U.S. patent application number 10/772985 was filed with the patent office on 2004-07-22 for mammalian cytokine-like factor 7.
Invention is credited to Gilbert, Teresa, Presnell, Scott R..
Application Number | 20040143096 10/772985 |
Document ID | / |
Family ID | 46276894 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040143096 |
Kind Code |
A1 |
Presnell, Scott R. ; et
al. |
July 22, 2004 |
Mammalian cytokine-like factor 7
Abstract
Novel mammalian zcyto7 polypeptides, polynucleotides encoding
the polypeptides, and related compositions and methods including
antibodies and anti-idiotypic antibodies.
Inventors: |
Presnell, Scott R.; (Tacoma,
WA) ; Gilbert, Teresa; (Seattle, WA) |
Correspondence
Address: |
Michelle L. Johnson
Patent Department
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Family ID: |
46276894 |
Appl. No.: |
10/772985 |
Filed: |
February 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10772985 |
Feb 5, 2004 |
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09633452 |
Aug 7, 2000 |
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09633452 |
Aug 7, 2000 |
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09066745 |
Apr 24, 1998 |
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60071676 |
Jan 16, 1998 |
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60044886 |
Apr 25, 1997 |
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Current U.S.
Class: |
530/351 |
Current CPC
Class: |
C07K 14/52 20130101;
C07K 2319/02 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
530/351 |
International
Class: |
C07K 014/52 |
Claims
1. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:12 or the amino acid sequence of SEQ ID NO:39.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/633,452, filed Aug. 7, 2000, which is a continuation of U.S.
application Ser. No. 09/066,745, filed Apr. 24, 1998, which claims
the benefit of U.S. Provisional Application Serial No. 60/071,676
filed Jan. 16, 1998; and U.S. Provisional Application Serial No.
60/044,886 filed Apr. 25, 1997.
BACKGROUND OF THE INVENTION
[0002] Proliferation and differentiation of cells of multicellular
organisms are controlled by hormones and polypeptide growth
factors. These diffusable molecules allow cells to communicate with
each other and act in concert to form cells and organs, and to
repair and regenerate damaged tissue. Examples of hormones and
growth factors include the steroid hormones (e.g. estrogen,
testosterone), parathyroid hormone, follicle stimulating hormone,
the interleukins, platelet derived growth factor (PDGF), epidermal
growth factor (EGF), granulocyte-macrophage colony stimulating
factor (GM-CSF), erythropoietin (EPO) and calcitonin.
[0003] Hormones and growth factors influence cellular metabolism by
binding to proteins. Proteins may be integral membrane proteins
that are linked to signaling pathways within the cell, such as
second messenger systems. Other classes of proteins are soluble
molecules, such as the transcription factors.
[0004] Of particular interest are cytokines, molecules that promote
the proliferation and/or differentiation of cells. Examples of
cytokines include erythropoietin (EPO), which stimulates the
development of red blood cells; thrombopoietin (TPO), which
stimulates development of cells of the megakaryocyte lineage; and
granulocyte-colony stimulating factor (G-CSF), which stimulates
development of neutrophils. These cytokines are useful in restoring
normal blood cell levels in patients suffering from anemia or
receiving chemotherapy for cancer. The demonstrated in vivo
activities of these cytokines illustrates the enormous clinical
potential of, and need for, other cytokines, cytokine agonists, and
cytokine antagonists.
SUMMARY OF THE INVENTION
[0005] The present invention addresses this need by providing a
novel polypeptide called cytokine-like factor 7,hereinafter
referred to as Zcyto7 and related compositions and methods.
[0006] Thus, one aspect of the present invention provides for an
isolated Zcyto7 polypeptide having amino acid sequences as follows.
Both the human and the mouse cDNAs have been discovered. The human
sequences are defined by SEQ ID NOs: 1 and 2. The murine nucleotide
and amino acid sequences are defined by SEQ ID NOs: 11 and 12.
[0007] The nucleotide sequence of SEQ ID NO:1 contains an open
reading frame encoding a polypeptide of about 180 amino acids with
the initial Met as shown in SEQ ID NO:1 and SEQ ID NO:2. A
predicted signal sequence is comprised of amino acid residues 1-20,
and the resultant predicted mature Zcyto7 polypeptide is
represented by the amino acid sequence extending from amino acid
residue 21, a glutamine to and including amino acid residue 180 a
phenylalanine, also represented by SEQ ID NO: 14. Peptide mapping
data indicate that mature Zcyto7 can be comprised of a number of
N-terminal mature variants including the amino acid sequence
extending from amino acid residue 23, an arginine to and including
amino acid residue 180 of SEQ ID NO: 2, also defined by SEQ ID NO:
36; amino acid sequence extending from amino acid residue 27, a
serine to and including amino acid residue 180 of SEQ ID NO: 2,
also defined by SEQ ID NO: 37; the amino acid sequence defined by
amino acid residue 30, a lysine, to and including amino acid
residue 180 of SEQ ID NO: 2, also defined by SEQ ID NO: 38; amino
acid sequence extending from amino acid 28, a lysine, to and
including amino acid residue 180 of SEQ ID NO:2, also defined by
SEQ ID NO: 41 and the amino acid sequence extending from amino acid
residue 53, a methionine, to and including amino acid residue 180,
also defined by SEQ ID NO: 42. The only observed cleavage at the
carboxyl terminus is the phenylalanine at position 180 can be
cleaved off. This can occur in all of the above-defined mature
Zcyto7 polypeptides an example of which is shown by SEQ ID NO:43.
Additional variants of human Zcyto7 are defined by SEQ ID NOs:
15-25. Within an additional embodiment, the polypeptide further
comprises an affinity tag.
[0008] SEQ ID NOs: 11 and 12 define murine Zcyto7 wherein the
mature protein extends from amino acid residues amino acid residue
21, a histidine, to and including amino acid residue 180 a
phenylalanine, also defined by SEQ ID NO: 39; or as an alternative
splice site from amino acid residue 23, an arginine, to and
including amino acid 180 also defined by SEQ ID NO: 40. The present
invention is also comprised of polypeptides having an amino acid
sequence at least 90% identical, more preferably 95%, 97% or 99%
identical to those Zcyto7 polypeptides defined in above.
[0009] An additional embodiment of the present invention relates to
a peptide or polypeptide which has the amino acid sequence of an
epitope-bearing portion of a Zcyto7 polypeptide having an amino
acid sequence described above. Peptides or polypeptides having the
amino acid sequence of an epitope-bearing portion of a Zcyto7
polypeptide of the present invention include portions of such
polypeptides with at least nine, preferably at least 15 and more
preferably at least 30 to 50 amino acids, although epitope-bearing
polypeptides of any length up to and including the entire amino
acid sequence of a polypeptide of the present invention described
above are also included in the present invention. Examples of said
polypeptides are defined by the amino acid sequences of SEQ ID NOs:
25-35. Also claimed are any of these polypeptides that are fused to
another polypeptide or carrier molecule.
[0010] The present invention further comprises a polypeptide
defined by SEQ ID NOs: 15-25 wherein the amino termini of said
polypeptides are modified and begin at either amino acid residue 3,
an arginine; amino acid residue 7, a serine; amino acid residue 8,
a lysine; amino acid residue 10, a lysine or amino acid residue 33
methionine.
[0011] The present invention is further comprised of a polypeptide
wherein the polypeptide is a polypeptide defined by SEQ ID NOs: 2,
12, 14-25 and 36 to 42 wherein the amino acid sequences end at the
isoleucines at amino acid residue 179 of SEQ ID NO: 2, at amino
acid residue 159 of SEQ ID NOs: 14-25, which corresponds to amino
acid residue 157 of SEQ ID NO:36, amino acid residue 153 of SEQ ID
NO:37, amino acid residue 150 of SEQ ID NO:38, amino acid residue
159 of SEQ ID NO: 39, amino acid residue 157 of SEQ ID NO:40, amino
acid residue 152 SEQ ID NO:42, amino acid residue 127 of SEQ ID
NO:42.
[0012] The present invention is further comprised of an isolated
peptide or polypeptide of the above-described peptides or
polypeptide having an amino acid sequence modified by addition,
deletion and/or replacement of one or more amino acid residues and
which maintains the biological activity of said peptide or
polypeptide.
[0013] Within a further aspect of the invention there is provided a
chimeric polypeptide consisting essentially of a first portion and
a second portion joined by a peptide bond. The first portion of the
chimeric polypeptide consists essentially of (a) a Zcyto7
polypeptide as described above (b) allelic variants of the
polypeptides described above. The second portion of the chimeric
polypeptide consists essentially of another polypeptide such as an
affinity tag. Within one embodiment the affinity tag is an
immunoglobulin F.sub.c polypeptide. The invention also provides
expression vectors encoding the chimeric polypeptides and host
cells transfected to produce the chimeric polypeptides.
