Note: Descriptions are shown in the official language in which they were submitted.
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Description
HELICAL POLYPEPT>DE ZALPHA29
BACKGROUND OF THE INVENTION
Cytokines are polypeptide hormones that are produced by a cell and
affect the growth or metabolism of that cell or another cell. In multicellular
animals,
cytokines control cell growth, migration, differentiation, and maturation.
Cytokines
play a role in both normal development and pathogenesis, including the
development of
solid tumors.
Cytokines are physicochemically diverse, ranging in size from 5 kDa
(TGF-a) to 140 kDa (Mullerian-inhibiting substance). They include single
polypeptide
chains, as well as disulfide-linked homodimers and heterodimers.
Cytokines influence cellular events by binding to cell-surface receptors.
Binding initiates a chain of signalling events within the cell, which
ultimately results in
phenotypic changes such as cell division, protease production, cell migration,
expression of cell surface proteins, and production of additional growth
factors.
2 0 Cell differentiation and maturation are also under control of cytokines.
For example, the hematopoietic factors erythropoietin, thrombopoietin, and G-
CSF
stimulate the production of erythrocytes, platelets, and neutrophils,
respectively, from
precursor cells in the bone marrow. Development of mature cells from
pluripotent
progenitors may require the presence of a plurality of factors.
2 5 The role of cytokines in controlling cellular processes makes them likely
candidates and targets for therapeutic intervention; indeed, a number of
cytokines have
been approved for clinical use. Interferon-alpha (IFN-a), for example, is used
in the
treatment of hairy cell leukemia, chronic myeloid leukemia, Kaposi's sarcoma,
condylomata acuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal
and
3 0 Puri, "Common and Uncommon Features of Cytokines and Cytokine Receptors:
An
Overview", in Aggarwal and Puri, eds., Human C~tokines: Their Role in Disease
and
Therapy, Blackwell Science, Cambridge, MA, 1995, 3-24). Platelet-derived
growth
factor (PDGF) has been approved in the United States and other countries for
the
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treatment of dermal ulcers in diabetic patients. The hematopoietic cytokine
erythropoietin has been developed for the treatment of anemias (e.g., EP
613,683). G-
CSF, GM-CSF, IFN-(3, IFN-y, and IL-2 have also been approved for use in humans
(Aggarwal and Puri, ibid.). Experimental evidence supports additional
therapeutic uses
of cytokines and their inhibitors. Inhibition of PDGF receptor activity has
been shown
to reduce intimal hyperplasia in injured baboon arteries (Giese et al.,
Restenosis
Summit VIII, Poster Session #23, 1996; U.S. Patent No. 5,620,687). Vascular
endothelial growth factors (VEGFs) have been shown to promote the growth of
blood
vessels in ischemic limbs (Isner et al., The Lancet 348:370-374, 1996), and
have been
proposed for use as wound-healing agents, for treatment of periodontal
disease, for
promoting endothelialization in vascular graft surgery, and for promoting
collateral
circulation following myocardial infarction (WIPO Publication No. WO 95/24473;
U.S.
Patent No. 5,219,739). A soluble VEGF receptor (soluble flt-1) has been found
to
block binding of VEGF to cell-surface receptors and to inhibit the growth of
vascular
tissue in vitro (Biotechnology News 16( 17):5-6, 1996). Experimental evidence
suggests
that inhibition of angiogenesis may be used to block tumor development
(Biotechnology News, Nov. 13, 1997) and that angiogenesis is an early
indicator of
cervical cancer (Br. J. Cancer 76:1410-1415, 1997). More recently,
thrombopoietin
has been shown to stimulate the production of platelets in vivo (Kaushansky et
al.,
2 0 Nature 369:568-571, 1994) and has been the subject of several clinical
trials (reviewed
by von dem Borne et al., Bailliere's Clin. Haematol. 11:427-445, 1998).
In view of the proven clinical utility of cytokines, there is a need in the
art for additional such molecules for use as both therapeutic agents and
research tools
and reagents. Cytokines are used in the laboratory to study developmental
processes,
2 5 and in laboratory and industry settings as components of cell culture
media.
SUMMARY OF THE INVENTION
Within one aspect of the invention there is provided an isolated
polypeptide comprising a sequence of amino acid residues selected from the
group
3 o consisting of residues 48-62 of SEQ ID N0:2, residues 47-61 of SEQ ID
N0:4,
residues 63-104 of SEQ ID N0:2, residues 62-103 of SEQ ID N0:4, residues 105-
119
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of SEQ >D N0:2, residues 104-118 of SEQ >D N0:4, residues 120-137 of SEQ >D
N0:2, residues 119-136 of SEQ >D N0:4, residues 138-152 of SEQ ID N0:2,
residues
137-151 of SEQ >D N0:4, residues 153-170 of SEQ 1D N0:2, residues 152-169 of
SEQ
>D N0:4, residues 171-185 of SEQ >D N0:2, and residues 170-184 of SEQ >D N0:4.
Within one embodiment, the isolated polypeptide has from 15 to 1500 amino acid
residues. Within a related embodiment, the sequence of amino acid residues is
operably
linked via a peptide bond or polypeptide linker to a second polypeptide
selected from
the group consisting of maltose binding protein, an immunoglobulin constant
region, a
polyhistidine tag, and a peptide as shown in SEQ ID NO:S. Within another
1 o embodiment, the isolated polypeptide comprises at least 30 contiguous
residues of SEQ
>D N0:2 or SEQ >D N0:4. Within other embodiments, the isolated polypeptide
comprises residues 48-185 or residues 27-190 of SEQ >D N0:6. Within further
embodiments, the isolated polypeptide comprises residues 48-185 of SEQ >D
N0:2,
residues 47-184 of SEQ >D N0:4, residues 27-190 of SEQ >D N0:2, or residues 26-
188
of SEQ >D N0:4.
Within a second aspect of the invention there is provided an expression
vector comprising the following operably linked elements: a transcription
promoter; a
DNA segment encoding a polypeptide as disclosed above; and a transcription
terminator. Within one embodiment, the DNA segment comprises nucleotides 79 to
2 0 570 of SEQ >D N0:7. Within another embodiment, the expression vector
further
comprises a secretory signal sequence operably linked to the DNA segment.
Within a third aspect the invention provides a cultured cell into which
has been introduced an expression vector as disclosed above, wherein the cell
expresses
the DNA segment. Within one embodiment, the expression vector further
comprises a
2 5 secretory signal sequence operably linked to the DNA segment, and the
polypeptide is
secreted by the cell.
Within a fourth aspect the invention provides a method of making a
protein comprising culturing a cell into which has been introduced an
expression vector
as disclosed above under conditions whereby the DNA segment is expressed and
the
3 0 polypeptide is produced, and recovering the protein. When the expression
vector
further comprises a secretory signal sequence operably linked to the DNA
segment, the
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polypeptide is secreted by the cell and recovered from a medium in which the
cell is
cultured.
Within a fifth aspect the invention provides a protein produced by the
method disclosed above.
Within a sixth aspect of the invention there is provided an antibody that
specifically binds to the protein disclosed above.
Within a seventh aspect of the invention there is provided method of
detecting, in a test sample, the presence of an antagonist of zalpha29
activity. The
method comprises the steps of (a) culturing a cell that is responsive to
zalpha29; (b)
exposing the cell to a zalpha29 polypeptide in the presence and absence of a
test
sample; (c) comparing levels of response to the zalpha29 polypeptide, in the
presence
and absence of the test sample, by a biological or biochemical assay; and (d)
determining from the comparison the presence of an antagonist of zalpha29
activity in
the test sample.
These and other aspects of the invention will become evident upon
reference to the following detailed description of the invention and the
attached
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. lA-1D are a Hopp/Woods hydrophilicity profile of the amino acid
sequence shown in SEQ ID N0:2. The profile is based on a sliding six-residue
window. Buried G, S, and T residues and exposed H, Y, and W residues were
ignored.
These residues are indicated in the figure by lower case letters.
Fig. 2 is an alignment of representative human (SEQ B~ N0:2) and
2 5 mouse (SEQ >D N0:4) zalpha29 amino acid sequences.
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention in detail, it may be helpful to the
understanding thereof to define the following terms:
3 0 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
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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
5 Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene
67:31,
1988), maltose binding protein (Kellerman and Ferenci, Methods Enzymol. 90:459-
463,
1982; Guan et al., Gene 67:21-30, 1987), Glu-Glu affinity tag (Grussenmeyer et
al.,
Proc. Natl. Acid. Sci. USA 82:7952-4, 1985; see SEQ >D NO:S), substance P,
FIagTM
peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidin binding
peptide,
thioredoxin, ubiquitin, cellulose binding protein, T7 polymerise, 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 and other reagents
are
available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ;
New
England Biolabs, Beverly, MA; and Eastman Kodak, New Haven, CT).
