 2 samples. Two of three day 7 samples generated a strong
band while the third day 7 sample generated a very strong band. The
three day 10 samples generated a strong band. Finally, the two
`normal` control samples didn't generate any band. These results
suggest that there may be an upregulation of IL-TIF in certain
types of inflammatory responses in the colon, including those
associated with IBD, UC, and CD. The data is summarized in Table 13
below where Relative Expression was scored as follows: 0=No band,
1=faint band, 2=strong band, 3=very strong band.
TABLE-US-00014 TABLE 13 Tissue Relative Expression (0-3) Normal
Colon 0 Normal Colon 0 Day 2 Post Treatment 1 Day 2 Post Treatment
1 Day 7 Post Treatment 3 Day 7 Post Treatment 2 Day 7 Post
Treatment 2 Day 10 Post Treatment 2 Day 10 Post Treatment 2 Day 10
Post Treatment 2
Example 28
Construct for Generating Hzcytor11/hCRF2-4 Heterodimer
[0582] A cell line expressing a secreted hzcytor11/hCRF2-4
heterodimer was constructed. In this construct, the extracellular
domain of hzcytor11 (SEQ ID NO:46) was fused to the heavy chain of
IgG gamma1 (Fc4) (SEQ ID NO:5) with a Glu-Glu tag (SEQ ID NO:59) at
the C-terminus, while the extracellular domain of CRF2-4 (SEQ ID
NO:47) was fused to Fc4 with a His tag (SEQ ID NO:60) at the
C-terminus. For both of the hzcytor11 and hCRF2-4 arms of the
heterodimer, a Gly-Ser spacer of 8 amino acids (SEQ ID NO:48) was
engineered between the extracellular portion of the receptor and
the n-terminus of Fc4. In addition, a thrombin cleavage site was
engineered between the Fc4 domain and the c-terminal tag to enable
possible proteolytic removal of the tag.
[0583] For construction of the hzcytor11/Fc4-CEE portion of the
heterodimer, the extracellular portion of hzcytor11 was PCRed from
a vector containing human zcytor11 fused ot Fc4 (hzcytor11/IgG)
with oligos ZC39335 (SEQ ID NO:49) and ZC39434 (SEQ ID NO:50) with
EcoRI and BamHI restriction sites engineered at the 5' and 3' ends,
respectively, under conditions as follows: 25 cycles of 94.degree.
C. for 60 sec., 57.degree. C. for 60 sec., and 72.degree. C. for
120 sec.; and 72.degree. C. for 7 min. PCR products were purified
using QIAquick PCR Purification Kit (Qiagen), digested with EcoRI
and BamHI (Boerhinger-Mannheim), separated by gel electrophoresis
and purified using a QIAquick gel extraction kit (Qiagen). The
hzcytor11 EcoRI/BamHI fragment was ligated into pZP-9
hzcytor7/Fc4-TCS-CEE that had been digested with EcoRI and BamHI.
This vector has the extracellular portion of hzcytor7 (U.S. Pat.
No. 5,945,511) fused to Fc4 (SEQ ID NO:5) with a CEE tag (SEQ ID
NO:10), and digesting with EcoRI and BamHI removes the
extracellular portion of hzcytor7 and allows substitution of
hzcytor11. Minipreps of the resulting ligation were screened for an
EcoRI/BamHI insert of the correct size and positive minipreps were
sequenced to confirm accuracy of the PCR reaction. The polypeptide
sequence of the hzcytor11/Fc4-CEE fusion polypeptide is shown in
SEQ ID NO:61.
[0584] For construction of the hCRF2-4/Fc4-cHIS portion of the
heterodimer, the extracellular portion of hCRF2-4 was PCRed from
pZP-9 CRF with oligos ZC39,319 (SEQ ID NO:51) and ZC39,325 (SEQ ID
NO:52) under conditions as follows: 30 cycles of 94.degree. C. for
60 sec., 57.degree. C. for 60 sec., and 72.degree. C. for 120 sec;
and 72.degree. C. for 7 min. PCR product were purified as described
above and then digested with EcoRI and BamHI. Because the PCR
product had an internal EcoRI site two bands were obtained upon
digestion; a 0.101 kB EcoRI/EcoRI fragment and a 0.574 kB
EcoRI/BamHI fragment. The 0.574 EcoRI/BamHI fragment was ligated
into vector pHZ-1 DR1/Fc4-TCS-cHIS that had been digested with
EcoRI and BamHI. This vector has the extracellular portion of hDR-1
fused to Fc4 with a C-HIS tag (SEQ ID NO:12), and digesting with
EcoRI and BamHI removes the extracellular portion of hDR-1 and
allows substitution of hCRF2-4. Minipreps of the resulting ligation
were screened for an EcoRI/BamHI insert of the correct size, and
positive minipreps, were EcoRI digested and band purified for
further construction. The 0.101 kB EcoRI/EcoRI fragment was ligated
into the EcoRI digested minipreps and clones were screened for
proper orientation of insertion by KpnI/NdeI restriction digestion.
Clones with the correct size insertion were submitted for DNA
sequencing to confirm the accuracy of the PCR reaction. The
polypeptide sequence of the hzcytor11/Fc4-CEE fusion polypeptide is
shown in SEQ ID NO:62.
[0585] About 16 .mu.g each of the hzcytor11/Fc4-cEE and
hCRF2-4/Fc-4-cHIS were co-transfected into BHK-570 (ATCC No.
CRL-10314) cells using Lipofectamine (Gibco/BRL), as per
manufacturer's instructions. The transfected cells were selected
for 10 days in DMEM+5% FBS (Gibco/BRL) containing 1 .mu.M
methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5 mg/ml G418
(Gibco/BRL) for 10 days. The resulting pool of transfectants was
selected again in 10 .mu.M MTX and 0.5 mg./ml G418 for 10 days.
Example 29
Purification of zcytor11/CRF2-4 Heterodimer Receptor
[0586] Conditioned culture media zcytor11/CRF2-4 heterodimer was
filtered through 0.2 .mu.m filter and 0.02% (w/v) Sodium Azide was
added. The conditioned media was directly loaded a Poros Protein A
50 Column at 10-20 ml/min. Following load the column was washed
with PBS and the bound protein eluted with 0.1M Glycine pH 3.0. The
eluted fractions containing protein were adjusted to pH 7.2 and
Concentrated to <80 ml using YM30 Stirred Cell Membrane
(Millipore).
[0587] The 80 ml eluate from the Protein A column was loaded onto a
318 ml Superdex 200 HiLoad 26/60 Column (Pharmacia). The column was
eluted with PBS pH 7.2 at 3 ml min. Protein containing fractions
were pooled to eliminate aggregates. The Superdex 200 pool was
adjusted to 0.5M NaCl, 10 mM Imidazole using solid NaCl and
Imidazole and the pH was adjusted to 7.5 with NaOH. The adjusted
protein solution was loaded onto a 200 ml NiNTA column (Qiagen) at
2 CV/hr. The bound protein was eluted, following PBS wash of the
column, with five concentration steps of Imidazole: 40 mM, 100 mM,
150 mM, 250 mM, 500 mM. The fractions eluted at each step of
imidizole were pooled and analyzed by N-terminal sequencing. Pools
containing heterodimer, determined by sequencing were pooled and
concentrated to 50 ml using a YM30 Stirred Cell Membrane
(Millipore). The 50 ml eluate from the NiNTA column was loaded onto
a 318 ml Superdex 200 HiLoad 26/60 Column (Pharmacia). The column
was eluted with PBS pH 7.2 at 3 ml/min. Protein containing
fractions were pooled to eliminate aggregates, as determined by SEC
MALS analysis.
[0588] Purified proteins were analyzed by N-terminal sequencing,
amino acid analysis, and SEC-MALS. Binding affinities and
biological activities were determined.
Example 30
Comparison of Zcytor16-Fc4 Activity with CRF2-4/Zcytor11-Fc4
Activity Using BaF3/CRF2-4/zcytor11 Cells in an Alamar Blue
Proliferation Assay
[0589] BaF3/CRF2-4/zcytor11 cells described herein were spun down
and washed in PBS 2 times to ensure the removal of the mIL-3, and
then spun a third time and re-suspended in the complete media (RPMI
1640, 10% FBS, 1% GlutaMAX, 1% Sodium Pyruvate), but without mIL-3
(hereinafter referred to as "m/L-3 free media"). Cells were then
counted in a hemocytometer. 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.
[0590] IL-TIF protein (SEQ ID NO:15) was diluted to 200 .mu.g/ml in
mIL-3 free media. Zcytor16-Fc4 fusion protein (described herein)
was diluted to 1 .mu.g/ml in the mIL-3 free/IL-TIF media on the top
row of the plate, and then diluted serially 1:2 down the remaining
7 rows on the 96-well plate, leaving a volume of 100 .mu.l in each
well. This was then added to the 100 .mu.l of cells, for a final
IL-TIF concentration of 100 pg/ml in all wells, and final
Zcytor16-Fc4 concentrations of approximately 1, 0.5, 0.25, 0.125,
0.063, 0.31, 0.016, and 0.008 .mu.g/ml in a total assay volume of
200 .mu.l. CRF2-4/zcytor11-Fc4 was diluted to 8 .mu.g/ml in the
mIL-3 free/IL-TIF media on the top row of the plate, and then
diluted serially 1:2 down the remaining 7 rows on the 96-well
plate, leaving a volume of 100 .mu.l in each well. This was then
added to the 100 .mu.l of cells, for a final IL-TIF concentration
of 100 pg/ml in all wells, and final CRF2-4/zcytor11-Fc4
concentrations of approximately 8, 4, 2, 1, 0.05, 0.25, 0.125 and
0.063 .mu.g/ml, in a total assay volume of 200 .mu.l. The assay
plates were incubated at 37.degree. C., 5% CO.sub.2 for 4 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 16 hours. Alamar Blue gives a fluourometric readout
based on number of live cells, and is thus a direct measurement of
cell proliferation in comparison to a negative control. Plates were
read on the Wallac Victor 2 1420 Multilabel Counter (Wallac, Turku,
Finland) at wavelengths 530 (Excitation) and 590 (Emmssion).
Results showed a strong dose-dependant inhibition of the
proliferative effect of IL-TIF on BaF3/CRF2-4/zcytor11 cells by
Zcytor16-Fc4. CRF2-4/zcytor11-Fc4 showed a much weaker inhibition
of IL-TIF. IL-TIF alone stimulated the cells 13-fold over
background. Zcytor16 completely inhibited that proliferation at
concentrations from 0.025-1 .mu.g/ml, and partially inhibited
proliferation at all the remaining concentrations down to 8 ng/ml.
CRF2-4/zcytor11-Fc4 was only able to completely inhibit
proliferation at the highest concentration of 8 .mu.g/ml, it
partially inhibited proliferation at 0.125-4 .mu.g/ml, and
inhibition was barely detectable at the lowest concentration of 63
ng/ml.
Example 31
Zcytor16 Decreases IL-6 and SAA Levels in Mouse Collagen Induced
Arthritis (CIA) Model
A. Mouse Collagen Induced Arthritis (CIA) Model
[0591] Ten week old male DBA/1J mice (Jackson Labs) were divided
into 3 groups of 13 mice/group. On day-21, animals were given a
subcutaneous injection of 50-100 .mu.l of 1 mg/ml chick Type II
collagen formulated in Complete Freund's Adjuvant (prepared by
Chondrex, Redmond, Wash.), and three weeks later on Day 0 they were
given a 100 .mu.l (25 .mu.g) injection of LPS from E. coli 0111:B4,
prepared as 250 .mu.g/ml from a lyophilized aliquot (Sigma, St.