[0014] Another aspect of the present invention provides for
isolated nucleic acid molecules comprising a polynucleotide
selected from the group consisting of: (a) a nucleotide sequence
encoding the Zcyto7 polypeptides described above; (b)a nucleotide
sequence encoding the polypeptides of SEQ ID NOs: 14-40 and (c) a
nucleotide sequence complementary to any of to any of the
nucleotide sequences in (a) or (b).
[0015] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably
95%, 97%, 98%, or 99% identical to any of the nucleotide sequences
in (a), (b) or (c) above, or a polynucleotide which hybridizes
under stringent hybridization conditions to a polynucleotide having
a nucleotide sequence of (a) (b) or (c) above. An additional
nucleic acid embodiment of the present invention relates to an
isolated nucleic acid molecule comprising an amino acid of an
epitope-bearing portion of a Zcyto7 polypeptide.
[0016] Within another aspect of the invention there is provided an
expression vector comprising (a) a transcription promoter; (b) a
DNA segment encoding a polypeptide described above, and (c) a
transcription terminator, wherein the promoter, DNA segment, and
terminator are operably linked.
[0017] Within a third aspect of the invention there is provided a
cultured eukaryotic cell into which has been introduced an
expression vector as disclosed above, wherein said cell expresses a
protein polypeptide encoded by the DNA segment.
[0018] In another embodiment of the present invention is an
isolated antibody that binds specifically to a Zcyto7 polypeptide
described above. Also claimed is a method for producing antibodies
which bind to a Zcyto7 polypeptide comprising inoculating a mammal
with a Zcyto7 polypeptide or Zcyto7 epitope-bearing polypeptide so
that the mammal produces antibodies to the polypeptide; and
isolating said antibodies.
[0019] These and other aspects of the invention will become evident
upon reference to the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The teachings of all of the references cited herein are
incorporated in their entirety herein by reference.
[0021] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A [Nilsson et al, EMBO J. 4:1075 (1985); Nilsson et al.,
Methods Enzymol. 198:3 (1991)], glutathione S transferase [Smith
and Johnson, Gene 67:31 (1988)], Glu-Glu affinity tag [Grussenmeyer
et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985)], substance P,
FLAG.TM. peptide (Hopp et al., Biotechnology 6:1204-10 (1988),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2: 95-107 (1991). DNAs encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0022] The term "allelic variant" denotes 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.
[0023] The term "expression vector" denotes a DNA molecule, linear
or circular, that comprises a segment encoding a polypeptide of
interest operably linked to additional segments that provide for
its transcription. Such additional segments may include promoter
and terminator sequences, and may optionally include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, and the like. Expression
vectors are generally derived from plasmid or viral DNA, or may
contain elements of both.
[0024] The term "isolated", when applied to a polynucleotide
molecule, denotes that the polynucleotide has been removed from its
natural genetic milieu and is thus free of other extraneous or
unwanted coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art. See for example, Dynan
and Tijan, Nature 316:774-78 (1985). When applied to a protein, the
term "isolated" indicates that the protein is found in a condition
other than its native environment, such as apart from blood and
animal tissue. In a preferred form, the isolated protein is
substantially free of other proteins, particularly other proteins
of animal origin. It is preferred to provide the protein in a
highly purified form, i.e., greater than 95% pure, more preferably
greater than 99% pure.
[0025] The term "operably linked", when referring to DNA segments,
denotes that the segments are arranged so that they function in
concert for their intended purposes, e.g. transcription initiates
in the promoter and proceeds through the coding segment to the
terminator
[0026] The term "polynucleotide" denotes a single- or
double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. Polynucleotides include RNA
and DNA, and may be isolated from natural sources, synthesized in
vitro, or prepared from a combination of natural and synthetic
molecules.
[0027] The term "complements of polynucleotide molecules" denotes
polynucleotide molecules having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to 5'
CCCGTGCAT 3'.
[0028] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0029] The term "promoter" denotes a portion of a gene containing
DNA sequences that provide for the binding of RNA polymerase and
initiation of transcription. Promoter sequences are commonly, but
not always, found in the 5' non-coding regions of genes.
[0030] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger peptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0031] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-domain structure comprising an
extracellular ligand-binding domain and an intracellular effector
domain that is typically involved in signal transduction. Binding
of ligand to receptor results in a conformational change in the
receptor that causes an interaction between the effector domain and
other molecule(s) in the cell. This interaction in turn leads to an
alteration in the metabolism of the cell. Metabolic events that are
linked to receptor-ligand interactions include gene transcription,
phosphorylation, dephosphorylation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. Most nuclear receptors also exhibit a
multi-domain structure, including an amino-terminal,
transactivating domain, a DNA binding domain and a ligand binding
domain. In general, receptors can be membrane bound, cytosolic or
nuclear; monomeric (e.g., thyroid stimulating hormone receptor,
beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,
growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF
receptor, erythropoietin receptor and IL-6 receptor).
[0032] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complem- ent pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0033] A "soluble protein" is a protein polypeptide that is not
bound to a cell membrane.
[0034] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of the DNA
of SEQ ID NO:1, or a sequence complementary thereto, under
stringent conditions. In general, stringent conditions are selected
to be about 5.degree. C. lower than the thermal melting point
(T.sub.m) for the specific sequence at a defined ionic strength and
pH. The T.sub.m is the temperature (under defined ionic strength
and pH) at which 50% of the target sequence hybridizes to a
perfectly matched probe. Typical stringent conditions are those in
which the salt concentration is about 0.02 M or less at pH 7 and
the temperature is at least about 60.degree. C. As previously
noted, the isolated polynucleotides of the present invention
include DNA and RNA. Methods for isolating DNA and RNA are well
known in the art. Total RNA can be prepared using guanidine HCl
extraction followed by isolation by centrifugation in a CsCl
gradient [Chirgwin et al., Biochemistry 18:52-94 (1979)]. Poly
(A).sup.+ RNA is prepared from total RNA using the method of Aviv
and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972).
Complementary DNA (cDNA) is prepared from poly(A).sup.+ RNA using
known methods. Polynucleotides encoding Zcyto7 polypeptides are
then identified and isolated by, for example, hybridization or
PCR.
[0035] Additionally, the polynucleotides of the present invention
can be synthesized using a DNA synthesizer. Currently the method of
choice is the phosphoramidite method. If chemically synthesized
double stranded DNA is required for an application such as the
synthesis of a gene or a gene fragment, then each complementary
strand is made separately. The production of short genes (60 to 80
bp) is technically straightforward and can be accomplished by
synthesizing the complementary strands and then annealing them. For
the production of longer genes (>300 bp), however, special
strategies must be invoked, 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. In addition to the protein coding sequence,
synthetic genes can be designed with terminal sequences that
facilitate insertion into a restriction endonuclease sites of a
cloning vector and other sequences should also be added that
contain signals for the proper initiation and termination of
transcription and translation.
[0036] See Glick, Bernard R. and Jack J. Pasternak, Molecular
Biotechnology, Principles & Applications of Recombinant
DNA,(ASM Press, Washington, D.C. 1994), Itakura, K. et al.
Synthesis and use of synthetic oligonucleotides. Annu. Rev.
Biochem. 53: 323-356 (1984), and Climie, S. et al. Chemical
synthesis of the thymidylate synthase gene. Proc. Natl. Acad. Sci.
USA 87:633-637 (1990).
[0037] Those skilled in the art will recognize that the sequences
disclosed in SEQ ID NOS:1 and 2 represent a single allele of the
human. There are a number of naturally occurring mature N-terminal
variants having the leader sequence cleaved at differing positions.
They include the sequences defined by SEQ ID NOs 14, 36, 37 and 38.
Allelic variants of these sequences can be cloned by probing cDNA
or genomic libraries from different individuals according to
standard procedures. Examples of variants of human Zcyto7 are
represented by the polypeptides of SEQ ID NOs: 15-25.
[0038] The murine Zcyto7 cDNA and protein are disclosed by SEQ ID
NOs: 11 and 12. The mature Zcyto7 polypeptide is defined by SEQ ID
NOs: 39 and 40.
[0039] The present invention further provides counterpart proteins
and polynucleotides from other species ("species orthologs"). Of
particular interest are Zcyto7 polypeptides from other mammalian
species, including murine, porcine, ovine, bovine, canine, feline,
equine, and other primates. Species orthologs of the human Zcyto7
protein can be cloned using information and compositions provided
by the present invention in combination with conventional cloning
techniques. For example, a cDNA can be cloned using mRNA obtained
from a tissue or cell type that expresses the protein. Suitable
sources of mRNA can be identified by probing Northern blots with
probes designed from the sequences disclosed herein. A library is
then prepared from mRNA of a positive tissue or cell line. A
protein-encoding cDNA can then be isolated by a variety of methods,
such as by probing with a complete or partial human or mouse cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using the polymerase
chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using
primers designed from the sequences disclosed herein. Within an
additional method, the cDNA library can be used to transform or
transfect host cells, and expression of the cDNA of interest can be
detected with an antibody to the protein. Similar techniques can
also be applied to the isolation of genomic clones. As used and
claimed the language "an isolated polynucleotide which encodes a
polypeptide, said polynucleotide being defined by SEQ ID NO: 2"
includes all allelic variants and species orthologs of the
polypeptide of SEQ ID NO:2.