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
2 0 is also used herein to denote a protein encoded by an allelic variant of a
gene.
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
2 5 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.
"Angiogenic" denotes the ability of a compound to stimulate the
formation of new blood vessels from existing vessels, acting alone or in
concert with
3 0 one or more additional compounds. Angiogenic activity is measurable as
endothelial
cell activation, stimulation of protease secretion by endothelial cells,
endothelial cell
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migration, capillary sprout formation, and endothelial cell proliferation.
Angiogenesis
can also be measured using any of several in vivo assays as disclosed herein.
A "complement" of a polynucleotide molecule is a polynucleotide
molecule having a complementary base sequence and reverse orientation as
compared
to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is
complementary to 5' CCCGTGCAT 3'.
The term "corresponding to", when applied to positions of amino acid
residues in sequences, means corresponding positions in a plurality of
sequences when
the sequences are optimally aligned.
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).
The term "expression vector" is used to denote a DNA molecule, linear
or circular, that comprises a segment encoding a polypeptide of interest
operably linked
to additional segments that provide for its transcription. Such additional
segments
include promoter and terminator sequences, and may also include one or more
origins
of replication, one or more selectable markers, an enhancer, a polyadenylation
signal,
2 o etc. Expression vectors are generally derived from plasmid or viral DNA,
or may
contain elements of both.
The term "isolated", when applied to a polynucleotide, denotes that the
polynucleotide has been removed from its natural genetic milieu and is thus
free of
other extraneous or unwanted coding sequences, and is in a form suitable for
use within
2 5 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
3 0 associated regions will be evident to one of ordinary skill in the art
(see for example,
Dynan and Tijan, Nature 316:774-78, 1985).
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An "isolated" polypeptide or protein is a polypeptide or protein that is
found in a condition other than its native environment, such as apart from
blood and
animal tissue. The isolated polypeptide may be substantially free of other
polypeptides,
particularly other polypeptides of animal origin. The polypeptides may be
prepared in a
highly purified form, i.e. greater than 95% pure or greater than 99% pure.
When used
in this context, the term "isolated" does not exclude the presence of the same
polypeptide in alternative physical forms, such as dimers or alternatively
glycosylated
or derivatized forms.
"Operably linked" means that two or more entities are joined together
such that they function in concert for their intended purposes. When referring
to DNA
segments, the phrase indicates, for example, that coding sequences are joined
in the
correct reading frame, and transcription initiates in the promoter and
proceeds through
the coding segments) to the terminator. When referring to polypeptides,
"operably
linked" includes both covalently (e.g., by disulfide bonding) and non-
covalently (e.g.,
by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions)
linked
sequences, wherein the desired functions) of the sequences are retained.
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.
2 0 A "polynucleotide" is a single- or double-stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
Polynucleotides include RNA and DNA, and may be isolated from natural sources,
synthesized in vitro, or prepared from a combination of natural and synthetic
molecules.
Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides
2 5 ("nt"), or kilobases ("kb"). Where the context allows, the latter two
terms may describe
polynucleotides that are single-stranded or double-stranded. When the term is
applied
to double-stranded molecules it is used to denote overall length and will be
understood
to be equivalent to the term "base pairs". It will be recognized by those
skilled in the
art that the two strands of a double-stranded polynucleotide may differ
slightly in length
3 o and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all
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nucleotides within a double-stranded polynucleotide molecule may not be
paired. Such
unpaired ends will in general not exceed 20 nt in length.
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".
The term "promoter" is used herein for its art-recognized meaning to
denote a portion of a gene containing DNA sequences that provide for the
binding of
RNA polymerise and initiation of transcription. Promoter sequences are
commonly,
but not always, found in the 5' non-coding regions of genes.
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.
A "secretory signal sequence" is a DNA sequence that encodes a
polypeptide (a "secretory peptide") that, as a component of a larger
polypeptide, directs
the larger polypeptide through a secretory pathway of a cell in which it is
synthesized.
2 0 The larger polypeptide is commonly cleaved to remove the secretory peptide
during
transit through the secretory pathway.
A "segment" is a portion of a larger molecule (e.g., polynucleotide or
polypeptide) having specified attributes. For example, a DNA segment encoding
a
specified polypeptide is a portion of a longer DNA molecule, such as a plasmid
or
2 5 plasmid fragment, that, when read from the 5' to the 3' direction, encodes
the sequence
of amino acids of the specified polypeptide.
Molecular weights and lengths of polymers determined by imprecise
analytical methods (e.g., gel electrophoresis) will be understood to be
approximate
values. When such a value is expressed as "about" X or "approximately" X, the
stated
3 o value of X will be understood to be accurate to ~10%.
All references cited herein are incorporated by reference in their entirety.
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The present invention provides novel cytokine polypeptides and
proteins. This novel cytokine, termed "zalpha29", was identified by the
presence of
polypeptide and polynucleotide features characteristic of four-helix-bundle
cytokines
(e.g., erythropoeitin, thrombopoietin, G-CSF, IL-2, IL,-4, leptin, and growth
hormone).
Analysis of the human zalpha29 amino acid sequence shown in SEQ m N0:2
indicates
the presence of four amphipathic, alpha-helical regions. These regions include
at least
amino acid residues 48 through 62 (helix A), 105 through 119 (helix B), 138
through
152 (helix C), and 171 through 185 (helix D). Within these helical regions,
residues
that are expected to lie within the core of the four-helix bundle occur at
positions 48,
51, 52, 55, 58, 59, 62, 105, 108, 109, 112, 115, 116, 119, 138, 141, 142, 145,
148, 149,
152, 171, 174, 175, 178, 181, 182, and 185 of SEQ ID N0:2. Residues 49, 50,
53, 54,
56, 57, 60, 61, 106, 107, 110, 111, 113, 114, 117, 118, 139, 140, 143, 144,
146, 147,
150, 151, 172, 173, 176, 177, 179, 180, 183, and 184 are expected to lie on
the exposed
surface of the bundle. Inter-helix loops comprise approximately residues 63
through
104 (loop A-B), 120 through 137 (loop B-C), and 153 through 170 (loop C-D).
The
human zalpha29 cDNA (SEQ ID NO:1 ) encodes a polypeptide of 190 amino acid
residues. While not wishing to be bound by theory, this sequence is predicted
to
include a secretory peptide of 26 residues. Cleavage after residue 26 will
result in a
mature polypeptide (residues 27-190 of SEQ ID N0:2) having a calculated
molecular
2 0 weight (exclusive of glycosylation) of 18,558 Da. Those skilled in the art
will
recognize, however, that some cytokines (e.g., endothelial cell growth factor,
basic
FGF, and IL-1(3) do not comprise conventional secretory peptides and are
secreted by a
mechanism that is not understood. There is a single consensus N-linked
glycosylation
site in SEQ ID N0:2 at residues 111-113. The cDNA also includes a clear
2 5 polyadenylation signal.
The mouse zalpha29 polypeptide (SEQ ID N0:4) is predicted to include
helices and loops at analogous positions, including helices at residues 47-61,
104-118,
137-151, and 170-184; and loops at residues 62-103, 119-136, and 152-169. See
Fig. 2.
Those skilled in the art will recognize that predicted domain boundaries
3 0 are somewhat imprecise and may vary by up to ~ 5 amino acid residues.
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Polypeptides of the present invention comprise at least 15 contiguous
amino acid residues of SEQ ID N0:2. Within certain embodiments of the
invention,
the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of
SEQ ID
N0:2, up to the entire predicted mature polypeptide (residues 27 to 190 of SEQ
ID
5 N0:2) or the primary translation product (residues 1 to 190 of SEQ ID N0:2).
Corresponding mouse zalpha29 polypeptides (see SEQ ID N0:4) are also provided
by
the invention. As disclosed in more detail below, these polypeptides can
further
comprise additional, non-zalpha29, polypeptide sequence(s).
Within the polypeptides of the present invention are polypeptides that
10 comprise an epitope-bearing portion of a protein as shown in SEQ ID N0:2 or
SEQ ID
N0:4. An "epitope" is a region of a protein to which an antibody can bind.
See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984.
Epitopes can
be linear or conformational, the latter being composed of discontinuous
regions of the
protein that form an epitope upon folding of the protein. Linear epitopes are
generally
at least 6 amino acid residues in length. 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 et al., Science 219:660-666,
1983.
Antibodies that recognize short, linear epitopes are particularly useful in
analytic and
diagnostic applications that employ denatured protein, such as Western
blotting (Tobin,
2 0 Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or in the analysis of
fixed cells or
tissue samples. Antibodies to linear epitopes are also useful for detecting
fragments of
zalpha29, such as might occur in body fluids or cell culture media.