Louis, Mo.). Zcytor16 was administered as an intraperitoneal
injection 3 times a week for 4 weeks, from Day 0 to Day 25. The
first two groups received either 100 or 10 .mu.g of zcytor16 per
animal per dose, and the third group received the vehicle control,
PBS (Life Technologies, Rockville, Md.). Animals began to show
symptoms of arthritis following the LPS injection, with most
animals developing inflammation within 2-3 weeks. The extent of
disease was evaluated in each paw by using a caliper to measure paw
thickness, and by assigning a clinical score (0-3) to each paw:
0=Normal, 0.5=Toe(s) inflamed, 1=Mild paw inflammation, 2=Moderate
paw inflammation, and 3=Severe paw inflammation as detailed
below.
Monitoring Disease:
[0592] Animals can begin to show signs of paw inflammation soon
after the second collagen injection, and some animals may even
begin to have signs of toe inflammation prior to the second
collagen injection. Most animals develop arthritis within 2-3 weeks
of the boost injection, but some may require a longer period of
time. Incidence of disease in this model is typically 95-100%, and
0-2 non-responders (determined after 6 weeks of observation) are
typically seen in a study using 40 animals. Note that as
inflammation begins, a common transient occurrence of variable
low-grade paw or toe inflammation can occur. For this reason, an
animal is not considered to have established disease until marked,
persistent paw swelling has developed.
[0593] All animals were observed daily to assess the status of the
disease in their paws, which was done by assigning a qualitative
clinical score to each of the paws. Every day, each animal has its
4 paws scored according to its state of clinical disease. To
determine the clinical score, the paw can be thought of as having 3
zones, the toes, the paw itself (manus or pes), and the wrist or
ankle joint. The extent and severity of the inflammation relative
to these zones was noted including observation all the toes for any
joint swelling, torn nails, or redness, notation of any evidence of
edema or redness in any of the paws, and notation any loss of fine
anatomic demarcation of tendons or bones, and evaluation the wrist
or ankle for any edema or redness, and notation if the inflammation
extends proximally up the leg. A paw a score of 1, 2, or 3 was
based first on the overall impression of severity, and second on
how many zones were involved. The scale used for clinical scoring
is shown below.
Clinical Score:
[0594] 0=Normal [0595] 0.5=One or more toes involved, but only the
toes are inflamed [0596] 1=mild inflammation involving the paw (1
zone), and may include a toe or toes [0597] 2=moderate inflammation
in the paw & may include some of the toes and/or the
wrist/ankle (2 zones) [0598] 3=severe inflammation in the paw,
wrist/ankle, and some or all of the toes (3 zones)
[0599] Established disease is defined as a qualitative score of paw
inflammation ranking 2 or more, that persists overnight (two days
in a row). Once established disease is present, the date is
recorded and designated as that animal's first day with
"established disease".
[0600] Blood was collected throughout the experiment to monitor
serum levels of anti-collagen antibodies. Animals were euthanized
on Day 21, and blood was collected for serum and for CBC's. From
each animal, one affected paw was collected in 10% NBF for
histology and one was frozen in liquid nitrogen and stored at
-80.degree. C. for mRNA analysis. Also, 1/2 spleen, 1/2 thymus, 1/2
mesenteric lymph node, one liver lobe and the left kidney were
collected in RNAlater for RNA analysis, and .1/2 spleen, 1/2
thymus, 1/2 mesenteric lymph node, the remaining liver, and the
right kidney were collected in 10% NBF for histology. Serum was
collected and frozen at -80.degree. C. for immunoglobulin and
cytokine assays.
[0601] No statistically significant differences were found between
the groups when the paw scores and measurements data were analyzed,
although there was a suggestion that one treatment group receiving
zcytor16 may have had a delay in the onset and progression of paw
inflammation. There were no significant differences between the
groups for changes in body weight, CBC parameters, or anti-collagen
antibody levels. These early results indicate that zcytor16 does
not adversely effect body weight, red or white blood cells, or
antibody production, but may be able to reduce inflammation.
Further investigations into dosing, mechanism of action, and
efficacy are under way.
B. Anti-Collagen ELISA Data in Mouse CIA Model
[0602] Serum samples were collected on days 0, 7, 14, 21 and 28
relative to date of LPS challenge (day 0) from the murine model of
collagen induced arthritis (Example 31A above). The serum samples
were screened by ELISA for anti-collagen antibody titers. There
were no statistically significant effects of zcytor16 treatment in
100 .mu.g or 10 .mu.g treatment groups on levels of anti-collagen
antibodies compared with PBS controls. Below is a description of
anti-collagen ELISA methods and materials.
[0603] Reagents used for anti-collagen ELISAs were Maxisorp 96-well
microtiter plates (NUNC, Rochester, N.Y.), chick type-II collagen
(Chondrex, Redmond, Wash.), Super Block (Pierce, Rockford, Ill.),
horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG+A+M (H+
L) (Zymed, South San Francisco, Calif.) and o-phenylenediamine
dihydrochloride substrate (Pierce, Rockford, Ill.). Buffers used in
all assays were ELISA B diluent buffer (PBS+0.1% BSA+0.05% Tween
(Sigma, St. Louis, Mo.)), ELISA C wash buffer (PBS+0.05% Tween) and
NovoD developing buffer (0.063M sodium citrate, 0.037M citric
acid), H.sub.2O.sub.2 (Sigma) and 1N H.sub.2SO.sub.4 (VWR,
Tukwilla, Wash.).
[0604] Approximately 100 .mu.L of peripheral blood was collected by
retro-orbital bleed into serum separator tubes (Becton Dickinson).
Serum was collected by centrifugation (2-3 min, 16,000.times.g,
4-6.degree. C.) and stored at -20.degree. C. until analyzed. To
determine anti-collagen Ig antibody levels, NUNC plates were coated
with 10 .mu.g/mL chick type-II collagen (Chondrex, Redmond Wash.)
and incubated overnight at 4.degree. C. Plates were washed with
ELISA C, blocked (5 minutes, room temperature) with Super Block
(Pierce, Rockford, Ill.), and washed with ELISA C. Diluted serum
samples (diluted in ELISA B 5-fold from 1:5000 to 1:625,000) were
added to ELISA plates in triplicate and the plates were incubated
overnight at 4.degree. C. After incubation, the plates were washed
with ELISA C, and peroxidase-labeled goat anti-mouse Ig Fc (Zymed,
1:2000 in ELISA B) was added. The plates were incubated (room
temperature, 90 minutes), rinsed again using ELISA C, and HRP
activity was developed using o-phenylenediamine dihydrochloride
substrate (10 mL NovoD+1 tablet OPD+10 .mu.L H.sub.2O.sub.2,
Pierce). The reaction was stopped with 1N H.sub.2SO.sub.4. Relative
optical density measurements of serum samples at 1:25,000 dilution
were taken at 490 nm using a Spectra MAX 190, and data were
analyzed using SoftMax Pro software (Molecular Devices Corporation,
Palo Alto, Calif.).
C. IL-6 and SAA Analysis in Mouse CIA Model
[0605] Day 0 serum samples were harvested from CIA mice (Example
31A above) 4 hr post administration of 25 .mu.g LPS
intraperitoneally. Samples were screened for IL-6 and serum amyloid
A (SAA) concentrations by commercial ELISA kits purchased for
Biosource International (Camarillo, Calif.) as per manufacturer's
instructions.
[0606] The IL-6 levels were 9651+/-1563 pg/ml, 10,865+/-1478 pg/ml
and 15,006+/-2,099 pg/ml in the mice groups subjected to 100 .mu.g
zcytor16, 10 .mu.g zcytor16 and PBS control, respectively. The IL-6
concentration in the group of CIA mice exposed to the 100 .mu.g
dose of zcytor16 was significantly lower compared to PBS control
mice with p=0351. Statistical significance was calculated using
Fisher's PLSD with a significance level of 5% (ABACUS Concepts,
INC, Berkeley, Calif.).
[0607] In addition, SAA concentrations were 381+/-40 .mu.g/ml,
348+/-37 .mu.g/ml and 490+/-50 .mu.g/ml in the mice groups
subjected to 100 .mu.g zcytor16, 10 .mu.g zcytor16 and PBS control
groups, respectively. The SAA concentration in the group of CIA
mice exposed to the 10 .mu.g dose of zcytor16 was significantly
lower compared with PBS control mice with p=0.0257. Statistical
significance was calculated using Fisher's PLSD with a significance
level of 5% (ABACUS Concepts, INC, Berkeley, Calif.).
Example 32
Expression of IL-TIF Receptor, Zcytor11, in the DSS Mouse Model
[0608] Quantitative RT-PCR was performed to measure expression
levels of mouse zcytor11 in the colons of mice with DSS-induced IBD
(Example 27). RNA was isolated from normal mouse colon and from the
distal colons of DSS-treated mice from treatment days 2, 7 and 10.
RT-PCR was performed using Applied Biosystems 7700 TaqMan
instrument and protocols. Briefly, "Primer Express" software was
used to designed primers against the mouse zcytor11 sequence
(ZC39776 (SEQ ID NO:53) and ZC39777 (SEQ ID NO:54)) and a FAM/TAMRA
labeled TaqMan probe (ZC38752 (SEQ ID NO:55)) according to Applied
Biosystems guidelines. 25 ng of RNA was added to each reaction,
along with PE/Applied Biosystems TaqMan EZ RT-PCR Core Reagents and
the above mentioned primers and probe. RT-PCR reactions were run in
duplicate under the following conditions: 50.degree. C. for 2
minutes, 60.degree. C. for 30 minutes, 95.degree. C. for 5 minutes,
40 cycles of 94.degree. C. for 20 seconds and 60.degree. C. for 1
minute. Expression values were compared to a standard curve of
known numbers of molecules of a synthetic mouse zcytor11 RNA
transcript, and expression is reported as absolute number of
molecules of mouse zcytor11 per reaction. Preliminary data suggests
that mouse zcytor11 expression may be slightly down-regulated in
the distal colons of day 7 and day 10 mice with DSS-induced IBD
when compared to expression levels in normal mouse colon.
Example 33
IL-TIF and Proinflammatory Indicators in Mild Endotoxemia Model:
LPS-Induced Endotoxemia Mouse Model
A. LPS-Induced Endotoxemia Mouse Model: Assessment Proinflammatory
Cytokines and Body Temperature in the LPS-Induced Endotoxemia Mouse
Model
[0609] An in vivo experiment was designed to examine the effect of
zcytor16 in a mouse LPS model of mild endotoxemia. To initially
assess the model, we measured proinflammatory cytokines and body
temperature to collect reference data for the model.
[0610] Briefly, six month Balb/c (CRL) female mice were injected
with 25 .mu.g LPS (Sigma) in sterile PBS intraperitoneally (IP).
Serum samples were collected at 0, 1, 4, 8, 16, 24, 48 and 72 hr
from groups of 8 mice for each time point. Serum samples were
assayed for inflammatory cytokine levels. IL-1b, IL-6, TNFa, IL-10
and serum amyloid A protein (SAA) levels were measured using
commercial ELISA kits purchased from Biosource International
(Camarillo, Calif.).