[0040] The present invention also provides isolated protein
polypeptides that are substantially homologous to the protein
polypeptides of SEQ ID NO: 2 and its species orthologs. By
"isolated" is meant a protein or polypeptide that is found in a
condition other than its native environment, such as apart from
blood and animal tissue. In a preferred form, the isolated
polypeptide is substantially free of other polypeptides,
particularly other polypeptides of animal origin. It is preferred
to provide the polypeptides in a highly purified form, i.e. greater
than 95% pure, more preferably greater than 99% pure. The term
"substantially homologous" is used herein to denote polypeptides
having 50%, preferably 60%, more preferably at least 80%, sequence
identity to the sequence shown in SEQ ID NO:2,or its species
orthologs. Such polypeptides will more preferably be at least 90%
identical, and most preferably 95% or more identical to SEQ ID
NO:2,or its species orthologs. Percent sequence identity is
determined by conventional methods. See, for example, Altschul et
al., Bull. Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992). Briefly, two
amino acid sequences are aligned to optimize the alignment scores
using a gap opening penalty of 10, a gap extension penalty of 1,
and the "blossom 62" scoring matrix of Henikoff and Henikoff
(ibid.) as shown in Table 2 (amino acids are indicated by the
standard one-letter codes). The percent identity is then calculated
as: 1 Total number of identical matches [ length of the longer
sequence plus the number of gaps introduced into the longer
sequence in order to align the two sequences ] .times. 100
1TABLE 2 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
[0041] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0042] Substantially homologous proteins and polypeptides are
characterized as having one or more amino acid substitutions,
deletions or additions. These changes are preferably of a minor
nature, that is conservative amino acid substitutions (see Table 3)
and other substitutions that do not significantly affect the
folding or activity of the protein or polypeptide; small deletions,
typically of one to about 30 amino acids; and small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue, a small linker peptide of up to about 20-25 residues, or a
small extension that facilitates purification (an affinity tag),
such as 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)],
or other antigenic epitope or binding domain. See, in general Ford
et al., Protein Expression and Purification 2: 95-107 (1991. DNAs
encoding affinity tags are available from commercial suppliers
(e.g., Pharmacia Biotech, Piscataway, N.J.).
2TABLE 3 Conservative amino acid substitutions Basic: arginine
lysine histidine Acidic: glutamic acid aspartic acid Polar:
glutamine asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine
serine threonine methionine
[0043] 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: 1081-1085 (1989);
Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502 (1991)]. In
the latter technique, single alanine mutations are introduced at
every residue in the molecule, and the resultant mutant molecules
are tested for biological activity (e.g., ligand binding and signal
transduction) to identify amino acid residues that are critical to
the activity of the molecule. Sites of ligand-protein interaction
can also be determined by analysis of crystal structure as
determined by such techniques as nuclear magnetic resonance,
crystallography or photoaffinity labeling. See, for example, de Vos
et al., Science 255:306-312 (1992); Smith et al., J. Mol. Biol.
224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64 (1992).
The identities of essential amino acids can also be inferred from
analysis of homologies with related proteins.
[0044] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer, Science 241:53-57 (1988) or
Bowie and Sauer, Proc. Natl. Acad. Sci. USA 86:2152-2156 (1989).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display, e.g., Lowman et al., Biochem. 30:10832-10837 (1991);
Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO
92/06204) and region-directed mutagenesis, Derbyshire et al., Gene
46:145 (1986); Ner et al., DNA 7:127 (1988).
[0045] Mutagenesis methods as disclosed above can be combined with
high-throughput screening methods to detect activity of cloned,
mutagenized proteins in host cells. Preferred assays in this regard
include cell proliferation assays and biosensor-based
ligand-binding assays, which are described below. Mutagenized DNA
molecules that encode active proteins or portions thereof (e.g.,
ligand-binding fragments) 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.
[0046] Using the methods discussed above, one of ordinary skill in
the art can prepare a variety of polypeptides that are
substantially homologous to SEQ ID NO:2 or allelic variants thereof
and retain the properties of the wild-type protein. As expressed
and claimed herein the language, "a polypeptide as defined by SEQ
ID NO: 2" includes all allelic variants and species orthologs of
the polypeptide.
[0047] Another embodiment of the present invention provides for a
peptide or polypeptide comprising an epitope-bearing portion of a
polypeptide of the invention. The epitope of the this polypeptide
portion is an immunogenic or antigenic epitope of a polypeptide of
the invention. A region of a protein to which an antibody can bind
is defined as an "antigenic epitope". See for instance, Geysen, H.
M. et al., Proc. Natl. Acad Sci. USA 81:3998-4002 (1984).
[0048] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in the
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See Sutcliffe, J.
G. et al. Science 219:660-666 (1983). Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals. Peptides that are extremely hydrophobic and
those of six or fewer residues generally are ineffective at
inducing antibodies that bind to the mimicked protein; longer
soluble peptides, especially those containing proline residues,
usually are effective.
[0049] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. Antigenic epitope-bearing peptides and polypeptides
of the present invention contain a sequence of at least nine,
preferably between 15 to about 30 amino acids contained within the
amino acid sequence of a polypeptide of the invention. 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 react with the protein. Preferably, 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 and hydrophobic
residues are preferably avoided); and sequences containing proline
residues are particularly preferred. All of the polypeptides shown
in the sequence listing contain antigenic epitopes to be used
according to the present invention, however, specifically designed
antigenic epitopes include the peptides defined by SEQ ID NOs:
27-35.
[0050] Polynucleotides, generally a cDNA sequence, of the present
invention encode the above-described polypeptides. A cDNA sequence
which encodes a polypeptide of the present invention is comprised
of a series of codons, each amino acid residue of the polypeptide
being encoded by a codon and each codon being comprised of three
nucleotides. The amino acid residues are encoded by their
respective codons as follows.
[0051] Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;
[0052] Cysteine (Cys) is encoded by TGC or TGT;
[0053] Aspartic acid (Asp) is encoded by GAC or GAT;
[0054] Glutamic acid (Glu) is encoded by GAA or GAG;
[0055] Phenylalanine (Phe) is encoded by TTC or TTT;
[0056] Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;
[0057] Histidine (His) is encoded by CAC or CAT;
[0058] Isoleucine (Ile) is encoded by ATA, ATC or ATT;
[0059] Lysine (Lys) is encoded by AAA, or AAG;
[0060] Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or
CTT;
[0061] Methionine (Met) is encoded by ATG;
[0062] Asparagine (Asn) is encoded by AAC or AAT;
[0063] Proline (Pro) is encoded by CCA, CCC, CCG or CCT;
[0064] Glutamine (Gln) is encoded by CAA or CAG;
[0065] Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or
CGT;
[0066] Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or
TCT;
[0067] Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;
[0068] Valine (Val) is encoded by GTA, GTC, GTG or GTT;
[0069] Tryptophan (Trp) is encoded by TGG; and
[0070] Tyrosine (Tyr) is encoded by TAC or TAT.
[0071] It is to be recognized that according to the present
invention, when a cDNA is claimed as described above, it is
understood that what is claimed are both the sense strand, the
anti-sense strand, and the DNA as double-stranded having both the
sense and anti-sense strand annealed together by their respective
hydrogen bonds. Also claimed is the messenger RNA (mRNA) which
encodes the polypeptides of the present invention, and which mRNA
is encoded by the above-described cDNA. A messenger RNA (mRNA) will
encode a polypeptide using the same codons as those defined above,
with the exception that each thymine nucleotide (T) is replaced by
a uracil nucleotide (U).
[0072] The protein polypeptides of the present invention, including
full-length proteins, protein fragments (e.g. receptor-binding
fragments), and fusion polypeptides can be produced in genetically
engineered host cells according to conventional techniques.
Suitable host cells are those cell types that can be transformed or
transfected with exogenous DNA and grown in culture, and include
bacteria, fungal cells, and cultured higher eukaryotic cells.
Eukaryotic cells, particularly cultured cells of multicellular
organisms, are preferred. Techniques for manipulating cloned DNA
molecules and introducing exogenous DNA into a variety of host
cells are disclosed by Sambrook et al., Molecular Cloning: A
Laboratory Manual, (2nd ed.) (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989), and Ausubel et al., ibid.