Antigenic, epitope-bearing polypeptides of the present invention are
useful for raising antibodies, including monoclonal antibodies, that
specifically bind to
2 5 a zalpha29 protein. Antigenic, epitope-bearing polypeptides contain a
sequence of at
least six, often at least nine, commonly from 15 to about 30 contiguous amino
acid
residues of a zalpha29 protein (e.g., SEQ ID N0:2). Polypeptides comprising a
larger
portion of a zalpha29 protein, i.e. from 30 to 50 residues up to the entire
sequence, are
included. It is preferred that the amino acid sequence of the epitope-bearing
3 0 polypeptide is selected to provide substantial solubility in aqueous
solvents, that is the
sequence includes relatively hydrophilic residues, and hydrophobic residues
are
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substantially avoided. Such regions include the interdomain loops of zalpha29
and
fragments thereof, in particular loop B-C (residues 120-137 of SEQ ID N0:2),
which is
markedly hydrophilic (see Fig. 1C). Polypeptides in this regard include those
comprising residues 99-104, 129-134, and 162-167 of SEQ ID N0:2.
Of particular interest within the present invention are polypeptides that
comprise the entire four-helix bundle of a zalpha29 polypeptide (e.g.,
residues 48-185
of SEQ >D N0:2). Such polypeptides may further comprise all or part of one or
both of
the native zalpha29 amino-terminal (residues 27-47 of SEQ ID N0:2) and
carboxyl
terminal (residues 186-190 of SEQ ID N0:2) regions, as well as non-zalpha29
amino
acid residues or polypeptide sequences as disclosed in more detail below.
Polypeptides of the present invention can be prepared with one or more
amino acid substitutions, deletions or additions as compared to SEQ m N0:2.
These
changes will usually be of a minor nature, that is conservative amino acid
substitutions
and other changes that do not significantly affect the folding or activity of
the protein or
polypeptide, and include amino- or carboxyl-terminal extensions, such as an
amino-
terminal methionine residue, an amino or carboxyl-terminal cysteine residue to
facilitate subsequent linking to maleimide-activated keyhole limpet
hemocyanin, a
small linker peptide of up to about 20-25 residues, or an extension that
facilitates
purification (an affinity tag) as disclosed above. Two or more affinity tags
may be used
2 0 in combination. Polypeptides comprising affinity tags can further comprise
a
polypeptide linker and/or a proteolytic cleavage site between the zalpha29
polypeptide
and the affinity tag. Exemplary cleavage sites include thrombin cleavage sites
and
factor Xa cleavage sites.
The present invention further provides a variety of other polypeptide
2 5 fusions. For example, a zalpha29 polypeptide can be prepared as a fusion
to a
dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
Suitable
dimerizing proteins in this regard include immunoglobulin constant region
domains.
Immunoglobulin-zalpha29 polypeptide fusions can be expressed in genetically
engineered cells to produce a variety of multimeric zalpha29 analogs. In
addition, a
3 0 zalpha29 polypeptide can be joined to another bioactive molecule, such as
a cytokine,
to provide a multi-functional molecule. One or more helices of a zalpha29
polypeptide
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can be joined to another cytokine to enhance or otherwise modify its
biological
properties. Auxiliary domains can be fused to zalpha29 polypeptides to target
them to
specific cells, tissues, or macromolecules (e.g., collagen). For example, a
zalpha29
polypeptide or protein can be targeted to a predetermined cell type by fusing
a zalpha29
polypeptide to a ligand that specifically binds to a receptor on the surface
of the target
cell. In this way, polypeptides and proteins can be targeted for therapeutic
or diagnostic
purposes. A zalpha29 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.
Polypeptide fusions of the present invention will generally contain not
more than about 1,500 amino acid residues, often not more than about 1,200
residues,
usually not more than about 1,000 residues, and will in many cases be
considerably
smaller. For example, a zalpha29 polypeptide of 164 residues (residues 27-190
of SEQ
>D N0:2) can be fused to E. coli /3-galactosidase (1,021 residues; see
Casadaban et al.,
J. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue factor
Xa
cleavage site to yield a polypeptide of 1,199 residues. In a second example,
residues
27-190 of SEQ >D N0:2 can be fused to maltose binding protein (approximately
370
residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.
2 0 As disclosed above, the polypeptides of the present invention comprise
at least 15 contiguous residues of SEQ >D N0:2 or SEQ >D N0:4. These
polypeptides
may further comprise additional residues as shown in SEQ >Z7 N0:2, a variant
of SEQ
>D N0:2, or another protein as disclosed herein. "Variants of SEQ >D N0:2"
includes
polypeptides that are at least 85%, at least 90%, or at least 95% identical to
the
2 5 corresponding region of SEQ >D N0:2. 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 "BLOSUM62"
scoring
3 o matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids
are indicated
by the standard one-letter codes). The percent identity is then calculated as:
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Total number of identical matches
x 100
[length of the longer sequence plus the
number of gaps introduced into the longer
sequence in order to align the two sequences]
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14
N M
t!7N N O
M N N
~ i ; ~ M N
~3.~ ~
~p d'N N ~.,M
V>O N i ; i i ~
V7 ; M ~ O ~ M N N
N N O M N ~ N
~
_ N M ~ O M N i M i M
M M --'N N
x 0o , , , , i N ~ N N M
~pN ~ ~ N M M N O N N M M
i ~ i i i i i i i i i
w ~; N O M M ,-,N M ~ O ~ M N N
d ~nN N O M N ,..~p M ~ O ,-.N N
M ~ M M
U ~ ~ i i i i i m ; N M ;
~OM O N ~ ~ M d' ~ M M ~ O ~ d' M M
z ~ ~ M O O O ~' M M O N M N ,_,O ~ N M
N M ,-,O N O M N N ~ M N i ~ M N M
Q' ~ N N O i i O N ~ i i i N ; ~ O M N
~t O
Q x z n U a w ~ x ~ w x ~ w a.~ H
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The level of identity between amino acid sequences can be determined
using the "FASTA" similarity search algorithm disclosed by Pearson and Lipman
(Proc. Natl. Acad. Sci. USA 85:2444, 1988) and by Pearson (Meth. Enzymol.
183:63,
1990). Briefly, FASTA first characterizes sequence similarity by identifying
regions
5 shared by the query sequence (e.g., SEQ >D N0:2) and a test sequence that
have either
the highest density of identities (if the letup variable is 1 ) or pairs of
identities (if
letup=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
10 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 letup value), then the trimmed initial regions are examined
to
determine whether the regions can be joined to form an approximate alignment
with
15 gaps. Finally, the highest scoring regions of the two amino acid sequences
are aligned
using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and
Wunsch, J. Mol. Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787,
1974),
which allows for amino acid insertions and deletions. Preferred parameters for
FASTA analysis are: letup=l, gap opening penalty=10, gap extension penalty=1,
and
2 0 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, 1990 (ibid.).
FASTA can also be used to determine the sequence identity of nucleic
acid molecules using a ratio as disclosed above. For nucleotide sequence
2 5 comparisons, the letup value can range from one to six, preferably from
three to six,
most preferably three, with other parameters set as default.
The present invention includes polypeptides having one or more
conservative amino acid changes as compared with the amino acid sequence of
SEQ
ID N0:2. The BLOSUM62 matrix (Table 1) is an amino acid substitution matrix
3 0 derived from about 2,000 local multiple alignments of protein sequence
segments,
representing highly conserved regions of more than 500 groups of related
proteins
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16
(Henikoff and Henikoff, ibid.). Thus, 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. As used herein, the term
"conservative
amino acid substitution" 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, l, 2, or 3. Preferred
conservative amino acid substitutions are characterized by a BLOSUM62 value of
at
least one 1 (e.g., l, 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).
The proteins of the present invention can also comprise non-naturally
occuring amino acid residues. Non-naturally occuring amino acids include,
without
limitation, traps-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
traps-4-
hydroxyproline, N-methylglycine, allo-threonine, methylthreonine,
hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine,
pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-
azaphenylalanine, 4-
azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the
art
for incorporating non-naturally occuring 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
2 0 synthesizing amino acids and aminoacylating tRNA are known in the art.
Transcription and translation of plasmids containing nonsense mutations is
carried out
in a cell-free system comprising an E. coli S30 extract and commercially
available
enzymes and other reagents. Proteins are purified by chromatography. See, for
example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al.,
Methods
2 5 Enzymol. 202:301, 1991; Chung et al., Science 259:806-809, 1993; and Chung
et al.,
Proc. Natl. Acad. Sci. USA 90:10145-10149, 1993). In a second method,
translation is
carried out in Xenopus oocytes by microinjection of mutated mRNA and
chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-
19998,
1996). Within a third method, E. coli cells are cultured in the absence of a
natural
3 0 amino acid that is to be replaced (e.g., phenylalanine) and in the
presence of the
desired non-naturally occuring amino acids) (e.g., 2-azaphenylalanine, 3-
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17
azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-
naturally
occuring amino acid is incorporated into the protein in place of its natural
counterpart.