[0611] TNFa levels peaked to 400 pg/ml and IL-10 levels were 341
pg/ml at 1 hr post LPS injection. At 4 hr post LPS injection, IL-6,
IL-1b and IL-10 were 6,100 pg/ml, 299 pg/ml and 229 pg/ml,
respectively. The SAA levels in serum were 0.405 mg/ml by 4 hr post
LPS injection. SAA concentrations in serum continued to increase to
3.9 mg/ml by 24 hr post LPS, however SAA levels greater than 1 to 2
mg/ml in serum are difficult to measure accurately or reproducibly
with the existing ELISA kit due to interactions between SAA and
other serum components. These results indicated that
proinflammatory cytokines, in addition to IL-TIF (Example 33B),
were indeed produced in this model. Thus the following criteria
were established as biological markers for the LPS model of mild
endotoxemia: TNFa serum levels 1 hr post LPS, IL-6 serum levels 4
hr post LPS and SAA serum levels 4 and 8 hr post LPS.
[0612] Body temperatures in a separate group of animals were
monitored by surgically implanted telemetry devices over the course
of the 72 hr experiment. Body temperatures in mice dropped
maximally by 2.degree. C. from 37.07.degree. C. to 34.98.degree. C.
30 minutes after LPS injection.
[0613] Injection of 100 ug zcytor16-Fc fusion protein 30 minutes
prior to the LPS injection significantly reduced about 50% of the
SAA induction at 4 hr and 8 hr time point, while 10 ug zcytor16-Fc
did not have significant effect. There is no significant change to
the TNF-alpha and IL-6 level. Zcytor16-Fc injection reduced
neutrophil count in circulation at 1 hr time point. It showed the
administration of zcytor16-Fc can neutralize zcyto18 activity in
terms of SAA induction.
B. Detection of IL-TIF Activity in Mouse Serum from LPS-Induced
Endotoxemia Mouse Model Using BaF3/CRF2-4/zcytor11 Cells in an
Alamar Blue Proliferation Assay
[0614] BaF3/CRF2-4/zcytor11 cells, described herein, were spun down
and washed in PBS 2 times to ensure the removal of the mIL-3, and
then spun a third time and re-suspended in the complete media (RPMI
1640, 10% FBS, 1% GlutaMAX, 1% Sodium Pyruvate), but without mIL-3
(hereinafter referred to as "mIL-3 free media"). Cells were then
counted in a hemocytometer. 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.
[0615] Serum from the LPS-induced endotoxemia mice from the
experiment described in Example 33A above, was diluted to 2% in
mIL-3 free media on the top row of the plate and then diluted
serially 1:2 down the remaining 7 rows on the 96-well plate,
leaving a volume of 100 .mu.l in each well. This was then added to
the 100 .mu.l of cells, for final serum concentrations of 1%, 0.5%,
0.25%, 0.125%, 0.063%, 0.031%, 0.016%, and 0.018% in a total assay
volume of 200 .mu.l. The assay plates were incubated at 37.degree.
C., 5% CO.sub.2 for 4 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 16 hours. Alamar Blue
gives a fluourometric readout based on number of live cells, and is
thus a direct measurement of cell proliferation in comparison to a
negative control. Plates were read on the Wallac Victor 2 1420
Multilabel Counter (Wallac, Turku, Finland) at wavelengths 530
(Excitation) and 590 (Emmssion).
[0616] Results showed no significant proliferation above background
levels in the 0 hour, 1 hour, 8 hour, and 16 hour time points.
Serum samples from the 4 hour time point showed 4-fold to greater
than 10-fold increases in proliferation above background,
indicating the presence of IL-TIF in those samples.
C. LPS-Induced Endotoxemia Mouse Model: Experiment to Assess
Effects of Zcytor16
[0617] The ability of zcytor16 treatment to effect proinflammatory
indicators induced with a single 25 .mu.g LPS dose IP in mice was
tested. All samples were analyzed for SAA, IL-TIF and circulating
neutrophil counts. Subsets from each group were analyzed for
particular cytokine levels (1 hour samples were screened for TNF
alpha, 4 hour samples were analyzed for IL-6). Animals were
sacrificed at indicated time points in Table 14 below and whole
blood and serum were collected and aliquoted for analysis.
[0618] 72 B1/6 female mice (CRL) were given a single IP dose of
zcytor16 as described in Table 14, below. Control mice were C57B1/6
(CRL).
[0619] minutes later, they received another IP injection of 25
.mu.g LPS (Sigma) in 100 .mu.l, to initiate an endotoxemia cascade.
Mice in each group were sacrificed at corresponding time points as
indicated in Table 14, 50 .mu.l whole blood were collected to
measure total numbers of circulating neutrophils and the rest were
spun for serum and aliquoted for various assays described
herein.
TABLE-US-00015 TABLE 14 Group No Treatment LPS Sacrifice Samples A
8 100 .mu.g zcytor16 IP 25 .mu.g IP 1 hour Serum aliquots Blood for
30 min post tx CBC B 8 10 .mu.g zcytor16 IP 25 .mu.g IP 1 hour
Serum aliquots Blood for 30 min post tx CBC C 8 200 .mu.l PBS IP 25
.mu.g IP 1 hour Serum aliquots Blood for 30 min post tx CBC D 8 100
.mu.g zcytor16 IP 25 .mu.g IP 4 hours Serum aliquots Blood for 30
min post tx CBC E 8 10 .mu.g zcytor16 IP 25 .mu.g IP 4 hours Serum
aliquots Blood for 30 min post tx CBC F 8 200 .mu.l PBS IP 25 .mu.g
IP 4 hours Serum aliquots Blood for 30 min post tx CBC G 8 100
.mu.g zcytor16 IP 25 .mu.g IP 8 hours Serum aliquots Blood for 30
min post tx CBC H 8 10 .mu.g zcytor16 IP 25 .mu.g IP 8 hours Serum
aliquots Blood for 30 min post tx CBC J 8 200 .mu.l PBS IP 25 .mu.g
IP 8 hours Serum aliquots Blood for 30 min post tx CBC K 5 controls
none Pre LPS Serum aliquots Blood for CBC
D. Zcytor16/Fc4 Neutralizes SAA Induction In Vivo: SAA ELISA
Showing SAA Expression Induced by LPS in LPS-Induced Endotoxemia
Mouse Model is Inhibited by zcytor16-Fc4 Injection:
[0620] To assess whether zcytor16 could inhibit the SAA induction
in the LPS-induced endotoxemia mouse model, mice were injected with
Zcytor16, 30 minutes prior to LPS injection, as shown in Table 14
in Example 33C above.
[0621] An ELISA to determine SAA levels in the 4 hour and 8 hour
samples was performed using the Mouse SAA Immunoassay Kit
(BioSource International, California) following the manufacturer's
directions. At the 4 hour time point, mice treated with 100 .mu.g
or 10 .mu.g of Zcytor16 showed a dose-dependant, statistically
significant reduction in SAA levels relative to the PBS injected
mice. At the 8 hour time point, mice treated with 100 .mu.g,
continued to show a statistically significant reduction in SAA
levels relative to the PBS injected mice. This indicates that the
presence of Zcytor16 is able to inhibit the induction of SAA by LPS
in vivo.
Example 34
Baculovirus Expression of FlagTBXzCytor16
[0622] An expression vector, pzBV37L:egtNF(tbx)sCytor16, was
designed and prepared to express FlagTBXzCytor16 polypeptides in
insect cells.
Expression of FlagTBXzCytor16
[0623] An expression vector, pzBV37L:egtNF(tbx)sCytor16 was
designed to express zCytor16 polypeptide with an upstream 6 amino
acid thrombin cleavage site and an n-terminal Flag epitope tag
upstream of the enzyme cleavage site. This construct can be used to
express a flag tagged zCytor16 with an enzyme processing site
directly upstream of the soluble receptor sequence, after the
signal peptide has been cleaved off.
A. Construction of pzBV37LegtNF(tbx)sCytor16
[0624] A 698 bp, FlagTBXzCytor16 sequence fragment containing Bspe1
and Xba1 restriction sites on the 5' and 3' ends, respectively, was
generated by two rounds of PCR amplification from a zCytor16 cDNA
containing template. Primers ZC40,940 (SEQ ID NO:56) and ZC40,943
(SEQ ID NO:57) were used in the first round and primers ZC40942
(SEQ ID NO:58) and ZC40,943 (SEQ ID NO:57) in the second round. For
the first round of PCR, reaction conditions were as follows:
utilized the Expand High Fidelity PCR System (Boerhinger Mannheim)
for a 100 ul vol. reaction. 1 cycle at 94.degree. C. for 2 minutes;
35 cycles of 94.degree. C. for 15 seconds, 50.degree. C. for 30
seconds, and 72.degree. C. for 60 seconds; 1 cycle at 72.degree. C.
for 5 min; followed by 4.degree. C. soak. 5 ul of the first round
reaction mix was visualized by gel electrophoresis (1% NuSieve
agarose). Once the presence of a correct size PCR product was
confirmed, the second round of PCR was set up using 1 ul of the
first round reaction as template. Conditions of the second reaction
were the same as the first. 5 ul of the second round PCR was
visualized by gel electrophoresis (1% NuSieve agarose). The
remainder of the reaction mix was purified via Qiagen PCR
purification kit as per manufacturers instructions and eluted in 30
ul water. The cDNA was digested in a 35 ul vol. using Bspe1 and
Xba1 (New England Biolabs, Beverly, Mass.) in appropriate buffer
conditions at 37 degrees C. The digested PCR product band was run
through a 1% agarose TAE gel, excised and extracted using a
QIAquick.TM. Gel Extraction Kit (Qiagen, Cat. No. 28704) and eluted
in 30 ul of water. The digested FlagTBXzCytor16 PCR was ligated
into the MCS of vector pZBV37L at the Bspe1 and Xba1 sites. The
pZBV37L vector is a modification of the pFastBac1.TM. (Life
Technologies) expression vector, where the polyhedron promoter has
been removed and replaced with the late activating Basic Protein
Promoter and the EGT leader signal sequence upstream of the MCS. 5
ul of the restriction enzyme digested FlagTBXzCytor16 PCR fragment
and apx. 50 ng of the corresponding pZBV37L vector were ligated
overnight at 16.degree. C. in a 20 ul vol. in appropriate buffer
conditions. 5 ul of the ligation mix was transformed into 50 ul of
ElectoMAX.TM. DH12s.TM. cells (Life Technologies, Cat. No.
18312-017) by electroporation at 400 Ohms, 2V and 25 .mu.F in a 2
mm gap electroporation cuvette (BTX, Model No. 620). The
transformed cells were diluted in 350 .mu.l of SOC media (2% Bacto
Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10
mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM glucose) outgrown for 1
hr at 37 degrees C. and 50 .mu.l of the dilution were plated onto
LB plates containing 100 .mu.g/ml ampicillin. Clones were analyzed
by PCR and positive clones were selected, plated and submitted for
sequencing. Once proper sequence was confirmed, 25 ngs of positive
clone DNA was transformed into 100 .mu.l DH10Bac.TM. Max
Efficiency.RTM. competent cells (GIBCO-BRL Cat. No. 10361-012) by
heat shock for 45 seconds in a 42.degree. C. heat block. The
transformed DH10Bac.TM. cells were diluted in 900 .mu.l SOC media
(2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM
KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM glucose) outgrown
for 1 hr at 37 degrees C. and 100 .mu.l were plated onto Luria Agar
plates containing 50 .mu.g/ml kanamycin, 7 .mu.g/ml gentamicin, 10
.mu.g/ml tetracycline, 40 .mu.g/mL IPTG and 200 .mu.g/mL Bluo Gal.