[0073] In general, a DNA sequence encoding a Zcyto7 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.
[0074] To direct a Zcyto7 polypeptide into the secretory pathway of
a host cell, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) is provided in the
expression vector. The secretory signal sequence may be that of the
protein, or may be derived from another secreted protein (e.g.,
t-PA) or synthesized de novo. The secretory signal sequence is
joined to the Zcyto7 DNA sequence in the correct reading frame.
Secretory signal sequences are commonly positioned 5' to the DNA
sequence encoding the polypeptide of interest, although certain
signal sequences may be positioned elsewhere in the DNA sequence of
interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland
et al., U.S. Pat. No. 5,143,830).
[0075] Cultured mammalian cells are preferred hosts within the
present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection, Wigler et al., Cell 14:725 (1978); Corsaro and
Pearson, Somatic Cell Genetics 7:603 (1981): Graham and Van der Eb,
Virology 52:456 (1973), electroporation, Neumann et al., EMBO J.
1:841-845 (1982), DEAE-dextran mediated transfection, Ausubel et
al., eds., Current Protocols in Molecular Biology (John Wiley and
Sons, Inc., NY, 1987), and liposome-mediated transfection
(Hawley-Nelson et al., Focus 15:73 (1993); Ciccarone et al., Focus
15:80 (1993). The production of recombinant polypeptides in
cultured mammalian cells is disclosed, for example, by Levinson et
al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No.
4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold,
U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include
the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC
No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 [ATCC No. CRL
1573; Graham et al., J. Gen. Virol. 36:59-72 (1977)] and Chinese
hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional
suitable cell lines are known in the art and available from public
depositories such as the American Type Culture Collection,
Rockville, Md. In general, strong transcription promoters are
preferred, such as promoters from SV-40 or cytomegalovirus. See,
e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include
those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and
4,601,978) and the adenovirus major late promoter.
[0076] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems may also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g. hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
[0077] Other higher eukaryotic cells can also be used as hosts,
including insect cells, plant cells and avian cells. Transformation
of insect cells and production of foreign polypeptides therein is
disclosed by Guarino et al., U.S. Pat. No. 5,162,222; Bang et al.,
U.S. Pat. No. 4,775,624; and WIPO publication WO 94/06463. The use
of Agrobacterium rhizogenes as a vector for expressing genes in
plant cells has been reviewed by Sinkar et al., J. Biosci.
(Bangalore) 11:47-58 (1987).
[0078] Fungal cells, including yeast cells, and particularly cells
of the genus Saccharomyces, can also be used within the present
invention, such as for producing protein fragments or polypeptide
fusions. Methods for transforming yeast cells with exogenous DNA
and producing recombinant polypeptides therefrom are disclosed by,
for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al.,
U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et
al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No.
4,845,075. Transformed cells are selected by phenotype determined
by the selectable marker, commonly drug resistance or the ability
to grow in the absence of a particular nutrient (e.g., leucine). A
preferred vector system for use in yeast is the POT1 vector system
disclosed by Kawasaki et al., U.S. Pat. No. 4,931,373, which allows
transformed cells to be selected by growth in glucose-containing
media. Suitable promoters and terminators for use in yeast include
those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat.
No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and
Bitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes.
See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and
4,661,454. Transformation systems for other yeasts, including
Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces
lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris,
Pichia methanolica, Pichia guillermondii and Candida maltosa are
known in the art. See, for example, Gleeson et al., J. Gen.
Microbiol. 132:3459-3465 (1986) and Cregg, U.S. Pat. No. 4,882,279.
Aspergillus cells may be utilized according to the methods of
McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming
Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat.
No. 5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0079] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell.
[0080] Within one aspect of the present invention, a novel protein
is produced by a cultured cell, and the cell is used to screen for
a receptor or receptors for the protein, including the natural
receptor, as well as agonists and antagonists of the natural
ligand.
[0081] Protein Isolation:
[0082] Expressed recombinant polypeptides (or chimeric
polypeptides) can be purified using fractionation and/or
conventional purification methods and media. Ammonium sulfate
precipitation and acid or chaotrope extraction may be used for
fractionation of samples. Exemplary purification steps may include
hydroxyapatite, size exclusion, FPLC and reverse-phase high
performance liquid chromatography. Suitable anion exchange media
include derivatized dextrans, agarose, cellulose, polyacrylamide,
specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives
are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,
Piscataway, N.J.) being particularly preferred. Exemplary
chromatographic media include those media derivatized with phenyl,
butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),
Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.),
Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins,
such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid
supports include glass beads, silica-based resins, cellulosic
resins, agarose beads, cross-linked agarose beads, polystyrene
beads, cross-linked polyacrylamide resins and the like that are
insoluble under the conditions in which they are to be used. These
supports may be modified with reactive groups that allow attachment
of proteins by amino groups, carboxyl groups, sulfhydryl groups,
hydroxyl groups and/or carbohydrate moieties. Examples of coupling
chemistries include cyanogen bromide activation,
N-hydroxysuccinimide activation, epoxide activation, sulfhydryl
activation, hydrazide activation, and carboxyl and amino
derivatives for carboduimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Methods for binding receptor
polypeptides to support media are well known in the art. Selection
of a particular method is a matter of routine design and is
determined in part by the properties of the chosen support. See,
for example, Affinity Chromatography: Principles & Methods
(Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988).
[0083] The polypeptides of the present invention can be isolated by
exploitation of their properties. For example, immobilized metal
ion adsorption (IMAC) chromatography can be used to purify
histidine-rich proteins. Briefly, a gel is first charged with
divalent metal ions to form a chelate [E. Sulkowski, Trends in
Biochem. 3:1-7 (1985)]. Histidine-rich proteins will be adsorbed to
this matrix with differing affinities, depending upon the metal ion
used, and will be eluted by competitive elution, lowering the pH,
or use of strong chelating agents. Other methods of purification
include purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography [Methods in
Enzymol., Vol. 182:529-39, "Guide to Protein Purification", M.
Deutscher, (ed.), (Acad. Press, San Diego, 1990). Alternatively, a
fusion of the polypeptide of interest and an affinity tag (e.g.,
polyhistidine, maltose-binding protein, an immunoglobulin domain)
may be constructed to facilitate purification. Furthermore, to
facilitate purification of the secreted receptor polypeptide, an
amino or carboxyl-terminal extension, such as a poly-histidine tag,
substance P, FLAG.RTM. peptide [Hopp et al., Bio/Technology
6:1204-1210 (1988); available from Eastman Kodak Co., New Haven,
Conn.), a Glu-Glu affinity tag [Grussenmeyer et al., Proc. Natl.
Acad. Sci. USA 82:7952-4 (1985)], or another polypeptide or protein
for which an antibody or other specific binding agent is available,
can be fused to Zyto7 to aid in purification.
[0084] Uses
[0085] Northern blot analysis of the expression of Zcyto7 reveals
that Zcyto7 is specifically expressed in the spinal cord. In situ
analysis of the spinal cord reveals that this expression is
localized in the neurons and dorsal root ganglia. Therefore, Zcyto7
may play a role in the maintenance of spinal cord involving either
glial cells or neurons. This indicates that Zcyto7 can be used to
treat a variety of neurodegenerative diseases such as amyotrophic
lateral sclerosis (ALS), or dymyelinating diseases including
multiple sclerosis. Zcyto7 may also be used to treat sensory
neuropathis. The tissue specificity of Zcyto7 expression suggests
that Zcyto7 may be a growth and/or maintenance factor in the spinal
cord.
[0086] Zcyto7 gene's location on chromosome 5 indicates that zcyto7
is a cytokine which can be used to modulate the activities of cells
of the immune system. Zcyto7 can also be used as a chemoattractant
of neutrophils in the spinal column. This would be useful as an
anti-infective for infections in the spinal column. It could also
be used to help regulate other cytokines in the spinal cord. Zcyto7
may also be administered to treat peripheral neuropathies such as
Charcot-Marie-Tooth (CMT) disease which is localized to the same
chromosomal region of 5q as Zcyto7.
[0087] The fact that Zcyto7 inhibits the growth of BAF-3 and TF-1
cells as shown in examples 11 and 12 below indicates that Zcyto7
can be used to treat autoimmune diseases and possibly such cancers
such as leukemias.
[0088] The present invention also provides reagents which will find
use in diagnostic applications. For example, the Zcyto7 gene is
heavily expressed in the spinal cord. A probe comprising the Zcyto7
DNA or RNA or a subsequence thereof can be used to determine if the
Zcyto7 gene is present on chromosome 5 or if a mutation has
occurred.