See, Koide et al., Biochem. 33:7470-7476, 1994. Naturally occuring amino acid
residues can be converted to non-naturally occuring species by in vitro
chemical
modification. Chemical modification can be combined with site-directed
mutagenesis
to further expand the range of substitutions (Wynn and Richards, Protein Sci.
2:395-
403, 1993).
Amino acid sequence changes are made in zalpha29 polypeptides so as
to minimize disruption of higher order structure essential to biological
activity. For
example, changes in amino acid residues will be made so as not to disrupt the
four-
helix bundle characteristic of the protein family. The effects of amino acid
sequence
changes can be predicted by computer modeling as disclosed above or determined
by
analysis of crystal structure (see, e.g., Lapthorn et al., ibid.). A
hydrophilicity profile
of SEQ m N0:2 is shown in Figs. lA-1D. Those skilled in the art will recognize
that
this hydrophilicity will be taken into account when designing alterations in
the amino
acid sequence of a zalpha29 polypeptide, so as not to disrupt the overall
profile.
Residues within the core of the four-helix bundle can be replaced with other
residues
as shown in SEQ m N0:6. The residues predicted to be on the exposed surface of
the four-helix bundle will be relatively intolerant of substitution. Other
candidate
2 0 amino acid substitutions within human zalpha29 are suggested by alignment
of the
human (SEQ m N0:2) and mouse (SEQ m N0:4) sequences as shown in Fig. 2,
which sequences are approximately 85% identical overall. The cysteine residue
at
position 160 of SEQ 1D N0:2 (position 159 of SEQ m N0:4) lies in loop C-D,
suggesting its participation in an interchain disulfide bond. This residue is
thus
2 5 expected to be relatively intolerant of substitution.
One skilled in the art may employ many well known techniques,
independently or in combination, to analyze and compare the structural
features that
affect folding of a variant protein or polypeptide to a standard molecule to
determine
whether such modifications would be significant. One well known and accepted
3 0 method for measuring folding is circular dichroism (CD). Measuring and
comparing
the CD spectra generated by a modified molecule and standard molecule are
routine in
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18
the art (Johnson, Proteins 7:205-214, 1990). Crystallography is another well
known
and accepted method for analyzing folding and structure. Nuclear magnetic
resonance
(NMR), digestive peptide mapping and epitope mapping are other known methods
for
analyzing folding and structural similarities between proteins and
polypeptides
(Schaanan et al., Science 257:961-964, 1992).
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 as
disclosed below to identify amino acid residues that are critical to the
activity of the
molecule.
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, Briefly, authors disclose methodssimultaneously
1989). these for
randomizing two more positionsin a polypeptide, for functional
or selecting
polypeptide, sequencing mutagenized polypeptidesdetermine
and then the to the
2 0 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. Patent No. 5,223,409; Fuse, WIPO Publication WO 92/06204) and
region-
directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA
7:127,
1988).
2 5 Variants of the disclosed zalpha29 DNA and polypeptide sequences can
be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-
391,
1994 and Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751, 1994. Briefly,
variant genes are generated by in vitro homologous recombination by random
fragmentation of a parent gene followed by reassembly using PCR, resulting in
3 0 randomly introduced point mutations. This technique can be modified by
using a
family of parent genes, such as allelic variants or genes from different
species, to
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19
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.
In many cases, the structure of the final polypeptide product will result
from processing of the nascent polypeptide chain by the host cell, thus the
final
sequence of a zalpha29 polypeptide produced by a host cell will not always
correspond
to the full sequence encoded by the expressed polynucleotide. For example,
expressing the complete zalpha29 sequence in a cultured mammalian cell is
expected
to result in removal of at least the secretory peptide, while the same
polypeptide
produced in a prokaryotic host would not be expected to be cleaved.
Differential
processing of individual chains may result in heterogeneity of expressed
polypeptides.
Zalpha29 proteins of the present invention are characterized by their
activity, that is, modulation of the proliferation, differentiation,
migration, adhesion, or
metabolism of responsive cell types. Biological activity of zalpha29 proteins
is
assayed using in vitro or in vivo assays designed to detect cell
proliferation,
differentiation, migration or adhesion; or changes in cellular metabolism
(e.g.,
production of other growth factors or other macromolecules). Many suitable
assays
are known in the art, and representative assays are disclosed herein. Assays
using
2 0 cultured cells are most convenient for screening, such as for determining
the effects of
amino acid substitutions, deletions, or insertions. However, in view of the
complexity
of developmental processes (e.g., angiogenesis, wound healing), in vivo assays
will
generally be employed to confirm and further characterize biological activity.
Certain
in vitro models, such as the three-dimensional collagen gel matrix model of
Pepper et
al. (Biochem. Biophys. Res. Comm. 189:824-831, 1992), are sufficiently complex
to
assay histological effects. Assays can be performed using exogenously produced
proteins, or may be carried out in vivo or in vitro using cells expressing the
polypeptide(s) of interest. Assays can be conducted using zalpha29 proteins
alone or
in combination with other growth factors, such as members of the VEGF family
or
3 0 hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor).
Representative
assays are disclosed below.
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Mutagenesis methods as disclosed above can be combined with high
volume or high-throughput screening methods to detect biological activity of
zalpha29
variant polypeptides. Assays that can be scaled up for high throughput include
mitogenesis assays, which can be run in a 96-well format. Mutagenized DNA
5 molecules that encode active zalpha29 polypeptides 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.
Using the methods discussed above, one of ordinary skill in the art can
10 prepare a variety of polypeptide fragments or variants of SEQ >D N0:2 or
SEQ >D
N0:4 that retain the activity of wild-type zalpha29.
The present invention also provides polynucleotide molecules,
including DNA and RNA molecules, that encode the zalpha29 polypeptides
disclosed
above. A representative DNA sequence encoding the amino acid sequence of SEQ m
15 N0:2 is shown in SEQ m NO:l, and a representative DNA sequence encoding the
amino acid sequence of SEQ )D N0:4 is shown in SEQ >D N0:3. 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 m N0:7 is a degenerate DNA sequence that encompasses all DNAs that encode
2 0 the zalpha29 polypeptide of SEQ >D NO: 2. Those skilled in the art will
recognize
that the degenerate sequence of SEQ m N0:7 also provides all RNA sequences
encoding SEQ m N0:2 by substituting U for T. Thus, zalpha29 polypeptide-
encoding
polynucleotides comprising nucleotides 1-534 or nucleotides 52-534 of SEQ >D
N0:7,
and their RNA equivalents are contemplated by the present invention, as are
segments
2 5 of SEQ >D N0:7 encoding other zalpha29 polypeptides disclosed herein.
Table 2 sets
forth the one-letter codes used within SEQ >D N0:7 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
3 0 to T, and G being complementary to C.
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21
TABLE 2
Nucleotide Resolutions Complement Resolutions
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 CMG S CMG
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
The degenerate codons used in SEQ >D N0:7, encompassing all
possible codons for a given amino acid, are set forth in Table 3, below.
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22
TABLE 3
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 CA~1
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~AspB RAY
Glu~GlnZ SAR
Any X NNN
Gap
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23
One of ordinary skill in the art will appreciate that some ambiguity is
introduced in determining a degenerate codon, representative of all possible
codons
encoding each amino acid. For example, the degenerate codon for serine (WSN)
can,
in some circumstances, encode arginine (AGR), and the degenerate codon for
arginine
(MGN) can, in some circumstances, encode serine (AGY). A similar relationship
exists between codons encoding phenylalanine and leucine. Thus, some
polynucleotides encompassed by the degenerate sequence may encode variant
amino
acid sequences, but one of ordinary skill in the art can easily identify such
variant
sequences by reference to the amino acid sequence of SEQ m NO: 2. Variant
1 o sequences can be readily tested for functionality as described herein.
One of ordinary skill in the art will also appreciate that different
species can exhibit preferential codon usage. See, in general, Grantham et
al., Nuc.
Acids Res. 8:1893-912, 1980; Haas et al. Curr. Biol. 6:315-24, 1996; Wain-
Hobson et
al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm,
Nuc.
Acids Res. 14:3075-87, 1986; and Ikemura, J. Mol. Biol. 158:573-97, 1982.
Introduction of preferred codon sequences into recombinant DNA can, for
example,
enhance production of the protein by making protein translation more efficient
within
a particular cell type or species. Therefore, the degenerate codon sequence
disclosed
in SEQ ID N0:7 serves as a template for optimizing expression of
polynucleotides in
2 o various cell types and species commonly used in the art and disclosed
herein.
Within certain embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID NO:1, or a
sequence
complementary thereto, under stringent conditions. In general, stringent
conditions are
selected to be about 5°C lower than the thermal melting point (Tm) for
the specific
2 5 sequence at a defined ionic strength and pH. The Tm 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 up to about 0.03 M at pH 7 and the temperature is at least
about 60°C.