The plates were incubated for 48 hours at 37.degree. C. A color
selection was used to identify those cells having transposed viral
DNA (referred to as a "bacmid"). Those colonies, which were white
in color, were picked for analysis. Colonies were analyzed by PCR
and positive colonies (containing desired bacmid) were selected for
outgrow. Clones were screened for the correct M.W. insert by
amplifying DNA using primers to the transposable element in the
bacmid via PCR using primers ZC447 (SEQ ID NO:34) and ZC976 (SEQ ID
NO:7). The PCR reaction conditions were as follows: 1 cycle at
94.degree. C. for 2 minutes; 25 cycles of 94.degree. C. for 10
seconds, 50.degree. C. for 30 seconds, and 72.degree. C. for 120
seconds; 1 cycle at 72.degree. C. for 5 min; followed by 4.degree.
C. soak. The PCR product was run on a 1% agarose gel to check the
insert size. Those having the correct size insert were outgrown and
the bacmid DNA isolated and purified. This bacmid DNA was used to
transfect Spodoptera Frugiperda (Sf9) cells.
B. Transfection
[0625] Sf9 cells were seeded at 1.times.10.sup.6 cells per well in
a 6-well plate and allowed to attach for 1 hour at 27.degree. C.
Approximately five .mu.g. of bacmid DNA were diluted with 100 .mu.l
Sf-900 II SFM (Life Technologies). Twenty .mu.l of
Lipofectamine.TM. Reagent (Life Technologies, Cat. No. 18324-012)
were diluted with 100 .mu.l Sf-900 II SFM. The bacmid DNA and lipid
solutions were gently mixed and incubated 45 minutes at room
temperature. Eight hundred microliters of Sf-900 II SFM was added
to the lipid-DNA mixture. The media was aspirated from the well and
the 1 ml of DNA-lipid mix added to the cells. The cells were
incubated at 27.degree. C. overnight. The DNA-lipid mix was
aspirated and 2 ml of Sf-900 II media was added to each plate. The
plates were incubated at 27.degree. C., 90% humidity, for
approximately 7 days after which the virus was harvested.
C. Amplification
[0626] Sf9 cells were seeded at 1.times.10.sup.6 cells per well in
a 6-well plate in 2 mls SF-900II. 500 .mu.l of virus from the
transfection plate were placed in the well and the plate was
incubated at 27.degree. C., 90% humidity, for 96 hours after which
the virus was harvested (primary amplification).
[0627] A second round of amplification proceeded as follows: Sf9
cells were seeded at 1.times.10.sup.6 cells per well in a 6-well
plate in 2 mls SF-900II. 100 .mu.l of virus from the primary
amplification plate were placed in the well and the plate was
incubated at 27.degree. C., 90% humidity, for 144 hours after which
the virus was harvested (Secondary amplification).
[0628] An additional round of amplification was performed (3.sup.rd
round amp.) Sf9 cells were grown in 50 ml Sf-900 II SFM in a 250 ml
shake flask to an approximate density of 1.times.10.sup.6 cells/ml.
They were then infected with 1 mL of the viral stock from the above
plate and incubated at 27.degree. C. for 4 days after which time
the virus was harvested.
[0629] This viral stock was titered by a growth inhibition curve
and the titer culture that indicated a MOI of 1 was allowed to
proceed for a total of 48 hrs. The supernatant was analyzed via
Western blot using a primary monoclonal antibody specific for the
n-terminal Flag epitope and a HRP conjugated Gt anti Mu secondary
antibody. Results indicated a band of apx. 30 kDa. Supernatant was
also provided for activity analysis.
[0630] A large viral stock was then generated by the following
method: Sf9 cells were grown in 1 L Sf-900 II SFM in a 2800 ml
shake flask to an approximate density of 1.times.10.sup.6 cells/ml.
They were then infected with 5 mls of the viral stock from the
3.sup.rd round amp. and incubated at 27.degree. C. for 96 hrs after
which time the virus was harvested.
[0631] Larger scale infections were completed to provide material
for downstream purification.
[0632] 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
6212149DNAHomo sapiensCDS(1)...(693) 1atg atg cct aaa cat tgc ttt
cta ggc ttc ctc atc agt ttc ttc ctt 48Met Met Pro Lys His Cys Phe
Leu Gly Phe Leu Ile Ser Phe Phe Leu1 5 10 15act ggt gta gca gga act
cag tca acg cat gag tct ctg aag cct cag 96Thr Gly Val Ala Gly Thr
Gln Ser Thr His Glu Ser Leu Lys Pro Gln20 25 30agg gta caa ttt cag
tcc cga aat ttt cac aac att ttg caa tgg cag 144Arg Val Gln Phe Gln
Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln35 40 45cct ggg agg gca
ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192Pro Gly Arg Ala
Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr50 55 60aaa ata tat
gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240Lys Ile Tyr
Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly65 70 75 80act
caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag 288Thr
Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln85 90
95gaa cct tat tac ggg agg gtg agg gcg gcc tcg gct ggg agc tac tca
336Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr
Ser100 105 110gaa tgg agc atg acg ccg cgg ttc act ccc tgg tgg gaa
aca aaa ata 384Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu
Thr Lys Ile115 120 125gat cct cca gtc atg aat ata acc caa gtc aat
ggc tct ttg ttg gta 432Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn
Gly Ser Leu Leu Val130 135 140att ctc cat gct cca aat tta cca tat
aga tac caa aag gaa aaa aat 480Ile Leu His Ala Pro Asn Leu Pro Tyr
Arg Tyr Gln Lys Glu Lys Asn145 150 155 160gta tct ata gaa gat tac
tat gaa cta cta tac cga gtt ttt ata att 528Val Ser Ile Glu Asp Tyr
Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile165 170 175aac aat tca cta
gaa aag gag caa aag gtt tat gaa ggg gct cac aga 576Asn Asn Ser Leu
Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg180 185 190gcg gtt
gaa att gaa gct cta aca cca cac tcc agc tac tgt gta gtg 624Ala Val
Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val195 200
205gct gaa ata tat cag ccc atg tta gac aga aga agt cag aga agt gaa
672Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser
Glu210 215 220gag aga tgt gtg gaa att cca tgacttgtgg aatttggcat
tcagcaatgt 723Glu Arg Cys Val Glu Ile Pro225 230ggaaattcta
aagctccctg agaacaggat gactcgtgtt tgaaggatct tatttaaaat
783tgtttttgta ttttcttaaa gcaatattca ctgttacacc ttggggactt
ctttgtttat 843ccattctttt atcctttata tttcatttta aactatattt
gaacgacatt ccccccgaaa 903aattgaaatg taaagatgag gcagagaata
aagtgttcta tgaaattcag aactttattt 963ctgaatgtaa catccctaat
aacaaccttc attcttctaa tacagcaaaa taaaaattta 1023acaaccaagg
aatagtattt aagaaaatgt tgaaataatt tttttaaaat agcattacag
1083actgaggcgg tcctgaagca atggtttttc actctcttat tgagccaatt
aaattgacat 1143tgctttgaca atttaaaact tctataaagg tgaatatttt
tcatacattt ctattttata 1203tgaatatact ttttatatat ttattattat
taaatatttc tacttaatga atcaaaattt 1263tgttttaaag tctactttat
gtaaataaga acaggttttg gggaaaaaaa tcttatgatt 1323tctggattga
tatctgaatt aaaactatca acaacaagga agtctactct gtacaattgt
1383ccctcattta aaagatatat taagcttttc ttttctgttt gtttttgttt
tgtttagttt 1443ttaatcctgt cttagaagaa cttatcttta ttctcaaaat
taaatgtaat ttttttagtg 1503acaaagaaga aaggaaacct cattactcaa
tccttctggc caagagtgtc ttgcttgtgg 1563cgccttcctc atctctatat
aggaggatcc catgaatgat ggtttattgg gaactgctgg 1623ggtcgacccc
atacagagaa ctcagcttga agctggaagc acacagtggg tagcaggaga
1683aggaccggtg ttggtaggtg cctacagaga ctatagagct agacaaagcc
ctccaaactg 1743gcccctcctg ctcactgcct ctcctgagta gaaatctggt
gacctaaggc tcagtgcggt 1803caacagaaag ctgccttctt cacttgaggc
taagtcttca tatatgttta aggttgtctt 1863tctagtgagg agatacatat
cagagaacat ttgtacaatt ccccatgaaa attgctccaa 1923agttgataac
aatatagtcg gtgcttctag ttatatgcaa gtactcagtg ataaatggat
1983taaaaaatat tcagaaatgt attggggggt ggaggagaat aagaggcaga
gcaagagcta 2043gagaattggt ttccttgctt ccctgtatgc tcagaaaaca
ttgatttgag catagacgca 2103gagactgaaa aaaaaaaaat gctcgagcgg
ccgccatatc cttggt 21492231PRTHomo sapiens 2Met Met Pro Lys His Cys
Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu1 5 10 15Thr Gly Val Ala Gly
Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln20 25 30Arg Val Gln Phe
Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln35 40 45Pro Gly Arg
Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr50 55 60Lys Ile
Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly65 70 75
80Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln85
90 95Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr
Ser100 105 110Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu
Thr Lys Ile115 120 125Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn
Gly Ser Leu Leu Val130 135 140Ile Leu His Ala Pro Asn Leu Pro Tyr
Arg Tyr Gln Lys Glu Lys Asn145 150 155 160Val Ser Ile Glu Asp Tyr
Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile165 170 175Asn Asn Ser Leu
Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg180 185 190Ala Val
Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val195 200
205Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser
Glu210 215 220Glu Arg Cys Val Glu Ile Pro225 2303693DNAArtificial
SequenceThis degenerate nucleotide sequence encodes the amino acid
sequence of SEQ ID NO2. 3atgatgccna arcaytgytt yytnggntty
ytnathwsnt tyttyytnac nggngtngcn 60ggnacncarw snacncayga rwsnytnaar
ccncarmgng tncarttyca rwsnmgnaay 120ttycayaaya thytncartg
gcarccnggn mgngcnytna cnggnaayws nwsngtntay 180ttygtncart
ayaarathta yggncarmgn cartggaara ayaargarga ytgytggggn
240acncargary tnwsntgyga yytnacnwsn garacnwsng ayathcarga
rccntaytay 300ggnmgngtnm gngcngcnws ngcnggnwsn taywsngart
ggwsnatgac nccnmgntty 360acnccntggt gggaracnaa rathgayccn
ccngtnatga ayathacnca rgtnaayggn 420wsnytnytng tnathytnca
ygcnccnaay ytnccntaym gntaycaraa rgaraaraay 480gtnwsnathg
argaytayta ygarytnytn taymgngtnt tyathathaa yaaywsnytn
540garaargarc araargtnta ygarggngcn caymgngcng tngarathga
rgcnytnacn 600ccncaywsnw sntaytgygt ngtngcngar athtaycarc
cnatgytnga ymgnmgnwsn 660carmgnwsng argarmgntg ygtngarath ccn
693416PRTArtificial SequencePeptide linker. 4Gly Gly Ser Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 155699DNAHomo
sapiens 5gagcccagat 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 699632DNAArtificial
SequenceOligonucleotide primer ZC29181 6gcggatccac tcagtcaacg
catgagtctc tg 32733DNAArtificial SequenceOligonucleotide primer
ZC29182 7gcagatcttg gaatttccac acatctctct tca 338108DNAHomo
sapiensCDS(1)...(108) 8atg 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 gaa atc
cat gcc gag ttg aga cgc 96Ala Val Phe Val Ser Leu Ser Gln Glu Ile
His Ala Glu Leu Arg Arg20 25 30ttc cgt aga tcc 108Phe Arg Arg
Ser35936PRTHomo sapiens 9Met 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 Arg
Ser35106PRTArtificial SequenceGlu-Glu (CEE) Tag amino acid sequence
10Glu Tyr Met Pro Met Glu1 5118PRTArtificial SequenceFLAG Tag amino
acid sequence 11Asp Tyr Lys Asp Asp Asp Asp Lys1 5126PRTArtificial
SequenceHis Tag amino acid sequence 12His His His His His His1
513210PRTHomo sapiens 13Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
Arg Val Gln Phe Gln1 5 10 15Ser Arg Asn Phe His Asn Ile Leu Gln Trp
Gln Pro Gly Arg Ala Leu20 25 30Thr Gly Asn Ser Ser Val Tyr Phe Val
Gln Tyr Lys Ile Tyr Gly Gln35 40 45Arg Gln Trp Lys Asn Lys Glu Asp
Cys Trp Gly Thr Gln Glu Leu Ser50 55 60Cys Asp Leu Thr Ser Glu Thr
Ser Asp Ile Gln Glu Pro Tyr Tyr Gly65 70 75 80Arg Val Arg Ala Ala
Ser Ala Gly Ser Tyr Ser Glu Trp Ser Met Thr85 90 95Pro Arg Phe Thr
Pro Trp Trp Glu Thr Lys Ile Asp Pro Pro Val Met100 105 110Asn Ile
Thr Gln Val Asn Gly Ser Leu Leu Val Ile Leu His Ala Pro115 120
125Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn Val Ser Ile Glu
Asp130 135 140Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile Asn Asn
Ser Leu Glu145 150 155 160Lys Glu Gln Lys Val Tyr Glu Gly Ala His
Arg Ala Val Glu Ile Glu165 170 175Ala Leu Thr Pro His Ser Ser Tyr
Cys Val Val Ala Glu Ile Tyr Gln180 185 190Pro Met Leu Asp Arg Arg
Ser Gln Arg Ser Glu Glu Arg Cys Val Glu195 200 205Ile
Pro210141116DNAHomo sapiensCDS(21)...(557) 14tcgagttaga attgtctgca
atg gcc gcc ctg cag aaa tct gtg agc tct ttc 53Met Ala Ala Leu Gln
Lys Ser Val Ser Ser Phe1 5 10ctt atg ggg acc ctg gcc acc agc tgc
ctc ctt ctc ttg gcc ctc ttg 101Leu Met Gly Thr Leu Ala Thr Ser Cys
Leu Leu Leu Leu Ala Leu Leu15 20 25gta cag gga gga gca gct gcg ccc
atc agc tcc cac tgc agg ctt gac 149Val Gln Gly Gly Ala Ala Ala Pro
Ile Ser Ser His Cys Arg Leu Asp30 35 40aag tcc aac ttc cag cag ccc
tat atc acc aac cgc acc ttc atg ctg 197Lys Ser Asn Phe Gln Gln Pro
Tyr Ile Thr Asn Arg Thr Phe Met Leu45 50 55gct aag gag gct agc ttg
gct gat aac aac aca gac gtt cgt ctc att 245Ala Lys Glu Ala Ser Leu
Ala Asp Asn Asn Thr Asp Val Arg Leu Ile60 65 70 75ggg gag aaa ctg
ttc cac gga gtc agt atg agt gag cgc tgc tat ctg 293Gly Glu Lys Leu
Phe His Gly Val Ser Met Ser Glu Arg Cys Tyr Leu80 85 90atg aag cag
gtg ctg aac ttc acc ctt gaa gaa gtg ctg ttc cct caa 341Met Lys Gln
Val Leu Asn Phe Thr Leu Glu Glu Val Leu Phe Pro Gln95 100 105tct
gat agg ttc cag cct tat atg cag gag gtg gtg ccc ttc ctg gcc 389Ser
Asp Arg Phe Gln Pro Tyr Met Gln Glu Val Val Pro Phe Leu Ala110 115
120agg ctc agc aac agg cta agc aca tgt cat att gaa ggt gat gac ctg
437Arg Leu Ser Asn Arg Leu Ser Thr Cys His Ile Glu Gly Asp Asp
Leu125 130 135cat atc cag agg aat gtg caa aag ctg aag gac aca gtg
aaa aag ctt 485His Ile Gln Arg Asn Val Gln Lys Leu Lys Asp Thr Val
Lys Lys Leu140 145 150 155gga gag agt gga gag atc aaa gca att gga
gaa ctg gat ttg ctg ttt 533Gly Glu Ser Gly Glu Ile Lys Ala Ile Gly
Glu Leu Asp Leu Leu Phe160 165 170atg tct ctg aga aat gcc tgc att
tgaccagagc aaagctgaaa aatgaataac 587Met Ser Leu Arg Asn Ala Cys
Ile175taaccccctt tccctgctag aaataacaat tagatgcccc aaagcgattt
tttttaacca 647aaaggaagat gggaagccaa actccatcat gatgggtgga
ttccaaatga acccctgcgt 707tagttacaaa ggaaaccaat gccacttttg
tttataagac cagaaggtag actttctaag 767catagatatt tattgataac
atttcattgt aactggtgtt ctatacacag aaaacaattt 827attttttaaa
taattgtctt tttccataaa aaagattact ttccattcct ttaggggaaa
887aaacccctaa atagcttcat gtttccataa tcagtacttt atatttataa
atgtatttat 947tattattata agactgcatt ttatttatat cattttatta
atatggattt atttatagaa 1007acatcattcg atattgctac ttgagtgtaa
ggctaatatt gatatttatg acaataatta 1067tagagctata acatgtttat
ttgacctcaa taaacacttg gatatccta 111615179PRTHomo sapiens 15Met Ala
Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr Leu1 5 10 15Ala
Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Gly Gly Ala20 25
30Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn Phe Gln35
40 45Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu Ala Lys Glu Ala
Ser50 55 60Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys
Leu Phe65 70 75 80His Gly Val Ser Met Ser Glu Arg Cys Tyr Leu Met
Lys Gln Val Leu85 90 95Asn Phe Thr Leu Glu Glu Val Leu Phe Pro Gln
Ser Asp Arg Phe Gln100 105 110Pro Tyr Met Gln Glu Val Val Pro Phe
Leu Ala Arg Leu Ser Asn Arg115 120 125Leu Ser Thr Cys His Ile Glu
Gly Asp Asp Leu His Ile Gln Arg Asn130 135 140Val Gln Lys Leu Lys
Asp Thr Val Lys Lys Leu Gly Glu Ser Gly Glu145 150 155 160Ile Lys
Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn165 170
175Ala Cys Ile1623DNAArtificial SequenceOligonucleotide primer
ZC25963 16agtcaacgca tgagtctctg aag 231723DNAArtificial
SequenceOligonucleotide primer ZC28354 17accaacaaag agccattgac ttg
231823DNAArtificial SequenceOligonucleotide primer ZC21195
18gaggagacca taacccccga cag 231923DNAArtificial
SequenceOligonucleotide primer ZC21196 19catagctccc accacacgat ttt
232025DNAArtificial SequenceOligonucleotide primer ZC14063
20caccagacat aatagctgac agact 252121DNAArtificial
SequenceOligonucleotide primer ZC17574 21ggtrttgctc agcatgcaca c
212224DNAArtificial SequenceOligonucleotide primer ZC17600
22catgtaggcc atgaggtcca ccac 242322DNAArtificial
SequenceOligonucleotide primer ZC27659 23tcaagctgag ttctctgtat gg
22242831DNAHomo sapiensCDS(34)...(1755) 24tagaggccaa gggagggctc
tgtgccagcc ccg atg agg acg ctg ctg acc atc 54Met Arg Thr Leu Leu
Thr Ile1 5ttg act gtg gga tcc ctg gct gct cac gcc cct gag gac ccc
tcg gat 102Leu Thr Val Gly Ser Leu Ala Ala His Ala Pro Glu Asp Pro
Ser Asp10 15 20ctg ctc cag cac gtg aaa ttc cag tcc agc aac ttt gaa
aac atc ctg 150Leu Leu Gln His Val Lys Phe Gln Ser Ser Asn Phe Glu
Asn Ile Leu25 30 35acg tgg gac agc ggg cca gag ggc acc cca gac acg
gtc tac agc atc 198Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro Asp Thr
Val Tyr Ser Ile40 45 50 55gag tat aag acg tac gga gag agg gac tgg
gtg gca aag aag ggc tgt 246Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp
Val Ala Lys Lys Gly Cys60 65 70cag cgg atc acc cgg aag tcc tgc aac
ctg acg gtg gag acg ggc aac 294Gln Arg Ile Thr Arg Lys Ser Cys Asn
Leu Thr Val Glu Thr Gly Asn75 80 85ctc acg gag ctc tac tat gcc agg
gtc acc gct gtc agt gcg gga ggc 342Leu Thr Glu Leu Tyr Tyr Ala Arg
Val Thr Ala Val Ser Ala Gly Gly90 95 100cgg tca gcc acc aag atg act
gac agg ttc agc tct ctg cag cac act 390Arg Ser Ala Thr Lys Met Thr
Asp Arg Phe Ser Ser Leu Gln His Thr105 110 115acc ctc aag cca cct
gat gtg acc tgt atc tcc aaa gtg aga tcg att 438Thr Leu Lys Pro Pro
Asp Val Thr Cys Ile Ser Lys Val Arg Ser Ile120 125 130 135cag atg
att gtt cat cct acc ccc acg cca atc cgt gca ggc gat ggc 486Gln Met
Ile Val His Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly140 145
150cac cgg cta acc ctg gaa gac atc ttc cat gac ctg ttc tac cac tta
534His Arg Leu Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr His
Leu155 160 165gag ctc cag gtc aac cgc acc tac caa atg cac ctt gga
ggg aag cag 582Glu Leu Gln Val Asn Arg Thr Tyr Gln Met His Leu Gly
Gly Lys Gln170
175 180aga gaa tat gag ttc ttc ggc ctg acc cct gac aca gag ttc ctt
ggc 630Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu
Gly185 190 195acc atc atg att tgc gtt ccc acc tgg gcc aag gag agt