[0089] The present invention also provides reagents with
significant therapeutic value. The Zcyto7 polypeptide (naturally
occurring or recombinant), fragments thereof, antibodies and
anti-idiotypic antibodies thereto, along with compounds identified
as having binding affinity to the Zcyto7 polypeptide, should be
useful in the treatment of conditions associated with abnormal
physiology or development, including abnormal proliferation, e.g.,
cancerous conditions, or degenerative conditions. For example, a
disease or disorder associated with abnormal expression or abnormal
signaling by a Zcyto7 polypeptide should be a likely target for an
agonist or antagonist of the Zcyto7 polypeptide.
[0090] In particular, Zcyto7 can be used to treat inflammation.
Inflammation is a result of an immune response to an infection or
as an autoimmune response to a self-antigen.
[0091] Treatment dosages should be titrated to optimize safety and
efficacy. Methods for administration include intravenous,
peritoneal, intramuscular, subdural, into the spinal fluid or
transdermal administration. Pharmaceutically acceptable carriers
will include water, saline, buffers to name just a few. Dosage
ranges would ordinarily be expected from 0.1 .mu.g to 1 mg per
kilogram of body weight per day. Preferably, 1 .mu.g to 100 .mu.g
per day. However, the doses by be higher or lower as can be
determined by a medical doctor with ordinary skill in the art. For
a complete discussion of drug formulations and dosage ranges see
Remington's Pharmaceutical Sciences,17.sup.th Ed., (Mack Publishing
Co., Easton, Pa., 1990), and Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 9.sup.th Ed. (Pergamon Press
1996).
[0092] Use of Zcyto7 to Promote Bone and Cartilage Growth
[0093] It has been discovered that Zcyto7 stimulates the
proliferation of both chondrocytes and osteoblasts as is shown
below in Examples 7 and 9 respectively. In addition, Zcyto7 also
stimulates the steady state level of glycosaminoglycan present in
chondrocyte cultures as shown in Example 8. Thus Zcyto7 can be used
to stimulate both bone and cartilage growth in a variety of
different therapeutic settings.
[0094] Zcyto7 can be implanted in a mammalian body so that the
zcyto7 is in contact with osteoblasts such that osteoblast
proliferation occurs and bone growth is stimulated. For example,
zcyto7 can be placed in a matrix [with or without a bone
morphogenic protein (BMP)]. The BMP induces the migration of
mesenchymal osteoblast precursors to the site and further induces
differentiation of the mesenchymal cells into osteoblast. Zcyto7
will then stimulate the further proliferation of the osteoblasts. A
suitable matrix is made up of particles of porous materials. The
pores must be of a dimension to permit progenitor cell migration
and subsequent differentiation and proliferation. An ideal particle
size should be in the range of 70-850 mm, preferably 150-420 mm.
The matrix containing the zcyto7 can be molded into a shape
encompassing a bone defect. Examples of matrix materials are
particulate, demineralized, guanidine extracted, species-specific
bone. Other potentially useful matrix materials include collagen,
homopolymers and copolymers of glycolic acid and lactic acid,
hydroxyapatite, tricalcium phosphate and other calcium phosphates.
Zcyto7 can be applied into a matrix at a sufficient concentration
to promote the proliferation of osteoblasts, preferably at a
concentration of at least 1 .mu.g/ml of matrix. A solution of
zcyto7 can also be injected directly into the site of a bone
fracture or defect including areas of bone degeneration to expedite
healing of the fracture or defect site. Examples of BMPs and the
use of matrices to produce are disclosed in PCT application
publication number WO 92/07073, publication No. WO 91/05802,U.S.
Pat. No. 5,645,591 and U.S. Pat. No. 5,108,753.
[0095] Zcyto7 can be further used to treat osteoporosis by
administering a therapeutically effective amount of zcyto7 to an
individual. A preferred dosage would be 1 .mu.g of zcyto7 per
kilogram of body weight per day.
[0096] As stated above, it has also been determined that zcyto7 can
be used to promote the production of cartilage through its ability
to stimulate the proliferation of chondrocytes. Zcyto7 can be
injected directly into the site where cartilage is to be grown. For
example, zcyto7 can be injected directly in joints which have been
afflicted with osteoarthritis or other injured joints in which the
cartilage has been worn down. An example of a case in which
additional cartilage needs to be grown is shoulders and knees of
injured athletes.
[0097] Cartilage can also be grown by first removing chondrocytes
from an individual, culturing the chondrocytes with zcyto7 so that
they proliferate and reimplanting the chondrocytes back into the
individual where cartilage needs to be produced.
[0098] Zcyto7 can also be used to stimulate the regeneration of
dentin or bone which has been lost due to periodontal disease. To
do this, the surrounding tissue should be thoroughly cleaned and a
solution of Zcto7 be administered, preferably by injection, into
the site in which dentin regeneration is desired.
[0099] Antibodies to the zcyto7 polypeptide can be purified and
then administered to a patient. These reagents can be combined for
therapeutic use with additional active or inert ingredients, e.g.,
in pharmaceutically acceptable carriers or diluents along with
physiologically innocuous stabilizers and excipients. These
combinations can be sterile filtered and placed into dosage forms
as by lyophilization in dosage vials or storage in stabilized
aqueous preparations. This invention also contemplates use of
antibodies, binding fragments thereof or single-chain antibodies of
the antibodies including forms which are not complement
binding.
[0100] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, physiological state of the patient,
and other medications administered. Thus, treatment dosages should
be titrated to optimize safety and efficacy. Typically, dosages
used in vitro may provide useful guidance in the amounts useful for
in vivo administration of these reagents. Animal testing of
effective doses for treatment of particular disorders will provide
further predictive indication of human dosage. Methods for
administration include oral, intravenous, peritoneal,
intramuscular, or transdermal administration. Pharmaceutically
acceptable carriers will include water, saline, buffers to name
just a few. Dosage ranges would ordinarily be expected from 1 .mu.g
to 1000 .mu.g per kilogram of body weight per day. However, the
doses may be higher or lower as can be determined by a medical
doctor with ordinary skill in the art. For a complete discussion of
drug formulations and dosage ranges see Remington's Pharmaceutical
Sciences, 17.sup.th Ed., (Mack Publishing Co., Easton, Pa., 1990),
and Goodman and Gilman's: The Pharmacological Bases of
Therapeutics,9.sup.th Ed. (Pergamon Press 1996).
[0101] Nucleic Acid-Based Therapeutic Treatment
[0102] If a mammal has a mutated or lacks a Zcyto7 gene, the Zcyto7
gene can be introduced into the cells of the mammal. In one
embodiment, a gene encoding a Zcyto7 polypeptide is introduced in
vivo in a viral vector. Such vectors include an attenuated or
defective DNA virus, such as but not limited to herpes simplex
virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus,
adeno-associated virus (AAV), and the like. Defective viruses,
which entirely or almost entirely lack viral genes, are preferred.
A defective virus is not infective after introduction into a cell.
Use of defective viral vectors allows for administration to cells
in a specific, localized area, without concern that the vector can
infect other cells. Examples of particular vectors include, but are
not limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt
et al., Molec. Cell. Neurosci.,2:320-330 (1991)], an attenuated
adenovirus vector, such as the vector described by
Stratford-Perricaudet et al., J. Clin. Invest., 90:626-630 (1992),
and a defective adeno-associated virus vector [Samulski et al., J.
Virol., 61:3096-3101 (1987); Samulski et al. J. Virol.,
63:3822-3828 (1989)].
[0103] In another embodiment, the gene can be introduced in a
retroviral vector, e.g., as described in Anderson et al., U.S. Pat.
No. 5,399,346; Mann et al., Cell, 33:153 (1983); Temin et al., U.S.
Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;
Markowitz et al., J. Virol., 62:1120 (1988); Temin et al., U.S.
Pat. No. 5,124,263; International Patent Publication No. WO
95/07358, published Mar. 16, 1995 by Dougherty et al.; and Blood,
82:845 (1993).
[0104] Alternatively, the vector can be introduced by lipofection
in vivo using liposomes. Synthetic cationic lipids can be used to
prepare liposomes for in vivo transfection of a gene encoding a
marker [Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417
(1987); see Mackey et al., Proc. Natl. Acad. Sci. USA, 85:8027-8031
(1988)]. The use of lipofection to introduce exogenous genes into
specific organs in vivo has certain practical advantages. Molecular
targeting of liposomes to specific cells represents one area of
benefit. It is clear that directing transfection to particular
cells represents one area of benefit. It is clear that directing
transfection to particular cell types would be particularly
advantageous in a tissue with cellular heterogeneity, such as the
pancreas, liver, kidney, and brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting. Targeted
peptides, e.g., hormones or neurotransmitters, and proteins such as
antibodies, or non-peptide molecules could be coupled to liposomes
chemically.