As previously noted, the isolated polynucleotides of the present
3 0 invention include DNA and RNA. Methods for preparing DNA and RNA are well
known in the art. In general, RNA is isolated from a tissue or cell that
produces large
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24
amounts of zalpha29 RNA. Zalpha29 transcripts have also been detected in
numerous
tissues as disclosed below. Total RNA can be prepared using guanidine HCl
extraction followed by isolation by centrifugation in a CsCI gradient
(Chirgwin et al.,
Biochemistry 18:52-94, 1979). Poly (A)+ RNA is prepared from total RNA using
the
method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972).
Complementary DNA (cDNA) is prepared from poly(A)+ RNA using known methods.
In the alternative, genomic DNA can be isolated. Polynucleotides encoding
zalpha29
polypeptides are then identified and isolated by, for example, hybridization
or PCR.
Full-length clones encoding zalpha29 can be obtained by conventional
cloning procedures. Complementary DNA (cDNA) clones are commonly used within
protein production systems, although for some applications (e.g., expression
in
transgenic animals) it may be preferable to use a genomic clone, or to modify
a cDNA
clone to include at least one genomic intron. A partial human genomic zalpha29
sequence is shown in SEQ ID N0:14. This sequence comprises an exon from
nucleotide 1885 to nucleotide 2112 (corresponding to nucleotides 483-710 of
SEQ >D
NO:1 ). Partial mouse genomic sequences are shown in SEQ >D NO:15 and SEQ >D
N0:16. Within SEQ >D NO:15, nucleotides 6-165 are an exon corresponding to
nucleotides 40-199 of SEQ >D N0:3. Within SEQ >D N0:16, nucleotides 175-295
are
an exon corresponding to nucleotides 200-320 of SEQ >D N0:3. Methods for
2 0 preparing cDNA and genomic clones are well known and within the level of
ordinary
skill in the art, and include the use of the sequence disclosed herein, or
parts thereof,
for probing or priming a library. Expression libraries can be probed with
antibodies to
zalpha29, receptor fragments, or other specific binding partners.
Zalpha29 polynucleotide sequences disclosed herein can also be used
2 5 as probes or primers to clone 5' non-coding regions of a zalpha29 gene.
Promoter
elements from a zalpha29 gene can be used to direct the expression of
heterologous
genes in, for example, transgenic animals or patients treated with gene
therapy.
Cloning of 5' flanking sequences also facilitates production of zalpha29
proteins by
"gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly,
expression of an
3 0 endogenous zalpha29 gene in a cell is altered by introducing into the
zalpha29 locus a
DNA construct comprising at least a targeting sequence, a regulatory sequence,
an
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exon, and an unpaired splice donor site. The targeting sequence is a zalpha29
5' non-
coding sequence that permits homologous recombination of the construct with
the
endogenous zalpha29 locus, whereby the sequences within the construct become
operably linked with the endogenous zalpha29 coding sequence. In this way, an
5 endogenous zalpha29 promoter can be replaced or supplemented with other
regulatory
sequences to provide enhanced, tissue-specific, or otherwise regulated
expression.
Those skilled in the art will recognize that the sequences disclosed in
SEQ m NOS:1-2 and 3-4 represent single allele of human and mouse zalpha29,
respectively. Allelic variants of these sequences can be cloned by probing
cDNA or
10 genomic libraries from different individuals according to standard
procedures.
The present invention further provides counterpart polypeptides and
polynucleotides from other species ("orthologs"). Of particular interest are
zalpha29
polypeptides from other mammalian species, including murine, porcine, ovine,
bovine,
canine, feline, equine, and other primate polypeptides. Orthologs of human
zalpha29
15 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 zalpha29
as
disclosed above. A library is then prepared from mRNA of a positive tissue or
cell
line. A zalpha29-encoding cDNA can then be isolated by a variety of methods,
such
2 0 as by probing with a complete or partial human cDNA or with one or more
sets of
degenerate probes based on the disclosed sequence. A cDNA can also be cloned
using
the polymerise chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202),
using
primers designed from the representative human and mouse zalpha29 sequences
disclosed herein. Within an additional method, the cDNA library can be used to
2 5 transform or transfect host cells, and expression of the cDNA of interest
can be
detected with an antibody to a zalpha29 polypeptide. Similar techniques can
also be
applied to the isolation of genomic clones.
For any zalpha29 polypeptide, including variants and fusion proteins,
one of ordinary skill in the art can readily generate a fully degenerate
polynucleotide
3 0 sequence encoding that polypeptide using the information set forth in
Tables 3 and 4,
above. Moreover, those of skill in the art can use standard software to devise
zalpha29
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26
variants based upon the nucleotide and amino acid sequences described herein.
The
present invention thus provides a computer-readable medium encoded with a data
structure that provides at least one of the following sequences: SEQ ID NO:1,
SEQ ID
N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:7, and portions
thereof. 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
l0 digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
The zalpha29 polypeptides of the present invention, including full-
length polypeptides, biologically active fragments, and fusion polypeptides
can be
produced according to conventional techniques using cells into which have been
introduced an expression vector encoding the polypeptide. As used herein, a
"cell into
which has been introduced an expression vector" includes both cells that have
been
directly manipulated by the introduction of exogenous DNA molecules and
progeny
thereof that contain the introduced DNA. 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.
Techniques for
2 0 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, NY,
1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, John
Wiley
and Sons, Inc., NY, 1987.
2 5 In general, a DNA sequence encoding a zalpha29 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
3 0 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
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27
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.
To direct a zalpha29 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 zalpha29, or may be derived from another secreted protein (e.g., t-
PA; see,
U.S. Patent No. 5,641,655) or synthesized de novo. The secretory signal
sequence
is operably linked to the zalpha29 DNA sequence, i.e., the two sequences are
joined in
the correct reading frame and positioned to direct the newly sythesized
polypeptide
into the secretory pathway of the host cell. Secretory signal sequences are
commonly
positioned 5' to the DNA sequence encoding the polypeptide of interest,
although
certain signal sequences may be positioned elsewhere in the DNA sequence of
interest
(see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S.
Patent No.
5,143,830).
Expression of zalpha29 polypeptides via a host cell secretory pathway
is expected to result in the production of multimeric proteins. Such multimers
include
both homomultimers and heteromultimers, the latter including proteins
comprising
2 0 only zalpha29 polypeptides and proteins including zalpha29 and
heterologous
polypeptides (e.g., a second four-helix-bundle cytokine polypeptide). If a
mixture of
proteins results from expression, individual species are isolated by
conventional
methods. Monomers, dimers, and higher order multimers are separated by, for
example, size exclusion chromatography. Heteromultimers can be separated from
2 5 homomultimers by immunoaffinity chromatography using antibodies specific
for
individual dimers or by sequential immunoaffinity steps using antibodies
specific for
individual component polypeptides. See, in general, U.S. Patent No. 5,094,941.
Multimers may also be assembled in vitro upon incubation of component
polypeptides
under suitable conditions. In general, in vitro assembly will include
incubating the
3 o protein mixture under denaturing and reducing conditions followed by
refolding and
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28
reoxidation of the polypeptides to from homodimers and heterodimers. Recovery
and
assembly of proteins expressed in bacterial cells is disclosed below.
Cultured mammalian cells can be used as hosts within the present
invention. Methods for introducing exogenous DNA into mammalian host cells
include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725,
1978;
Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845,
1982),
DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-
mediated
transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus
15:80,
1993). The production of recombinant polypeptides in cultured mammalian cells
is
disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen
et al.,
U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and
Ringold,
U.S. Patent 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; or CHO DG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)
cell
lines. Additional suitable cell lines are known in the art and available from
public
depositories such as the American Type Culture Collection, Manassas, VA.
Promoters for use in cultured mammalian cells include promoters from SV-40 or
cytomegalovirus (see, e.g., U.S. Patent No. 4,956,288), metallothionein gene
promoters (U.S. Patent Nos. 4,579,821 and 4,601,978), and the adenovirus major
late
promoter. Expression vectors for use in mammalian cells include pZP-1 and pZP-
9,
which have been deposited with the American Type Culture Collection, Manassas,
VA
2 5 USA under accession numbers 98669 and 98668, respectively, and derivatives
thereof.
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
3 0 transfectants." An exemplary selectable marker is a gene encoding
resistance to the
antibiotic neomycin. Selection is carried out in the presence of a neomycin-
type drug,
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29
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. An exemplary
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.
The adenovirus system (disclosed in more detail below) can also be
used for protein production in vitro. By culturing adenovirus-infected non-293
cells
under conditions where the cells are not rapidly dividing, the cells can
produce
proteins for extended periods of time. For instance, BHK cells are grown to
confluence in cell factories, then exposed to the adenoviral vector encoding
the
secreted protein of interest. The cells are then grown under serum-free
conditions,
which allows infected cells to survive for several weeks without significant
cell
division. In an alternative method, adenovirus vector-infected 293 cells can
be grown
as adherent cells or in suspension culture at relatively high cell density to
produce
significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-55,
1994).