gcc ccc tac 678Thr Ile Met Ile Cys Val Pro Thr Trp Ala Lys Glu Ser
Ala Pro Tyr200 205 210 215atg tgc cga gtg aag aca ctg cca gac cgg
aca tgg acc tac tcc ttc 726Met Cys Arg Val Lys Thr Leu Pro Asp Arg
Thr Trp Thr Tyr Ser Phe220 225 230tcc gga gcc ttc ctg ttc tcc atg
ggc ttc ctc gtc gca gta ctc tgc 774Ser Gly Ala Phe Leu Phe Ser Met
Gly Phe Leu Val Ala Val Leu Cys235 240 245tac ctg agc tac aga tat
gtc acc aag ccg cct gca cct ccc aac tcc 822Tyr Leu Ser Tyr Arg Tyr
Val Thr Lys Pro Pro Ala Pro Pro Asn Ser250 255 260ctg aac gtc cag
cga gtc ctg act ttc cag ccg ctg cgc ttc atc cag 870Leu Asn Val Gln
Arg Val Leu Thr Phe Gln Pro Leu Arg Phe Ile Gln265 270 275gag cac
gtc ctg atc cct gtc ttt gac ctc agc ggc ccc agc agt ctg 918Glu His
Val Leu Ile Pro Val Phe Asp Leu Ser Gly Pro Ser Ser Leu280 285 290
295gcc cag cct gtc cag tac tcc cag atc agg gtg tct gga ccc agg gag
966Ala Gln Pro Val Gln Tyr Ser Gln Ile Arg Val Ser Gly Pro Arg
Glu300 305 310ccc gca gga gct cca cag cgg cat agc ctg tcc gag atc
acc tac tta 1014Pro Ala Gly Ala Pro Gln Arg His Ser Leu Ser Glu Ile
Thr Tyr Leu315 320 325ggg cag cca gac atc tcc atc ctc cag ccc tcc
aac gtg cca cct ccc 1062Gly Gln Pro Asp Ile Ser Ile Leu Gln Pro Ser
Asn Val Pro Pro Pro330 335 340cag atc ctc tcc cca ctg tcc tat gcc
cca aac gct gcc cct gag gtc 1110Gln Ile Leu Ser Pro Leu Ser Tyr Ala
Pro Asn Ala Ala Pro Glu Val345 350 355ggg ccc cca tcc tat gca cct
cag gtg acc ccc gaa gct caa ttc cca 1158Gly Pro Pro Ser Tyr Ala Pro
Gln Val Thr Pro Glu Ala Gln Phe Pro360 365 370 375ttc tac gcc cca
cag gcc atc tct aag gtc cag cct tcc tcc tat gcc 1206Phe Tyr Ala Pro
Gln Ala Ile Ser Lys Val Gln Pro Ser Ser Tyr Ala380 385 390cct caa
gcc act ccg gac agc tgg cct ccc tcc tat ggg gta tgc atg 1254Pro Gln
Ala Thr Pro Asp Ser Trp Pro Pro Ser Tyr Gly Val Cys Met395 400
405gaa ggt tct ggc aaa gac tcc ccc act ggg aca ctt tct agt cct aaa
1302Glu Gly Ser Gly Lys Asp Ser Pro Thr Gly Thr Leu Ser Ser Pro
Lys410 415 420cac ctt agg cct aaa ggt cag ctt cag aaa gag cca cca
gct gga agc 1350His Leu Arg Pro Lys Gly Gln Leu Gln Lys Glu Pro Pro
Ala Gly Ser425 430 435tgc atg tta ggt ggc ctt tct ctg cag gag gtg
acc tcc ttg gct atg 1398Cys Met Leu Gly Gly Leu Ser Leu Gln Glu Val
Thr Ser Leu Ala Met440 445 450 455gag gaa tcc caa gaa gca aaa tca
ttg cac cag ccc ctg ggg att tgc 1446Glu Glu Ser Gln Glu Ala Lys Ser
Leu His Gln Pro Leu Gly Ile Cys460 465 470aca gac aga aca tct gac
cca aat gtg cta cac agt ggg gag gaa ggg 1494Thr Asp Arg Thr Ser Asp
Pro Asn Val Leu His Ser Gly Glu Glu Gly475 480 485aca cca cag tac
cta aag ggc cag ctc ccc ctc ctc tcc tca gtc cag 1542Thr Pro Gln Tyr
Leu Lys Gly Gln Leu Pro Leu Leu Ser Ser Val Gln490 495 500atc gag
ggc cac ccc atg tcc ctc cct ttg caa cct cct tcc ggt cca 1590Ile Glu
Gly His Pro Met Ser Leu Pro Leu Gln Pro Pro Ser Gly Pro505 510
515tgt tcc ccc tcg gac caa ggt cca agt ccc tgg ggc ctg ctg gag tcc
1638Cys Ser Pro Ser Asp Gln Gly Pro Ser Pro Trp Gly Leu Leu Glu
Ser520 525 530 535ctt gtg tgt ccc aag gat gaa gcc aag agc cca gcc
cct gag acc tca 1686Leu Val Cys Pro Lys Asp Glu Ala Lys Ser Pro Ala
Pro Glu Thr Ser540 545 550gac ctg gag cag ccc aca gaa ctg gat tct
ctt ttc aga ggc ctg gcc 1734Asp Leu Glu Gln Pro Thr Glu Leu Asp Ser
Leu Phe Arg Gly Leu Ala555 560 565ctg act gtg cag tgg gag tcc
tgaggggaat gggaaaggct tggtgcttcc 1785Leu Thr Val Gln Trp Glu
Ser570tccctgtccc tacccagtgt cacatccttg gctgtcaatc ccatgcctgc
ccatgccaca 1845cactctgcga tctggcctca gacgggtgcc cttgagagaa
gcagagggag tggcatgcag 1905ggcccctgcc atgggtgcgc tcctcaccgg
aacaaagcag catgataagg actgcagcgg 1965gggagctctg gggagcagct
tgtgtagaca agcgcgtgct cgctgagccc tgcaaggcag 2025aaatgacagt
gcaaggagga aatgcaggga aactcccgag gtccagagcc ccacctccta
2085acaccatgga ttcaaagtgc tcagggaatt tgcctctcct tgccccattc
ctggccagtt 2145tcacaatcta gctcgacaga gcatgaggcc cctgcctctt
ctgtcattgt tcaaaggtgg 2205gaagagagcc tggaaaagaa ccaggcctgg
aaaagaacca gaaggaggct gggcagaacc 2265agaacaacct gcacttctgc
caaggccagg gccagcagga cggcaggact ctagggaggg 2325gtgtggcctg
cagctcattc ccagccaggg caactgcctg acgttgcacg atttcagctt
2385cattcctctg atagaacaaa gcgaaatgca ggtccaccag ggagggagac
acacaagcct 2445tttctgcagg caggagtttc agaccctatc ctgagaatgg
ggtttgaaag gaaggtgagg 2505gctgtggccc ctggacgggt acaataacac
actgtactga tgtcacaact ttgcaagctc 2565tgccttgggt tcagcccatc
tgggctcaaa ttccagcctc accactcaca agctgtgtga 2625cttcaaacaa
atgaaatcag tgcccagaac ctcggtttcc tcatctgtaa tgtggggatc
2685ataacaccta cctcatggag ttgtggtgaa gatgaaatga agtcatgtct
ttaaagtgct 2745taatagtgcc tggtacatgg gcagtgccca ataaacggta
gctatttaaa aaaaaaaaaa 2805aaaaaaaaaa atagcggccg cctcga
283125574PRTHomo sapiens 25Met Arg Thr Leu Leu Thr Ile Leu Thr Val
Gly Ser Leu Ala Ala His1 5 10 15Ala Pro Glu Asp Pro Ser Asp Leu Leu
Gln His Val Lys Phe Gln Ser20 25 30Ser Asn Phe Glu Asn Ile Leu Thr
Trp Asp Ser Gly Pro Glu Gly Thr35 40 45Pro Asp Thr Val Tyr Ser Ile
Glu Tyr Lys Thr Tyr Gly Glu Arg Asp50 55 60Trp Val Ala Lys Lys Gly
Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn65 70 75 80Leu Thr Val Glu
Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg Val85 90 95Thr Ala Val
Ser Ala Gly Gly Arg Ser Ala Thr Lys Met Thr Asp Arg100 105 110Phe
Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys115 120
125Ile Ser Lys Val Arg Ser Ile Gln Met Ile Val His Pro Thr Pro
Thr130 135 140Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu
Asp Ile Phe145 150 155 160His Asp Leu Phe Tyr His Leu Glu Leu Gln
Val Asn Arg Thr Tyr Gln165 170 175Met His Leu Gly Gly Lys Gln Arg
Glu Tyr Glu Phe Phe Gly Leu Thr180 185 190Pro Asp Thr Glu Phe Leu
Gly Thr Ile Met Ile Cys Val Pro Thr Trp195 200 205Ala Lys Glu Ser
Ala Pro Tyr Met Cys Arg Val Lys Thr Leu Pro Asp210 215 220Arg Thr
Trp Thr Tyr Ser Phe Ser Gly Ala Phe Leu Phe Ser Met Gly225 230 235
240Phe Leu Val Ala Val Leu Cys Tyr Leu Ser Tyr Arg Tyr Val Thr
Lys245 250 255Pro Pro Ala Pro Pro Asn Ser Leu Asn Val Gln Arg Val
Leu Thr Phe260 265 270Gln Pro Leu Arg Phe Ile Gln Glu His Val Leu
Ile Pro Val Phe Asp275 280 285Leu Ser Gly Pro Ser Ser Leu Ala Gln
Pro Val Gln Tyr Ser Gln Ile290 295 300Arg Val Ser Gly Pro Arg Glu
Pro Ala Gly Ala Pro Gln Arg His Ser305 310 315 320Leu Ser Glu Ile
Thr Tyr Leu Gly Gln Pro Asp Ile Ser Ile Leu Gln325 330 335Pro Ser
Asn Val Pro Pro Pro Gln Ile Leu Ser Pro Leu Ser Tyr Ala340 345
350Pro Asn Ala Ala Pro Glu Val Gly Pro Pro Ser Tyr Ala Pro Gln
Val355 360 365Thr Pro Glu Ala Gln Phe Pro Phe Tyr Ala Pro Gln Ala
Ile Ser Lys370 375 380Val Gln Pro Ser Ser Tyr Ala Pro Gln Ala Thr
Pro Asp Ser Trp Pro385 390 395 400Pro Ser Tyr Gly Val Cys Met Glu
Gly Ser Gly Lys Asp Ser Pro Thr405 410 415Gly Thr Leu Ser Ser Pro
Lys His Leu Arg Pro Lys Gly Gln Leu Gln420 425 430Lys Glu Pro Pro
Ala Gly Ser Cys Met Leu Gly Gly Leu Ser Leu Gln435 440 445Glu Val
Thr Ser Leu Ala Met Glu Glu Ser Gln Glu Ala Lys Ser Leu450 455
460His Gln Pro Leu Gly Ile Cys Thr Asp Arg Thr Ser Asp Pro Asn
Val465 470 475 480Leu His Ser Gly Glu Glu Gly Thr Pro Gln Tyr Leu
Lys Gly Gln Leu485 490 495Pro Leu Leu Ser Ser Val Gln Ile Glu Gly
His Pro Met Ser Leu Pro500 505 510Leu Gln Pro Pro Ser Gly Pro Cys
Ser Pro Ser Asp Gln Gly Pro Ser515 520 525Pro Trp Gly Leu Leu Glu
Ser Leu Val Cys Pro Lys Asp Glu Ala Lys530 535 540Ser Pro Ala Pro
Glu Thr Ser Asp Leu Glu Gln Pro Thr Glu Leu Asp545 550 555 560Ser
Leu Phe Arg Gly Leu Ala Leu Thr Val Gln Trp Glu Ser565
5702639DNAArtificial SequenceOligonucleotide linker ZC13252
26ggcctgaaag cttcggataa tgaaggtacc tgttagaaa 392727DNAArtificial
SequenceOligonucleotide linker ZC13453 27ttaggatccg gcccttcccc
agatact 272836DNAArtificial SequenceOligonucleotide primer ZC28590
28ttgggtacct ctgcaatggc cgccctgcag aaatct 362933DNAArtificial
SequenceOligonucleotide primer ZC28580 29ttgggatcca atgcaggcat
ttctcagaga cat 333023DNAArtificial SequenceOligonucleotide primer
ZC25963 30agtcaacgca tgagtctctg aag 233123DNAArtificial
SequenceOligonucleotide primer ZC25964 31gttcttgagt accccaacag tct
233218DNAArtificial SequenceOligonucleotide primer ZC14666
32agccaccaag atgactga 183322DNAArtificial SequenceOligonucleotide
primer ZC14742 33tgcatttggt aggtgcggtt ga 2234211PRTHomo sapiens
34Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser1
5 10 15Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr
Pro20 25 30Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg
Asp Trp35 40 45Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser
Cys Asn Leu50 55 60Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr
Ala Arg Val Thr65 70 75 80Ala Val Ser Ala Gly Gly Arg Ser Ala Thr
Lys Met Thr Asp Arg Phe85 90 95Ser Ser Leu Gln His Thr Thr Leu Lys
Pro Pro Asp Val Thr Cys Ile100 105 110Ser Lys Val Arg Ser Ile Gln
Met Ile Val His Pro Thr Pro Thr Pro115 120 125Ile Arg Ala Gly Asp
Gly His Arg Leu Thr Leu Glu Asp Ile Phe His130 135 140Asp Leu Phe
Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln Met145 150 155
160His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr
Pro165 170 175Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val Pro
Thr Trp Ala180 185 190Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys
Thr Leu Pro Asp Arg195 200 205Thr Trp Thr21035199PRTHomo sapiens
35Met Val Pro Pro Pro Glu Asn Val Arg Met Asn Ser Val Asn Phe Lys1
5 10 15Asn Ile Leu Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly Asn Leu
Thr20 25 30Phe Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp Lys
Cys Met35 40 45Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu Ser
Lys Tyr Gly50 55 60Asp His Thr Leu Arg Val Arg Ala Glu Phe Ala Asp
Glu His Ser Asp65 70 75 80Trp Val Asn Ile Thr Phe Cys Pro Val Asp
Asp Thr Ile Ile Gly Pro85 90 95Pro Gly Met Gln Val Glu Val Leu Ala
Asp Ser Leu His Met Arg Phe100 105 110Leu Ala Pro Lys Ile Glu Asn
Glu Tyr Glu Thr Trp Thr Met Lys Asn115 120 125Val Tyr Asn Ser Trp
Thr Tyr Asn Val Gln Tyr Trp Lys Asn Gly Thr130 135 140Asp Glu Lys
Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu Val Leu Arg145 150 155
160Asn Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg Gly Phe
Leu165 170 175Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro Val
Cys Glu Gln180 185 190Thr Thr His Asp Glu Thr Val19536211PRTHomo
sapiens 36Ser Asp Ala His Gly Thr Glu Leu Pro Ser Pro Pro Ser Val
Trp Phe1 5 10 15Glu Ala Glu Phe Phe His His Ile Leu His Trp Thr Pro
Ile Pro Asn20 25 30Gln Ser Glu Ser Thr Cys Tyr Glu Val Ala Leu Leu
Arg Tyr Gly Ile35 40 45Glu Ser Trp Asn Ser Ile Ser Asn Cys Ser Gln
Thr Leu Ser Tyr Asp50 55 60Leu Thr Ala Val Thr Leu Asp Leu Tyr His
Ser Asn Gly Tyr Arg Ala65 70 75 80Arg Val Arg Ala Val Asp Gly Ser
Arg His Ser Asn Trp Thr Val Thr85 90 95Asn Thr Arg Phe Ser Val Asp
Glu Val Thr Leu Thr Val Gly Ser Val100 105 110Asn Leu Glu Ile His
Asn Gly Phe Ile Leu Gly Lys Ile Gln Leu Pro115 120 125Arg Pro Lys
Met Ala Pro Ala Asn Asp Thr Tyr Glu Ser Ile Phe Ser130 135 140His
Phe Arg Glu Tyr Glu Ile Ala Ile Arg Lys Val Pro Gly Asn Phe145 150
155 160Thr Phe Thr His Lys Lys Val Lys His Glu Asn Phe Ser Leu Leu
Thr165 170 175Ser Gly Glu Val Gly Glu Phe Cys Val Gln Val Lys Pro
Ser Val Ala180 185 190Ser Arg Ser Asn Lys Gly Met Trp Ser Lys Glu
Glu Cys Ile Ser Leu195 200 205Thr Arg Gln210371618DNAHomo
sapiensCDS(237)...(932) 37agtttcttca tctgtaacat caaatgaata
ataataccaa tctcctagac ttcataagag 60gattaacaaa gacaaaatat gggaaaaaca
taacatggtg tcccataatt attagatctt 120attattgaca ctaaaatggc
attaaaatta ccaaaaggaa gacagcatct gtttcctctt 180tggtcctgag
ctggttaaaa ggaacactgg ttgcctgaac agtcacactt gcaacc atg 239Met1atg
cct aaa cat tgc ttt cta ggc ttc ctc atc agt ttc ttc ctt act 287Met
Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu Thr5 10
15ggt gta gca gga act cag tca acg cat gag tct ctg aag cct cag agg
335Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
Arg20 25 30gta caa ttt cag tcc cga aat ttt cac aac att ttg caa tgg
cag cct 383Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp
Gln Pro35 40 45ggg agg gca ctt act ggc aac agc agt gtc tat ttt gtg
cag tac aaa 431Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val
Gln Tyr Lys50 55 60 65ata tat gga cag aga caa tgg aaa aat aaa gaa
gac tgt tgg ggt act 479Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu
Asp Cys Trp Gly Thr70 75 80caa gaa ctc tct tgt gac ctt acc agt gaa
acc tca gac ata cag gaa 527Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu
Thr Ser Asp Ile Gln Glu85 90 95cct tat tac ggg agg gtg agg gcg gcc
tcg gct ggg agc tac tca gaa 575Pro Tyr Tyr Gly Arg Val Arg Ala Ala
Ser Ala Gly Ser Tyr Ser Glu100 105 110tgg agc atg acg ccg cgg ttc
act ccc tgg tgg gaa aca aaa ata gat 623Trp Ser Met Thr Pro Arg Phe
Thr Pro Trp Trp Glu Thr Lys Ile Asp115 120 125cct cca gtc atg aat
ata acc caa gtc aat ggc tct ttg ttg gta att 671Pro Pro Val Met Asn
Ile Thr Gln Val Asn Gly Ser Leu Leu Val Ile130 135 140 145ctc cat
gct cca aat tta cca tat aga tac caa aag gaa aaa aat gta 719Leu His
Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn Val150 155
160tct ata gaa gat tac tat gaa cta cta tac cga gtt ttt ata att aac
767Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile
Asn165 170 175aat tca cta gaa aag gag caa aag gtt tat gaa ggg gct
cac aga gcg 815Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala
His Arg Ala180 185 190gtt gaa att gaa gct cta aca cca cac tcc agc
tac tgt gta gtg gct 863Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser
Tyr Cys Val Val Ala195 200 205gaa ata tat cag ccc atg tta gac aga
aga agt cag aga agt gaa gag 911Glu Ile Tyr Gln Pro Met Leu Asp Arg
Arg Ser Gln Arg Ser Glu Glu210 215 220 225aga tgt gtg gaa att cca
tga cttgtggaat ttggcattca gcaatgtgga 962Arg Cys Val Glu Ile Pro
*230aattctaaag ctccctgaga acaggatgac tcgtgtttga aggatcttat
ttaaaattgt
1022ttttgtattt tcttaaagca atattcactg ttacaccttg gggacttctt
tgtttatcca 1082ttcttttatc ctttatattt catttgtaaa ctatatttga
acgacattcc ccccgaaaaa 1142ttgaaatgta aagatgaggc agagaataaa
gtgttctatg aaattcagaa ctttatttct 1202gaatgtaaca tccctaataa
caaccttcat tcttctaata cagcaaaata aaaatttaac 1262aaccaaggaa
tagtatttaa gaaaatgttg aaataatttt tttaaaatag cattacagac
1322tgaggcggtc ctgaagcaat ggtttttcac tctcttattg agccaattaa
attgacattg 1382ctttgacaat ttaaaacttc tataaaggtg aatatttttc
atacatttct attttatatg 1442aatatacttt ttatatattt attattatta
aatatttcta cttaatgaat caaaattttg 1502ttttaaagtc tactttatgt
aaataagaac aggttttggg gaaaaaaatc ttatgatttc 1562tggattgata
tctgaattaa aactatcaac aacaaggaaa aaaaaaaaaa aaaaaa 161838231PRTHomo
sapiens 38Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe
Phe Leu1 5 10 15Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu
Lys Pro Gln20 25 30Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile
Leu Gln Trp Gln35 40 45Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val
Tyr Phe Val Gln Tyr50 55 60Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn
Lys Glu Asp Cys Trp Gly65 70 75 80Thr Gln Glu Leu Ser Cys Asp Leu
Thr Ser Glu Thr Ser Asp Ile Gln85 90 95Glu Pro Tyr Tyr Gly Arg Val
Arg Ala Ala Ser Ala Gly Ser Tyr Ser100 105 110Glu Trp Ser Met Thr
Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile115 120 125Asp Pro Pro
Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val130 135 140Ile
Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn145 150
155 160Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile
Ile165 170 175Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly
Ala His Arg180 185 190Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser
Ser Tyr Cys Val Val195 200 205Ala Glu Ile Tyr Gln Pro Met Leu Asp
Arg Arg Ser Gln Arg Ser Glu210 215 220Glu Arg Cys Val Glu Ile
Pro225 230394PRTArtificial SequenceSXWS polypeptide motif 39Ser Xaa
Trp Ser14018DNAArtificial SequenceOligonucleotide primer ZC27713
40agctgccttc ttcacttg 184118DNAArtificial SequenceOligonucleotide
primer ZC27714 41ttgctctgcc tcttattc 18421050DNAMus
musculusCDS(5)...(589) 42aaca ggc tct cct ctc act tat caa ctt ttg
aca ctt gtg cga tcg gtg 49Gly Ser Pro Leu Thr Tyr Gln Leu Leu Thr
Leu Val Arg Ser Val1 5 10 15atg gct gtc ctg cag aaa tct atg agt ttt
tcc ctt atg ggg act ttg 97Met Ala Val Leu Gln Lys Ser Met Ser Phe
Ser Leu Met Gly Thr Leu20 25 30gcc gcc agc tgc ctg ctt ctc att gcc
ctg tgg gcc cag gag gca aat 145Ala Ala Ser Cys Leu Leu Leu Ile Ala
Leu Trp Ala Gln Glu Ala Asn35 40 45gcg ctg ccc atc aac acc cgg tgc
aag ctt gag gtg tcc aac ttc cag 193Ala Leu Pro Ile Asn Thr Arg Cys
Lys Leu Glu Val Ser Asn Phe Gln50 55 60cag ccg tac atc gtc aac cgc
acc ttt atg ctg gcc aag gag gcc agc 241Gln Pro Tyr Ile Val Asn Arg
Thr Phe Met Leu Ala Lys Glu Ala Ser65 70 75ctt gca gat aac aac aca
gac gtc cgg ctc atc ggg gag aaa ctg ttc 289Leu Ala Asp Asn Asn Thr
Asp Val Arg Leu Ile Gly Glu Lys Leu Phe80 85 90 95cga gga gtc agt
gct aag gat cag tgc tac ctg atg aag cag gtg ctc 337Arg Gly Val Ser
Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val Leu100 105 110aac ttc
acc ctg gaa gac att ctg ctc ccc cag tca gac agg ttc cgg 385Asn Phe
Thr Leu Glu Asp Ile Leu Leu Pro Gln Ser Asp Arg Phe Arg115 120
125ccc tac atg cag gag gtg gtg cct ttc ctg acc aaa ctc agc aat cag
433Pro Tyr Met Gln Glu Val Val Pro Phe Leu Thr Lys Leu Ser Asn
Gln130 135 140ctc agc tcc tgt cac atc agt ggt gac gac cag aac atc
cag aag aat 481Leu Ser Ser Cys His Ile Ser Gly Asp Asp Gln Asn Ile
Gln Lys Asn145 150 155gtc aga agg ctg aag gag aca gtg aaa aag ctt
gga gag agc gga gag 529Val Arg Arg Leu Lys Glu Thr Val Lys Lys Leu
Gly Glu Ser Gly Glu160 165 170 175atc aaa gcg atc ggg gaa ctg gac
ctg ctg ttt atg tct ctg aga aat 577Ile Lys Ala Ile Gly Glu Leu Asp
Leu Leu Phe Met Ser Leu Arg Asn180 185 190gct tgc gtc tga
gcgagaagaa gctagaaaac gaagaactgc tccttcctgc 629Ala Cys Val
*cttctaaaaa gaacaataag atccctgaat ggactttttt actaaaggaa agtgagaagc
689taacgtccac catcattaga agatttcaca tgaaacctgg ctcagttgaa
agagaaaata 749gtgtcaagtt gtccatgaga ccagaggtag acttgataac