[0105] It is possible to remove the cells from the body and
introduce the vector as a naked DNA plasmid and then re-implant the
transformed cells into the body. Naked DNA vector for gene therapy
can be introduced into the desired host cells by methods known in
the art, e.g., transfection, electroporation, microinjection,
transduction, cell fusion, DEAE dextran, calcium phosphate
precipitation, use of a gene gun or use of a DNA vector transporter
[see, e.g., Wu et al., J. Biol. Chem., 267:963-967 (1992); Wu et
al., J. Biol. Chem., 263:14621-14624 (1988)].
[0106] Zcyto7 polypeptides can also be used to prepare antibodies
that specifically bind to Zcyto7 polypeptides. These antibodies can
then be used to manufacture anti-idiotypic antibodies. As used
herein, the term "antibodies" includes polyclonal antibodies,
monoclonal antibodies, antigen-binding fragments thereof such as
F(ab').sub.2 and Fab fragments, and the like, including genetically
engineered antibodies. Antibodies are defined to be specifically
binding if they bind to a Zcyto7 polypeptide with a K.sub.a of
greater than or equal to 10.sup.7/M. The affinity of a monoclonal
antibody can be readily determined by one of ordinary skill in the
art (see, for example, Scatchard, ibid.).
[0107] Methods for preparing polyclonal and monoclonal antibodies
are well known in the art (see for example, 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). As would be evident to one of ordinary
skill in the art, polyclonal antibodies can be generated from a
variety of warm-blooded animals such as horses, cows, goats, sheep,
dogs, chickens, rabbits, mice, and rats. The immunogenicity of a
Zcyto7 polypeptide may be increased through the use of an adjuvant
such as Freund's complete or incomplete adjuvant. A variety of
assays known to those skilled in the art can be utilized to detect
antibodies which specifically bind to Zcyto7 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, radio-immunoassays,
radio-immunoprecipitations, enzyme-linked immunosorbent assays
(ELISA), dot blot assays, inhibition or competition assays, and
sandwich assays.
[0108] As would be evident to one of ordinary skill in the art,
polyclonal antibodies can be generated by inoculating a variety of
warm-blooded animals such as horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, hamsters, guinea pigs and rats with a
Zcyto7 polypeptide or a fragment thereof. The immunogenicity of a
Zcyto7 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 Zcyto7 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.
[0109] 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.
[0110] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to Zcyto7 protein or peptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled Zcyto7 protein or peptide). Genes encoding
polypeptides having potential Zcyto7 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 Zcyto7 sequences disclosed
herein to identify proteins which bind to Zcyto7. These "binding
proteins" which interact with Zcyto7 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 proteins
can also be used in analytical methods such as for screening
expression libraries and neutralizing activity. The binding
proteins can also be used for diagnostic assays for determining
circulating levels of polypeptides; for detecting or quantitating
soluble polypeptides as marker of underlying pathology or disease.
These binding proteins can also act as Zcyto7 "antagonists" to
block Zcyto7 binding and signal transduction in vitro and in
vivo.
[0111] Antibodies can also be generated gene therapy. The animal is
administered the DNA or RNA which encodes Zcyto7 or an immunogenic
fragment thereof so that cells of the animals are transfected with
the nucleic acid and express the protein which in turn elicits an
immunogenic response. Antibodies which then are produced by the
animal are isolated in the form of polyclonal or monoclonal
antibodies.
[0112] Antibodies to Zcyto7 may be used for tagging cells that
express the protein, for affinity purification, within diagnostic
assays for determining circulating levels of soluble protein
polypeptides, and as antagonists to block ligand binding and signal
transduction in vitro and in vivo.
[0113] Radiation hybrid mapping is a somatic cell genetic technique
developed for constructing high-resolution, contiguous maps of
mammalian chromosomes [Cox et al., Science 250:245-250 (1990)].
Partial or full knowledge of a gene's sequence allows the designing
of PCR primers suitable for use with chromosomal radiation hybrid
mapping panels. Commercially available radiation hybrid mapping
panels which cover the entire human genome, such as the Stanford G3
RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc.,
Huntsville, Ala.), are available. These panels enable rapid, PCR
based, chromosomal localizations and ordering of genes,
sequence-tagged sites (STSs), and other nonpolymorphic- and
polymorphic markers within a region of interest. This includes
establishing directly proportional physical distances between newly
discovered genes of interest and previously mapped markers. The
precise knowledge of a gene's position can be useful in a number of
ways including: 1) determining if a sequence is part of an existing
contig and obtaining additional surrounding genetic sequences in
various forms such as YAC-, BAC- or cDNA clones, 2) providing a
possible candidate gene for an inheritable disease which shows
linkage to the same chromosomal region, and 3) for
cross-referencing model organisms such as mouse which may be
beneficial in helping to determine what function a particular gene
might have.
[0114] The present invention also provides reagents which will find
use in diagnostic applications. For example, the Zcyto7 gene has
been mapped on chromosome 5q31. A Zcyto7 nucleic acid probe could
to used to check for abnormalities on chromosome 5. For example, a
probe comprising Zcyto7 DNA or RNA or a subsequence thereof can be
used to determine if the Zcyto7 gene is present on chromosome 5q31
or if a mutation has occurred. Detectable chromosomal aberrations
at the Zcyto7 gene locus include but are not limited to aneuploidy,
gene copy number changes, insertions, deletions, restriction site
changes and rearrangements. Such aberrations can be detected using
polynucleotides of the present invention by employing molecular
genetic techniques, such as restriction fragment length
polymorphism (RFLP) analysis, short tandem repeat (STR) analysis
employing PCR techniques, and other genetic linkage analysis
techniques known in the art [Sambrook et al., ibid.; Ausubel, et.
al., ibid.; Marian, A. J., Chest, 108: 255-265, (1995)].
[0115] Zcyto7 maps at the 5q31 region which is a "gene cluster"
which contains a group of cytokines and cytokine receptors. The
cytokines clustered there include IL-3, IL-4, IL-5, IL-13, GM-CSF,
and M-CSF. This result authenticates zcyto7 as a cytokine.
[0116] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
Cloning of Zcyto7
[0117] Zcyto7 was identified from expressed sequence tag defined
(EST) 582069 (SEQ ID NO: 3) by its homology to Interleukin-17. The
EST 582069 cDNA clone was obtained from a human fetal heart cDNA
library from the IMAGE consortium, Lawrence Livermore National
Laboratory through Genome Systems, Inc. The cDNA was supplied as an
agar stab containing E. coli transfected with the plasmid having
the cDNA of interest. The plasmid containing the cDNA was streaked
out on an LB 100 .mu.g/ml ampicillin and 100 .mu.g/ml methicillin
plate. The cDNA insert was sequenced. The insert was determined to
be 717 base pairs long with a 180 amino acid open reading frame and
a putative 20 amino acid signal peptide.
EXAMPLE 2
Northern Blot Analysis
[0118] Human multiple tissue blots 1,2,3 (Clontech)were probed to
determine the tissue distribution of Zcyto7. A EcoRI/NotI fragment
containing the entire Zcyto7 coding region was generated from the
EST582069 clone and used for the probe. A plasmid prep of EST582069
was prepared from a 5 ml LB 100 .mu.g/ml ampicillin overnight
culture at 37.degree. using the QIAprep Spin Miniprep Kit (Qiagen).
12 .mu.l out of 100 .mu.l were digested with 5 .mu.l of H buffer
(Boehringer Mannheim), 12.5 units of EcoRI (Gibco BRL) and 12.5
units Notl (New England Biolabs) in a 50 .mu.l reaction at
37.degree. C. for 2 hours. The digest was electrophoresed on a 0.7%
TBE agarose gel and the fragment was cut out. To obtain additional
material the digest was repeated under the same conditions as above
except 24 .mu.l of EST582069 was used in the second digest. The
second digest was electrophoresed on a 0.7% TBE agarose gel and the
fragment was cut out. The DNA was extracted from both gel slabs
with a QIAquick Gel Extraction Kit (Qiagen). 135 ng of this DNA was
labeled with P.sup.32 using the Multiprime DNA Labeling System
(Amersham) and unincorporated radioactivity was removed with a
NucTrap Probe Purification Column (Stratagene). Multiple tissue
northerns and a human RNA master blot were prehybridized 3 hours
with 10 ml ExpressHyb Solution (Clontech) containing 1 mg salmon
sperm DNA which was boiled 5 minutes and then iced 1 minute and
added to 10 ml of ExpressHyb Solution, mixed and added to blots.
Hybridization was carried out overnight at 65.degree. C. Initial
wash conditions were as follows: 2.times.SSC, 0.05% SDS RT for 40
minutes with several changes of solution then 0.1.times.SSC, 0.1%
SDS at 50.degree. C. for 40 minutes, 1 solution change. Blots were
than exposed to film a -80.degree. C. for 5 hours. There was cross
hybridization/background so blots were further washed at 55.degree.