With either protocol, an expressed, secreted heterologous protein can be
repeatedly
2 0 isolated from the cell culture supernatant, lysate, or membrane fractions
depending on
the disposition of the expressed protein in the cell. Within the infected 293
cell
production protocol, non-secreted proteins can also be effectively obtained.
Insect cells can be infected with recombinant baculovirus, commonly
derived from Autographa californica nuclear polyhedrosis virus (AcNPV)
according
2 5 to methods known in the art. Within one method, recombinant baculovirus is
produced through the use of a transposon-based system described by Luckow et
al. (J.
Virol. 67:4566-4579, 1993). This system, which utilizes transfer vectors, is
commercially available in kit form (Bac-to-BacT"" kit; Life Technologies,
Rockville,
MD). The transfer vector (e.g., pFastBaclTM; Life Technologies) contains a Tn7
3 0 transposon to move the DNA encoding the protein of interest into a
baculovirus
genome maintained in E. coli as a large plasmid called a "bacmid." See, Hill-
Perkins
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and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol.
75:1551-
1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.
In
addition, transfer vectors can include an in-frame fusion with DNA encoding a
polypeptide extension or affinity tag as disclosed above. Using techniques
known in
5 the art, a transfer vector containing a zalpha29-encoding sequence is
transformed into
E. coli host cells, and the cells are screened for bacmids which contain an
interrupted
lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the
recombinant baculovirus genome is isolated, using common techniques, and used
to
transfect Spodoptera frugiperda cells, such as Sf9 cells. Recombinant virus
that
1 o expresses zalpha29 protein is subsequently produced. Recombinant viral
stocks are
made by methods commonly used the art.
For protein production, the recombinant virus is used to infect host
cells, typically a cell line derived from the fall armyworm, Spodoptera
frugiperda
(e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High FiveT~~ cells;
Invitrogen,
15 Carlsbad, CA). See, for example, U.S. Patent No. 5,300,435. Serum-free
media are
used to grow and maintain the cells. Suitable media formulations are known in
the art
and can be obtained from commercial suppliers. The cells are grown up from an
inoculation density of approximately 2-5 x 105 cells to a density of 1-2 x 106
cells, at
which time a recombinant viral stock is added at a multiplicity of infection
(MOI) of
2 0 0.1 to 10, more typically near 3. Procedures used are generally known in
the art.
Other higher eukaryotic cells can also be used as hosts, including plant
cells and avian cells. The use of Agrobacterium rhizogenes as a vector for
expressing
genes in plant cells has been reviewed by Sinkar et al., J. Biosci.
(Bangalore) 11:47-
58, 1987.
2 5 Fungal cells, including yeast cells, can also be used within the present
invention. Yeast species of particular interest in this regard include
Saccharomyces
cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming
S.
cerevisiae cells with exogenous DNA and producing recombinant polypeptides
therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311;
3 0 Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No.
4,870,008; Welch
et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No.
4,845,075.
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31
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). An exemplary vector system for use in Saccharomyces
cerevisiae is
the POTl vector system disclosed by Kawasaki et al. (U.S. Patent 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. Patent No. 4,599,311; Kingsman et al.,
U.S.
Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol
dehydrogenase genes. See also U.S. Patents 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; Cregg, U.S. Patent No. 4,882,279; and Raymond
et
al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilized according to the
methods
of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming
Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No.
5,162,228.
Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent
No.
4,486,533. Production of recombinant proteins in Pichia methanolica is
disclosed in
2 0 U.S. Patents No. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.
Prokaryotic host cells, including strains of the bacteria Escherichia coli,
Bacillus and other genera are also useful host cells within the present
invention.
Techniques for transforming these hosts and expressing foreign DNA sequences
cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.).
When
2 5 expressing a zalpha29 polypeptide in bacteria such as E . cot i, 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
3 o 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
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32
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.
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
1 o 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. Liquid cultures are provided with
sufficient
aeration by conventional means, such as shaking of small flasks or sparging of
fermentors.
Depending upon the intended use, the polypeptides and proteins of the
present invention can be purified to __>80% purity, >_90% purity, >_95%
purity, or to a
pharmaceutically pure state, that is greater than 99.9% pure with respect to
2 0 contaminating macromolecules, particularly other proteins and nucleic
acids, and free
of infectious and pyrogenic agents. A purified polypeptide or protein can be
prepared
substantially free of other polypeptides or proteins, particularly those of
animal origin.
Expressed recombinant zalpha29 proteins (including chimeric
polypeptides and multimeric proteins) are purified by conventional protein
purification
2 5 methods, typically by a combination of chromatographic techniques. See, in
general,
Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology,
Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and
Practice,
Springer-Verlag, New York, 1994. Proteins comprising a polyhistidine affinity
tag
(typically about 6 histidine residues) are purified by affinity chromatography
on a
3 o nickel chelate resin. See, for example, Houchuli et al., Bioll'echnol. 6:
1321-1325,
1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity
CA 02377580 2001-12-20
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33
chromatography according to conventional procedures. See, for example,
Grussenmeyer et al., ibid. Maltose binding protein fusions are purified on an
amylose
column according to methods known in the art.
Zalpha29 polypeptides can also be prepared through chemical synthesis
according to methods known in the art, including exclusive solid phase
synthesis,
partial solid phase methods, fragment condensation or classical solution
synthesis.
See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al.,
Solid
Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL,
1984;
Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase
Peptide
Synthesis: A Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis
is
particularly advantageous for the preparation of smaller polypeptides.
Using methods known in the art, zalpha29 proteins can be prepared as
monomers or multimers; glycosylated or non-glycosylated; pegylated or non-
pegylated; and may or may not include an initial methionine amino acid
residue.
Target cells for use in zalpha29 activity assays include, without
limitation, vascular cells (especially endothelial cells and smooth muscle
cells),
hematopoietic (myeloid and lymphoid) cells, liver cells (including
hepatocytes,
fenestrated endothelial cells, Kupffer cells, and Ito cells), fibroblasts
(including human
dermal fibroblasts and lung fibroblasts), fetal lung cells, articular
synoviocytes,
2 o pericytes, chondrocytes, osteoblasts, and prostate epithelial cells.
Endothelial cells
and hematopoietic cells are derived from a common ancestral cell, the
hemangioblast
(Choi et al., Development 125:725-732, 1998).
Activity of zalpha29 proteins can be measured in vitro using cultured
cells or in vivo by administering molecules of the claimed invention to an
appropriate
2 5 animal model. Assays measuring cell proliferation or differentiation are
well known in
the art. For example, assays measuring proliferation include such assays as
chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New
Drugs
8:347-354, 1990), incorporation of radiolabelled nucleotides (as disclosed by,
e.g.,
Raines and Ross, Methods Enzymol. 109:749-773, 1985; Wahl et al., Mol. Cell
Biol.
3 0 8:5016-5025, 1988; and Cook et al., Analytical Biochem. 179:1-7, 1989),
incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating
cells
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34
(Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use of
tetrazolium
salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res.
48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et
al.,
Cancer Res. 48:4827-4833, 1988). Differentiation can be assayed using suitable
precursor cells that can be induced to differentiate into a more mature
phenotype.
Assays measuring differentiation include, for example, measuring cell-surface
markers
associated with stage-specific expression of a tissue, enzymatic activity,
functional
activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis,
Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol.
Bioprocesses,
l0 161-171, 1989; all incorporated herein by reference).
Zalpha29 activity may also be detected using assays designed to
measure zalpha29-induced production of one or more additional growth factors
or
other macromolecules. Such assays include those for determining the presence
of
hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming
growth
factor alpha (TGFa), interleukin-6 (IL,-6), VEGF, acidic fibroblast growth
factor
(aFGF), angiogenin, and other macromolecules produced by the liver. Suitable
assays
include mitogenesis assays using target cells responsive to the macromolecule
of
interest, receptor-binding assays, competition binding assays, immunological
assays
(e.g., ELISA), and other formats known in the art. Metalloprotease secretion
is
2 o measured from treated primary human dermal fibroblasts, synoviocytes and
chondrocytes. The relative levels of collagenase, gelatinase and stromalysin
produced
in response to culturing in the presence of a zalpha29 protein is measured
using
zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106, 1990).
Procollagen/collagen synthesis by dermal fibroblasts and chondrocytes in
response to a
2 5 test protein is measured using 3H-proline incorporation into nascent
secreted collagen.
3H-labeled collagen is visualized by SDS-PAGE followed by autoradiography
(Unemori and Amento, J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan
(GAG) secretion from dermal fibroblasts and chondrocytes is measured using a
1,9-
dimethylmethylene blue dye binding assay (Farndale et al., Biochim. Biophys.