cacaaagatt cattgacaat 809attttattgt cattgataat gcaacagaaa
aagtatgtac tttaaaaaat tgtttgaaag 869gaggttacct ctcattcctc
tagaagaaaa gcctatgtaa cttcatttcc ataaccaata 929ctttatatat
gtaagtttat ttattataag tatacatttt atttatgtca gtttattaat
989atggatttat ttatagaaaa attatctgat gttgatattt gagtataaag
caaataatat 1049t 105043194PRTMus musculus 43Gly Ser Pro Leu Thr Tyr
Gln Leu Leu Thr Leu Val Arg Ser Val Met1 5 10 15Ala Val Leu Gln Lys
Ser Met Ser Phe Ser Leu Met Gly Thr Leu Ala20 25 30Ala Ser Cys Leu
Leu Leu Ile Ala Leu Trp Ala Gln Glu Ala Asn Ala35 40 45Leu Pro Ile
Asn Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln Gln50 55 60Pro Tyr
Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser Leu65 70 75
80Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe Arg85
90 95Gly Val Ser Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val Leu
Asn100 105 110Phe Thr Leu Glu Asp Ile Leu Leu Pro Gln Ser Asp Arg
Phe Arg Pro115 120 125Tyr Met Gln Glu Val Val Pro Phe Leu Thr Lys
Leu Ser Asn Gln Leu130 135 140Ser Ser Cys His Ile Ser Gly Asp Asp
Gln Asn Ile Gln Lys Asn Val145 150 155 160Arg Arg Leu Lys Glu Thr
Val Lys Lys Leu Gly Glu Ser Gly Glu Ile165 170 175Lys Ala Ile Gly
Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn Ala180 185 190Cys
Val4420DNAArtificial SequenceOligonucleotide primer ZC39289
44tccgaggagt caatgctaag 204520DNAArtificial SequenceOligonucleotide
primer ZC39280 45tccaagcttt ttcactgtct 2046684DNAHomo Sapiens
46atgaggacgc tgctgaccat cttgactgtg ggatccctgg ctgctcacgc ccctgaggac
60ccctcggatc tgctccagca cgtgaaattc cagtccagca actttgaaaa catcctgacg
120tgggacagcg ggccagaggg caccccagac acggtctaca gcatcgagta
taagacgtac 180ggagagaggg actgggtggc aaagaagggc tgtcagcgga
tcacccggaa gtcctgcaac 240ctgacggtgg agacgggcaa cctcacggag
ctctactatg ccagggtcac cgctgtcagt 300gcgggaggcc ggtcagccac
caagatgact gacaggttca gctctctgca gcacactacc 360ctcaagccac
ctgatgtgac ctgtatctcc aaagtgagat cgattcagat gattgttcat
420cctaccccca cgccaatccg tgcaggcgat ggccaccggc taaccctgga
agacatcttc 480catgacctgt tctaccactt agagctccag gtcaaccgca
cctaccaaat gcaccttgga 540gggaagcaga gagaatatga gttcttcggc
ctgacccctg acacagagtt ccttggcacc 600atcatgattt gcgttcccac
ctgggccaag gagagtgccc cctacatgtg ccgagtgaag 660acactgccag
accggacatg gacc 68447660DNAHomo Sapiens 47atggcgtgga gtcttgggag
ctggctgggt ggctgcctgc tggtgtcagc attgggaatg 60gtaccacctc ccgaaaatgt
cagaatgaat tctgttaatt tcaagaacat tctacagtgg 120gagtcacctg
cttttgccaa agggaacctg actttcacag ctcagtacct aagttatagg
180atattccaag ataaatgcat gaatactacc ttgacggaat gtgatttctc
aagtctttcc 240aagtatggtg accacacctt gagagtcagg gctgaatttg
cagatgagca ttcagactgg 300gtaaacatca ccttctgtcc tgtggatgac
accattattg gaccccctgg aatgcaagta 360gaagtacttg atgattcttt
acatatgcgt ttcttagccc ctaaaattga gaatgaatac 420gaaacttgga
ctatgaagaa tgtgtataac tcatggactt ataatgtgca atactggaaa
480aacggtactg atgaaaagtt tcaaattact ccccagtatg actttgaggt
cctcagaaac 540ctggagccat ggacaactta ttgtgttcaa gttcgagggt
ttcttcctga tcggaacaaa 600gctggggaat ggagtgagcc tgtctgtgag
caaacaaccc atgacgaaac ggtcccctcc 660488PRTArtificial
SequenceGly-Ser spacer peptide 48Gly Ser Gly Ser Gly Ser Gly Ser1
54970DNAArtificial SequenceOligonucleotide Primer ZC39335
49atcggaattc gcagaagcca tgaggacgct gctgaccatc ttgactgtgg ggtccctggc
60tgctcacgcc 705031DNAArtificial SequenceOligonucleotide Primer
ZC39434 50cagtggatcc tggcagtgtc ttcactcggc a 315137DNAArtificial
SequenceOligonucleotide Primer ZC39319 51atcggaattc gcagaagcca
tggcgtggag ccttggg 375228DNAArtificial SequenceOligonucleotide
Primer ZC39325 52cagtggatcc ggaggggacc gtttcgtc 285316DNAArtificial
SequenceOligonucleotide Primer ZC39776 53gggcccgcta gcacct
165416DNAArtificial SequenceOligonucleotide Primer ZC39777
54gggtgatccg ctggca 165536DNAArtificial SequenceOligonucleotide
Primer ZC38752 55ccagccactt tctctctccg tatttcttat attcca
365639DNAArtificial SequenceOligonucleotide Primer ZC40940
56ttggtccctc gtggaagcac tcagtcaacg catgagtct 395739DNAArtificial
SequenceOligonucleotide Primer ZC40943 57atgcattcta gatcatggaa
tttccacaca tctctcttc 395857DNAArtificial SequenceOligonucleotide
Primer ZC40942 58atgcattccg gagattataa ggatgatgat gataagttgg
tccctcgtgg aagcact 575910PRTArtificial SequenceGlu-Glu (CEE) Tag
amino acid sequence with spacer 59Gly Ser Gly Gly Glu Tyr Met Pro
Met Glu1 5 106010PRTArtificial SequenceC-terminal HIS tag, with
spacer 60Gly Ser Gly Gly His His His His His His1 5
1061484PRTArtificial Sequencehuman zcytor11/Fc4-CEE fusion
polyeptide 61Met Arg Thr Leu Leu Thr Ile Leu Thr Val Gly Ser Leu
Ala Ala His1 5 10 15Ala Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val
Lys Phe Gln Ser20 25 30Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser
Gly Pro Glu Gly Thr35 40 45Pro Asp Thr Val Tyr Ser Ile Glu Tyr Lys
Thr Tyr Gly Glu Arg Asp50 55 60Trp Val Ala Lys Lys Gly Cys Gln Arg
Ile Thr Arg Lys Ser Cys Asn65 70 75 80Leu Thr Val Glu Thr Gly Asn
Leu Thr Glu Leu Tyr Tyr Ala Arg Val85 90 95Thr Ala Val Ser Ala Gly
Gly Arg Ser Ala Thr Lys Met Thr Asp Arg100 105 110Phe Ser Ser Leu
Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys115 120 125Ile Ser
Lys Val Arg Ser Ile Gln Met Ile Val His Pro Thr Pro Thr130 135
140Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile
Phe145 150 155 160His Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn
Arg Thr Tyr Gln165 170 175Met His Leu Gly Gly Lys Gln Arg Glu Tyr
Glu Phe Phe Gly Leu Thr180 185 190Pro Asp Thr Glu Phe Leu Gly Thr
Ile Met Ile Cys Val Pro Thr Trp195 200 205Ala Lys Glu Ser Ala Pro
Tyr Met Cys Arg Val Lys Thr Leu Pro Asp210 215 220Arg Thr Trp Thr
Gly Ser Gly Ser Gly Ser Gly Ser Glu Pro Arg Ser225 230 235 240Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu245 250
255Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu260 265 270Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser275 280 285His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu290 295 300Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr305 310 315 320Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn325 330 335Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser340 345 350Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln355 360
365Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val370 375 380Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val385 390 395 400Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro405 410 415Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr420 425 430Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val435 440 445Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu450 455 460Ser Pro
Gly Lys Leu Val Pro Arg Gly Ser Gly Ser Gly Gly Glu Tyr465 470 475
480Met Pro Met Glu62476PRTArtificial Sequencehuman CRF2-4/Fc4-CHIS
fusion polypeptide 62Met Ala Trp Ser Leu Gly Ser Trp Leu Gly Gly
Cys Leu Leu Val Ser1 5 10 15Ala Leu Gly Met Val Pro Pro Pro Glu Asn
Val Arg Met Asn Ser Val20 25 30Asn Phe Lys Asn Ile Leu Gln Trp Glu
Ser Pro Ala Phe Ala Lys Gly35 40 45Asn Leu Thr Phe Thr Ala Gln Tyr
Leu Ser Tyr Arg Ile Phe Gln Asp50 55 60Lys Cys Met Asn Thr Thr Leu
Thr Glu Cys Asp Phe Ser Ser Leu Ser65 70 75 80Lys Tyr Gly Asp His
Thr Leu Arg Val Arg Ala Glu Phe Ala Asp Glu85 90 95His Ser Asp Trp
Val Asn Ile Thr Phe Cys Pro Val Asp Asp Thr Ile100 105 110Ile Gly
Pro Pro Gly Met Gln Val Glu Val Leu Asp Asp Ser Leu His115 120
125Met Arg Phe Leu Ala Pro Lys Ile Glu Asn Glu Tyr Glu Thr Trp
Thr130 135 140Met Lys Asn Val Tyr Asn Ser Trp Thr Tyr Asn Val Gln
Tyr Trp Lys145 150 155 160Asn Gly Thr Asp Glu Lys Phe Gln Ile Thr
Pro Gln Tyr Asp Phe Glu165 170 175Val Leu Arg Asn Leu Glu Pro Trp
Thr Thr Tyr Cys Val Gln Val Arg180 185 190Gly Phe Leu Pro Asp Arg
Asn Lys Ala Gly Glu Trp Ser Glu Pro Val195 200 205Cys Glu Gln Thr
Thr His Asp Glu Thr Val Pro Ser Gly Ser Gly Ser210 215 220Gly Ser
Gly Ser Glu Pro Arg Ser Ser Asp Lys Thr His Thr Cys Pro225 230 235
240Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu
Phe245 250 255Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val260 265 270Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe275 280 285Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro290 295 300Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr305 310 315 320Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val325 330 335Ser Asn
Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala340 345
350Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg355 360 365Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly370 375 380Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro385 390 395 400Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser405 410 415Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln420 425 430Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His435 440 445Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Leu Val Pro Arg450 455
460Gly Ser Gly Ser Gly Gly His His His His His His465 470 475
* * * * *
References