C. then 65.degree. C. with 0.1%.times.SSC, 0.1% SDS for 1 hour
each. Spinal cord showed very high expression of Zcyto7 mRNA and
trachea showed weak expression of mRNA. The transcript size was
approximately 0.75 kb.
EXAMPLE 3
Chromosomal Assignment and Placement of Zcyto7
[0119] Zcyto7 was mapped to chromosome 5 using the commercially
available "GeneBridge 4 Radiation Hybrid Panel" (Research Genetics,
Inc., Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panel
contains PCRable DNAs from each of 93 radiation hybrid clones, plus
two control DNAs (the HFL donor and the A23 recipient). A publicly
available WWW server
(http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allows
mapping relative to the Whitehead Institute/MIT Center for Genome
Research's radiation hybrid map of the human genome (the "WICGR"
radiation hybrid map of the human genome) which was constructed
with the GeneBridge 4 Radiation Hybrid Panel.
[0120] For the mapping of Zcyto7 with the "GeneBridge 4 RH Panel",
20 1 reactions were set up in a PCRable 96-well microtiter plate
(Stratagene, La Jolla, Calif.) and used in a "RoboCycler Gradient
96" thermal cycler (Stratagene). Each of the 95 PCR reactions
consisted of 2 1 10.times. KlenTaq PCR reaction buffer (CLONTECH
Laboratories, Inc., Palo Alto, Calif.), 1.6 1 dNTPs mix (2.5 mM
each, PERKIN-ELMER, Foster City, Calif.), 1 1 sense primer SEQ ID
NO:4, 1 1 antisense primer SEQ ID NO:5, 2 1 "RediLoad" (Research
Genetics, Inc., Huntsville, Ala.), 0.4 1 50.times. Advantage
KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA
from an individual hybrid clone or control and x 1 ddH.sub.2O for a
total volume of 20 1. The reactions were overlaid with an equal
amount of mineral oil and sealed. The PCR cycler conditions were as
follows: an initial 1 cycle 5 minute denaturation at 95.degree. C.,
35 cycles of a 1 minute denaturation at 95.degree. C., 1 minute
annealing at 52.degree. C. and 1 minute extension at 72.degree. C.,
followed by a final 1 cycle extension of 7 minutes at 72.degree. C.
The reactions were separated by electrophoresis on a 3% NuSieve GTG
agarose gel (FMC Bioproducts, Rockland, Me.).
[0121] The results showed that Zcyto7 maps 490.89 cR from the top
of the human chromosome 5 linkage group on the WICGR radiation
hybrid map. Relative to the centromere, its nearest proximal marker
was D5S413 and its nearest distal maker was WI-5208. The use of
surrounding markers position Zcyto7 in the 5q31.3-q32 region on the
integrated LDB chromosome 5 map (The Genetic Location Database,
University of Southhampton, WWW server:
http://cedar.genetics.soton.ac.uk/public_html/).
EXAMPLE 4
Construction of Zcyto7 Expression Vectors
[0122] Two Zcyto7 construction vectors were made in a FLAG amino
acid sequence (SEQ ID NO: 10) was inserted onto the N-terminal or
C-terminal ends of the Zcyto7 polypeptide. For the construction in
which the FLAG amino acid sequence was attached to the N-terminus
of Zcyto7, a 473 bp Zcyto7 PCR DNA fragment was generated with 1
.mu.l of a dilution of the EST582069 plasmid prep of Example 2 and
20 picomoles (pm) of primer SEQ ID NO: 6 and 20 pm primer SEQ ID
NO: 7. The PCR reaction was incubated at 94.degree. C. for 1
minute, and then run for 5 cycles each individual cycle being
comprised of 20 seconds at 94.degree. C. and 2 minutes at
64.degree. C. This was followed by 22 cycles each cycle being
comprised of 20 seconds at 94.degree. C. and 2 minutes at
74.degree. C. The reaction was ended with an incubation for 10
minutes at 74.degree. C. 50 .mu.l of the PCR reaction mixture was
digested with 30 units of BamH1 (Boehringer Mannheim) and 120 units
of Xho1 (Boehringer Mannheim) for 2 hours at 37.degree. C. The
digested reaction mixture was electrophoresed on a 1% TBE gel; the
DNA band was excised with a razor blade and the DNA was extracted
from the gel with the Qiaquick<<Gel Extraction Kit (Qiagen).
The excised DNA was subcloned into plasmid nfpzp9 which had been
cut with Bam and Xho. Nfpzp9 is a mammalian cell expression vector
comprising an expression cassette containing the mouse
metallothionein-1 promoter, a sequence encoding the tissue
plasminogen activator (TPA) leader, then the FLAG peptide (SEQ ID
NO:10), then multiple restriction sites. These were followed by the
human growth hormone terminator, an E. coli origin of replication
and a mammalian selectable marker expression unit containing the
SV40 promoter, enhancer and origin of replication; a dihydrofolate
reductase gene (DHFR) and the SV40 terminator.
[0123] For the construction of the zcyto7 gene in which a
C-terminus FLAG was inserted onto the C-terminus of the zcyto7
polypeptide, a 543 bp zcyto7 PCR fragment was generated with 1
.mu.l of dilution of the EST582069 plasmid preparation described in
Example 1 and 20 pm each of primers SEQ ID NO: 8 and SEQ ID NO: 9.
The PCR reaction was incubated at 94.degree. C. for 1 minute, then
run for 5 cycles, each cycle being comprised 20 seconds at
94.degree. C. and 2 minutes at 55.degree. C. This was followed by
22 cycles each cycle comprised of 20 seconds at 94.degree. C. and 2
minutes at 74.degree. C. The reaction was ended with a final 10
minute extension at 74.degree. C. The entire reaction mixture was
run on a 1% TBE gel and the DNA was cut out with a razor blade and
the DNA was extracted using the QIAQUICK.TM. gel extraction kit. 20
.mu.l out of the recovered 35 .mu.l digested with 10 units of BamH1
(Boehringer Mannheim) and 10 units of EcoR1(Gibco BRL) for 2 hours
at 37.degree. C. The digested PCR mixture was electrophoresed on a
1% TBE gel. The DNA band was cut out with a razor blade and the DNA
was extracted from the gel using the QIAquick<<Gel Extraction
Kit (Qiagen). The extracted DNA was subcloned into plasmid cfpzp9
which had been cut with EcoR1 and BamH1. Plasmid cfpzp9 is a
mammalian expression vector containing an expression cassette
having the mouse metallothionein-1 promoter, multiple restriction
sites for insertion of coding sequences, a sequence encoding the
FLAG peptide, SEQ ID NO:10, a stop codon, a human growth hormone
terminator, 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.
[0124] Using antibodies to the FLAG polypeptides, one can separate
the FLAG-tagged Zcyto7 polypeptides from a cell supernatant
liquid.
EXAMPLE 5
Cloning of Murine Zcyto7
[0125] Mouse Zcyto7 was identified from EST 660242 SEQ ID NO:14 by
its homology to human Zcyto7. The cDNA clone was obtained from
Lawrence Livermore National Laboratory through Genome Systems from
a murine embryo cDNA library in which the embryos were between 13.5
and 14.5 days old. The cDNA was supplied as an agar stab containing
E. coli transfected with the plasmid having the cDNA of interest
and then streaked out on an LB 100 .mu.g/ml ampicillin, 25 .mu.g/ml
methicillin plate. The cDNA insert in EST660242 was sequenced. The
insert was determined to be 785 base pairs with an open reading
frame of 180 amino acids and a putative 20 amino acid signal
peptide. The sequences are defined by SEQ ID NO:11 and SEQ ID
NO:12.
EXAMPLE 6
Tissue Distribution of Murine Zcyto7
[0126] Mouse Multiple Tissue Northern Blot (Clontech, Palo Alto,
Calif.), mouse northern dot blot (Clontech), a mouse embryo
northern blot, and a mouse spinal cord dot blot were probed to
determine the tissue distribution of murine Zcyto7.
[0127] The mouse embryo RNA were isolated from mouse embryos which
were 6 to nine days from the date of fertilization using the POLY
(A) PURE.RTM. mRNA isolation kit (Ambion). 100 mg of each mouse
embryo was lysed in 1 ml of lysis buffer, homogenized and processed
in batch method according to the manufacturer's protocol. For the
northern blot, 2 .mu.g of RNA was loaded on 1.5% agarose, 2.2M
formaldehyde gel. The gel was run at 60V for four hours and 30
minutes. The RNA was transferred overnight onto a Nytran membrane
which had been pre-wetted in 20.times.SSC. The RNAs were
crosslinked onto the membrane by UV light and baked at 80.degree.