Acta
3 0 883:173-177, 1986). Collagen and GAG assays are also carried out in the
presence of
CA 02377580 2001-12-20
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IL-1(3 or TGF-~3 to examine the ability of zalpha29 protein to modify the
established
responses to these cytokines.
Monocyte activation assays are carried out ( 1 ) to look for the ability of
zalpha29 proteins to further stimulate monocyte activation, and (2) to examine
the
5 ability of zalpha29 proteins to modulate attachment-induced or endotoxin-
induced
monocyte activation (Fuhlbrigge et al., J. Immunol. 138: 3799-3802, 1987). IL-
1 (3 and
TNFa levels produced in response to activation are measured by ELISA
(Biosource,
Inc. Camarillo, CA). Monocyte/macrophage cells, by virtue of CD14 (LPS
receptor),
are exquisitely sensitive to endotoxin, and proteins with moderate levels of
endotoxin
10 like activity will activate these cells.
Hematopoietic activity of zalpha29 proteins can be assayed on various
hematopoietic cells in culture. Suitable assays include primary bone marrow
colony
assays and later stage lineage-restricted colony assays, which are known in
the art
(e.g., Holly et al., WIPO Publication WO 95/21920). Marrow cells plated on a
15 suitable semi-solid medium (e.g., 50% methylcellulose containing 15% fetal
bovine
serum, 10% bovine serum albumin, and 0.6% PSN antibiotic mix) are incubated in
the
presence of test polypeptide, then examined microscopically for colony
formation.
Known hematopoietic factors are used as controls. Mitogenic activity of
zalpha29
polypeptides on hematopoietic cell lines can be measured as disclosed above.
2 0 Cell migration is assayed essentially as disclosed by Kahler et al.
(Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). A
protein is
considered to be chemotactic if it induces migration of cells from an area of
low
protein concentration to an area of high protein concentration. A typical
assay is
performed using modified Boyden chambers with a polystryrene membrane
separating
2 5 the two chambers (e.g., Transwell~; Corning Costar Corp.). The test
sample, diluted
in medium containing 1 % BSA, is added to the lower chamber of a 24-well plate
containing Transwells. Cells are then placed on the Transwell insert that has
been
pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of
incubation at
37°C. Non-migrating cells are wiped off the top of the Transwell
membrane, and cells
3 0 attached to the lower face of the membrane are fixed and stained with 0.1
% crystal
violet. Stained cells are then extracted with 10% acetic acid and absorbance
is
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36
measured at 600 nm. Migration is then calculated from a standard calibration
curve.
Cell migration can also be measured using the matrigel method of Grant et al.
("Angiogenesis as a component of epithelial-mesenchymal interactions" in
Goldberg
and Rosen, Epithelial-Mesenchymal Interaction in Cancer, Birkhauser Verlag,
1995,
235-248; Baatout, Anticancer Research 17:451-456, 1997).
Cell adhesion activity is assayed essentially as disclosed by LaFleur et
al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter plates are
coated with
the test protein, non-specific sites are blocked with BSA, and cells (such as
smooth
muscle cells, leukocytes, or endothelial cells) are plated at a density of
approximately
104 - 105 cells/well. The wells are incubated at 37°C (typically for
about 60 minutes),
then non-adherent cells are removed by gentle washing. Adhered cells are
quantitated
by conventional methods (e.g., by staining with crystal violet, lysing the
cells, and
determining the optical density of the lysate). Control wells are coated with
a known
adhesive protein, such as fibronectin or vitronectin.
The activity of zalpha29 proteins can be measured with a silicon-based
biosensor microphysiometer that measures the extracellular acidification rate
or proton
excretion associated with receptor binding and subsequent physiologic cellular
responses. An exemplary such device is the CytosensorTM Microphysiometer
manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular
responses,
2 0 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 257:1906-1912, 1992; Pitchford et al.,
Meth.
Enzymol. 228:84-108, 1997; Arimilli et al., J. Immunol. Meth. 212:49-59, 1998;
and
Van Liefde et al., Eur. J. Pharmacol. 346:87-95, 1998. The microphysiometer
can be
2 5 used for assaying adherent or non-adherent eukaryotic or prokaryotic
cells. By
measuring extracellular acidification changes in cell media over time, the
microphysiometer directly measures cellular responses to various stimuli,
including
zalpha29 proteins, their agonists, and antagonists. The microphysiometer can
be used
to measure responses of a zalpha29-responsive eukaryotic cell, compared to a
control
3 0 eukaryotic cell that does not respond to zalpha29 polypeptide. Zalpha29-
responsive
eukaryotic cells comprise cells into which a receptor for zalpha29 has been
transfected
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37
creating a cell that is responsive to zalpha29, as well as cells naturally
responsive to
zalpha29. Differences, measured by a change in extracellular acidification, in
the
response of cells exposed to zalpha29 polypeptide relative to a control not
exposed to
zalpha29, are a direct measurement of zalpha29-modulated cellular responses.
Moreover, such zalpha29-modulated responses can be assayed under a variety of
stimuli. The present invention thus provides methods of identifying agonists
and
antagonists of zalpha29 proteins, comprising providing cells responsive to a
zalpha29
polypeptide, culturing a first portion of the cells in the absence of a test
compound,
culturing a second portion of the cells in the presence of a test compound,
and
1 o detecting a change in a cellular response of the second portion of the
cells as compared
to the first portion of the cells. The change in cellular response is shown as
a
measurable change in extracellular acidification rate. Culturing a third
portion of the
cells in the presence of a zalpha29 protein and the absence of a test compound
provides a positive control for the zalpha29-responsive cells and a control to
compare
the agonist activity of a test compound with that of the zalpha29 polypeptide.
Antagonists of zalpha29 can be identified by exposing the cells to zalpha29
protein in
the presence and absence of the test compound, whereby a reduction in zalpha29-
stimulated activity is indicative of antagonist activity in the test compound.
Expression of zalpha29 polynucleotides in animals provides models for
2 0 further study of the biological effects of overproduction or inhibition of
protein
activity in vivo. Zalpha29-encoding polynucleotides and antisense
polynucleotides can
be introduced into test animals, such as mice, using viral vectors or naked
DNA, or
transgenic animals can be produced.
One in vivo approach for assaying proteins of the present invention
2 5 utilizes viral delivery systems. Exemplary viruses for this purpose
include adenovirus,
herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
Adenovirus, a double-stranded DNA virus, is currently the best studied gene
transfer
vector for delivery of heterologous nucleic acids. For review, see Becker et
al., Meth.
Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-
53,
3 0 1997. The adenovirus system offers several advantages. Adenovirus can (i)
accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii)
infect a
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38
broad range of mammalian cell types; and (iv) be used with many different
promoters
including ubiquitous, tissue specific, and regulatable promoters. Because
adenoviruses are stable in the bloodstream, they can be administered by
intravenous
injection.
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. In an exemplary system, the essential E1 gene is deleted
from the
viral vector, and the virus will not replicate unless the El gene is provided
by the host
cell (e.g., the human 293 cell line). When intravenously administered to
intact
animals, adenovirus primarily targets the liver. If the adenoviral delivery
system has
an E1 gene deletion, the virus cannot replicate in the host cells. However,
the host's
tissue (e.g., liver) will express and process (and, if a signal sequence is
present,
secrete) the heterologous protein. Secreted proteins will enter the
circulation in the
highly vascularized liver, and effects on the infected animal can be
determined.
An alternative method of gene delivery comprises removing cells from
the body and introducing a vector into the cells as a naked DNA plasmid. The
transformed cells are then re-implanted in the body. Naked DNA vectors are
introduced into host cells by methods known in the art, including
transfection,
2 0 electroporation, microinjection, transduction, cell fusion, DEAF dextran,
calcium
phosphate precipitation, use of a gene gun, or use of a DNA vector
transporter. See,
Wu et al., J. Biol. Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem.
267:963-
967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
Transgenic mice, engineered to express a zalpha29 gene, and mice that
2 5 exhibit a complete absence of zalpha29 gene function, referred to as
"knockout mice"
(Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et
al.,
Nature 366:740-742, 1993). These mice can be employed to study the zalpha29
gene
and the protein encoded thereby in an in vivo system. Transgenic mice are
particularly
useful for investigating the role of zalpha29 proteins in early development in
that they
3 0 allow the identification of developmental abnormalities or blocks
resulting from the
over- or underexpression of a specific factor. See also, Maisonpierre et al.,
Science
CA 02377580 2001-12-20
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39
277:55-60, 1997 and Hanahan, Science 277:48-50, 1997. Promoters for transgenic
expression include promoters from metallothionein and albumin genes.
Antisense methodology can be used to inhibit zalpha29 gene
transcription to examine the effects of such inhibition in vivo.