C. for 1 hour.
[0128] The mouse spinal cord RNA was also prepared with the POLY
(A) PURE.RTM. mRNA isolation kit (Ambion). The mouse spinal cord
dot blot was made by spotting a dot with 1, 2 and 3 .mu.l of RNA at
1 .mu.g RNA/.mu.l concentration onto Nytran membrane.
[0129] A Not 1/EcoRI fragment containing the entire Zcyto7 coding
region was generated from the clone containing SEQ ID NO: 12
(hereinafter referred to as the SEQ ID NO:12 clone) and was used
for the probe. A plasmid prep of the SEQ ID NO:12 clone was
prepared from a 5 ml LB 100 .mu.g/ml ampicillin overnight culture
at 37.degree. C. using the QIAPREP SPIN MINIPREP kit (Qiagen). 4.66
.mu.g were digested with 8 .mu.l of high buffer (Boehringer
Mannheim), 20 units of Not1 (Biolabs) and 20 units of EcoRI (Gibco
BRL) in a 80 .mu.l reaction at 37.degree. C. for 2 hours. The
digest was electrophoresed on a 1.0% TBE gel and the fragment was
cut out. The DNA was extracted from the gel slab with a
QIAQUICK.RTM. gel extraction kit (Qiagen). 98.8 ng of this fragment
was labeled with P.sup.32 using the MULTIMPRIME.RTM. DNA labeling
system (Amersham) and unincorporated radioactivity was removed with
a NUCTRAP.RTM. probe purification column (Stratagene).
[0130] The two northern preps and the two dot blot preps were
prehybridized for 3 hours at 65.degree. C. as follows. 1 mg of
salmon sperm was boiled 5 minutes, iced 1 minute, mixed with 10 ml
of EXPRESSHYB.RTM. solution and added to the blots. Hybridization
was carried out overnight at 65.degree. C. Initial wash conditions
were as follows: 2.times.SSC, 0.1% SDS for 40 minutes at room
temperature then 0.1.times.SSC, 0.1% SDS for 40 minutes at
50.degree. C. Blots were exposed to film overnight at -80.degree.
C. The northern blots and the mouse dot blot were further washed
with 0.1%.times.SSC, 0.1% SDS at 60.degree. C. to remove
background. The mouse spinal cord dot blot was washed again at
higher stringency with 0.1.times.SSC, 0.1% SDS at 65.degree. C. to
confirm the earlier results.
[0131] Results: Mouse Zcyto7 expression was seen in the spinal
cord, submaxillary gland and epididymis. Mouse embryo showed
expression of Zcyto7 starting on day 12, peaking at day 16 and
ending day 17 from the date of fertilization. The transcript size
was approximately 1 kb.
EXAMPLE 7
Proliferation of Chondrocytes Using Zcyto7
[0132] A chondrocyte proliferation assay was done to determine the
effect that Zcyto7 would have on chondrocyte proliferation. The
assay was done with 20% confluent cultures. As a control vehicle,
bovine serum albumin was added to a culture of chondrocytes instead
of Zcyto7. The assay measured the 3H-thymidine incorporation of
nascent DNA in the chondrocytes, Wahl et al., Mol. Cell. Biol.
8:5016-5025 (1988).
[0133] Results: A 3.5--9 fold stimulation of primary chondrocyte
proliferation was seen upon exposure of the chondrocyte cultures to
1 .mu.g/ml of zcyto7. Chondrocyte stimulation by zcyto7 was seen
with multiple preparations of the protein and was seen across
species lines. It contrast to this, the control experiment using
BSA resulted in no stimulation of chondrocytes.
EXAMPLE 8
Production of Glycosaminoglycan by Zcyto7-treated Chondrocytes
[0134] A 20% confluent culture of chondrocytes was prepared and
zcyto7 was applied at a concentration of 1 .mu.g/ml. In a second
experiment in addition to zcyto7, IL-1.beta. was applied to the
cell culture. In a control group BSA was added to the chondrocyte
culture. The level of glycosaminoglycan (GAG) production by the
chondrocyte culture was then determined using a
1,9-dimethylmethlyene blue dye binding assay, Fardale et al.,
Biochem. Biophys. Acta 888:173-177 (1987).
[0135] Results: Chondrocytes which were cultured with zcyto7 showed
a 50% increase in the steady state presence of GAG in the
chondrocyte culture. Moreover, when the chondrocytes were
co-cultured with both zcyto7 and interleukin-1.beta. (IL-1)the GAG
production by the chondrocytes increased 2.5 fold as compared with
culturing of the chondrocytes with either zcyto7 or EL-1.beta.
alone. While the cultured cells to which BSA was added showed no
increased production of GAG.
EXAMPLE 9
Osteoblast Stimulation by Zcyto7
[0136] The CCC4 cell line is an osteoblast-like cell line derived
from p53 knockout mice. The CCC4 line was transfected with a
plasmid containing an inducible serum response element (SRE)
driving the expression of luciferase. The stimulation of the SRE
and thus the expression of luciferase indicates that the chemical
entity is likely to stimulate osteoblasts.
[0137] CCC4 cells were cultured in the presence of 1 .mu.g of
zcyto7/ml of culture medium. As a control BSA, fibroblast growth
factor (FGF) and platelet-derived growth factor (PDGF) were each
added to different cultures of CCC4 cells. BSA was a negative
control and FGF and PDGF were positive controls as they are known
to promote osteoblast proliferation. Luciferase activity was
detected by addition of 40 .mu.l of Promega luciferase substrate
using a 2 second integrated read on Labsystes
LUMINOSKAN.sup.<<.
[0138] Results
[0139] Zcyto7 as well as, FGF and PDGF stimulate the expression of
luciferase in this assay indicating that they stimulate
osteoblasts. The BSA vehicle control was negative in this
assay.
EXAMPLE 10
Effect of Zcyto7 on the Growth of Fibroblasts
[0140] Confluent cultures of human dermal, lung, and fetal lung
fibroblasts were inoculated with Zcyto7 to determine the effects of
Zcyto7 on the growth of fibroblasts. FGF was used as a positive
control and BSA as a negative control vehicle.
[0141] Results: Zcyto7 had no effect on the growth of
fibroblasts.
EXAMPLE 11
Effect of Zcyto7 on the Growth of BaF3 Cells
[0142] BaF3 cells, a murine pre-B cell line dependent on IL-3 to
proliferate, were washed several times with base medium and then
plated in a 96-well plate each well contained approximately 5500
cells/well. The cells were treated with either 1 .mu.g/ml of Zcyto7
or 1-2 pg/ml of IL-3 or with a combination of both Zcyto7 and IL-3.
Also in a separate experiment 0.1-10 ng/ml of TGF.beta. was added
to the wells instead of Zcyto7. After incubation of the assay plate
at 37.degree. C. and 5% CO2 for 3-6 days, 20 .mu.l of ALAMAR blue
was added to each well and the plate is incubated at 37.degree. C.
for 15-24 hours. The plate was then read with a fluorometer with
excitation wavelength of 544 m and emission wavelength of 590 m.
The assay was also scored by eye for stimulation or inhibition of
cell proliferation prior to the addition of the ALAMAR blue. The
base medium contained RPMI 1640+10% HIA-FBS+L-glutamine+Na
pyruvate.
[0143] Results: Zcyto7 and TGF.beta. significantly inhibited the
IL-3 driven proliferation of BaF3 cells. However, when neutralizing
antibodies to TGF.beta. were added in along with the Zcyto7, the
Zcyto7 inhibition of the proliferation of the BaF3 cells was
eliminated.
EXAMPLE 12
Effect of Zcyto7 on the Growth of TF-1 Cells
[0144] TF-1 cells, a human leukemia cell line which is GM-CSF or
IL-1.beta. dependent, were washed several times with base medium
and then plated in a 96-well plate each well containing
approximately 7000 cells/well. The cells were co-cultured with 1
.mu.g/ml of Zcyto7 and 100-200 .mu.g/ml of IL-1.beta.. Also in a
separate experiment TGF.beta. was added to the wells instead of
Zcyto7. After incubation of the assay plate at 37.degree. C. and 5%
CO2 for 3-6 days, 20 .mu.l of ALAMAR blue was added to each well
and the plate is incubated at 37.degree. C. for 15-24 hours. The
plate is then read with a fluorometer with excitation wavelength of
544 nm and emission wavelength of 590 nm. The assay was also scored
by eye for stimulation or inhibition of cell proliferation prior to
the addition of the ALAMAR blue. The base medium contained RPMI
1640+10% HIA-FBS+L-glutamine+Na pyruvate.
[0145] Results: The IL-1.beta. stimulation of TF-1 cells is
inhibited by both Zcyto7 and TGF-.beta.. The concentration of
Zcyto7 at which inhibition of proliferation occurred was greater
than 200 ng/ml; and the concentration of TGF-.beta. at which
inhibition of proliferation occurred was about 50 pg/ml.
* * * * *
References