Polynucleotides that are
complementary to a segment of a zalpha29-encoding polynucleotide (e.g., a
polynucleotide as set forth in SEQ ID NO:1 ) are designed to bind to zalpha29-
encoding mRNA and to inhibit translation of such mRNA. Such antisense
oligonucleotides can also be used to inhibit expression of zalpha29
polypeptide-
encoding genes in cell culture.
Most four-helix bundle cytokines as well as other proteins produced by
activated lymphocytes play an important biological role in cell
differentiation,
activation, recruitment and homeostasis of cells throughout the body. Zalpha29
and
inhibitors of zalpha29 activity are expected to have a variety of therapeutic
applications. These therapeutic applications include treatment of diseases
which
require immune regulation, including autoimmune diseases such as rheumatoid
arthritis, multiple sclerosis, myasthenia gravis, systemic lupus
erythematosis, and
diabetes. Zalpha29 may be important in the regulation of inflammation, and
therefore
would be useful in treating rheumatoid arthritis, asthma and sepsis. There may
be a
role of zalpha29 in mediating tumorgenesis, whereby a zalpha29 antagonist
would be
2 o useful in the treatment of cancer. Zalpha29 may be useful in modulating
the immune
system, whereby zalpha29 and zalpha29 antagonists may be used for reducing
graft
rejection, preventing graft-vs-host disease, boosting immunity to infectious
diseases,
treating immunocompromised patients (e.g., HIV+ patients), or in improving
vaccines.
Zalpha29 polypeptides can be administered alone or in combination
2 5 with other vasculogenic or angiogenic agents, including VEGF. When using
zalpha29 in combination with an additional agent, the two compounds can be
administered simultaneously or sequentially as appropriate for the specific
condition
being treated.
For pharmaceutical use, zalpha29 proteins are formulated for topical or
3 0 parenteral, particularly intravenous or subcutaneous, delivery according
to
conventional methods. In general, pharmaceutical formulations will include a
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zalpha29 polypeptide in combination with a pharmaceutically acceptable
vehicle, such
as saline, buffered saline, 5% dextrose in water, or the like. Formulations
may further
include one or more excipients, preservatives, solubilizers, buffering agents,
albumin
to prevent protein loss on vial surfaces, etc. Methods of formulation are well
known
5 in the art and are disclosed, for example, in Remington: The Science and
Practice of
Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995.
Zalpha29
will preferably be used in a concentration of about 10 to 100 ~g/ml of total
volume,
although concentrations in the range of 1 ng/ml to 1000 ~g/ml may be used. For
topical application, such as for the promotion of wound healing, the protein
will be
10 applied in the range of 0.1-10 p,g/cm2 of wound area, with the exact dose
determined
by the clinician according to accepted standards, taking into account the
nature and
severity of the condition to be treated, patient traits, etc. Determination of
dose is
within the level of ordinary skill in the art. Dosing is daily or
intermittently over the
period of treatment. Intravenous administration will be by bolus injection or
infusion
15 over a typical period of one to several hours. Sustained release
formulations can also
be employed. In general, a therapeutically effective amount of zalpha29 is an
amount
sufficient to produce a clinically significant change in the treated
condition, such as a
clinically significant change in hematopoietic or immune function, a
significant
reduction in morbidity, or a significantly increased histological score.
2 0 Zalpha29 proteins, agonists, and antagonists are useful for modulating
the expansion, proliferation, activation, differentiation, migration, or
metabolism of
responsive cell types, which include both primary cells and cultured cell
lines. Of
particular interest in this regard are hematopoietic cells (including stem
cells and
mature myeloid and lymphoid cells), endothelial cells, smooth muscle cells,
2 5 fibroblasts, and hepatocytes. Zalpha29 polypeptides are added to tissue
culture media
for these cell types at a concentration of about 10 pg/ml to about 100 ng/ml.
Those
skilled in the art will recognize that zalpha29 proteins can be advantageously
combined with other growth factors in culture media.
Within the laboratory research field, zalpha29 proteins can also be used
3 0 as molecular weight standards or as reagents in assays for determining
circulating
CA 02377580 2001-12-20
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41
levels of the protein, such as in the diagnosis of disorders characterized by
over- or
under-production of zalpha29 protein or in the analysis of cell phenotype.
Zalpha29 proteins can also be used to identify inhibitors of their
activity. Test compounds are added to the assays disclosed above to identify
compounds that inhibit the activity of zalpha29 protein. In addition to those
assays
disclosed above, samples can be tested for inhibition of zalpha29 activity
within a
variety of assays designed to measure receptor binding or the
stimulation/inhibition of
zalpha29-dependent cellular responses. For example, zalpha29-responsive cell
lines
can be transfected with a reporter gene construct that is responsive to a
zalpha29-
stimulated cellular pathway. Reporter gene constructs of this type are known
in the
art, and will generally comprise a zalpha29-activated serum response element
(SRE)
operably linked to a gene encoding an assayable protein, such as luciferase.
Candidate
compounds, solutions, mixtures or extracts are tested for the ability to
inhibit the
activity of zalpha29 on the target cells as evidenced by a decrease in
zalpha29
stimulation of reporter gene expression. Assays of this type will detect
compounds
that directly block zalpha29 binding to cell-surface receptors, as well as
compounds
that block processes in the cellular pathway subsequent to receptor-ligand
binding. In
the alternative, compounds or other samples can be tested for direct blocking
of
zalpha29 binding to receptor using zalpha29 tagged with a detectable label
(e.g., l2sl,
2 0 biotin, horseradish peroxidase, FTTC, and the like). Within assays of this
type, the
ability of a test sample to inhibit the binding of labeled zalpha29 to the
receptor is
indicative of inhibitory activity, which can be confirmed through secondary
assays.
Receptors used within binding assays may be cellular receptors or isolated,
immobilized receptors.
2 5 As used herein, the term "antibodies" includes polyclonal antibodies,
monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and
Fab
fragments, single chain antibodies, and the like, including genetically
engineered
antibodies. Non-human antibodies may be humanized by grafting non-human CDRs
onto human framework and constant regions, or by incorporating the entire non-
3 0 human variable domains (optionally "cloaking" them with a human-like
surface by
replacement of exposed residues, wherein the result is a "veneered" antibody).
In
CA 02377580 2001-12-20
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42
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.
One skilled in the art can generate humanized antibodies with specific and
different
constant domains (i.e., different Ig subclasses) to facilitate or inhibit
various immune
functions associated with particular antibody constant domains. Antibodies are
defined to be specifically binding if they bind to a zalpha29 polypeptide or
protein
with an affinity at least 10-fold greater than the binding affinity to control
(non-
zalpha29) polypeptide or protein. The affinity of a monoclonal antibody can be
readily determined by one of ordinary skill in the art (see, for example,
Scatchard,
Ann. NY Acad. Sci. 51: 660-672, 1949).
Methods for preparing polyclonal and monoclonal antibodies are well
known in the art (see for example, Hurrell, J. G. R., Ed., Monoclonal
Hybridoma
Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL,
1982,
which is incorporated herein by reference). 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 zalpha29 polypeptide may be increased through the use
of an
2 o 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 a zalpha29 polypeptide 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",
2 5 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.
Alternative techniques for generating or selecting antibodies include in
vitro exposure of lymphocytes to zalpha29 polypeptides, and selection of
antibody
3 0 display libraries in phage or similar vectors (e.g., through the use of
immobilized or
labeled zalpha29 polypeptide). Human antibodies can be produced in transgenic,
non-
CA 02377580 2001-12-20
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43
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.
A variety of assays known to those skilled in the art can be utilized to
detect antibodies which specifically bind to zalpha29 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-
1 o immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot
assays, Western blot assays, inhibition or competition assays, and sandwich
assays.
Antibodies to zalpha29 may be used for affinity purification of the
protein, within diagnostic assays for determining circulating levels of the
protein; for
detecting or quantitating soluble zalpha29 polypeptide as a marker of
underlying
pathology or disease; for immunolocalization within whole animals or tissue
sections,
including immunodiagnostic applications; for immunohistochemistry; and as
antagonists to block protein activity in vitro and in vivo. Antibodies to
zalpha29 may
also be used for tagging cells that express zalpha29; for affinity
purification of
zalpha29 polypeptides and proteins; in analytical methods employing FACS; for
2 0 screening expression libraries; and for generating anti-idiotypic
antibodies.
Antibodies can be linked to other compounds, including therapeutic and
diagnostic
agents, using known methods to provide for targetting of those compounds to
cells
expressing receptors for zalpha29. For certain applications, including in
vitro and in
vivo diagnostic uses, it is advantageous to employ labeled antibodies.
Suitable direct
2 5 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 of the present invention may
also be
directly or indirectly conjugated to drugs, toxins, radionuclides and the
like, and these
3 0 conjugates used for in vivo diagnostic or therapeutic applications (e.g.,
inhibition of
CA 02377580 2001-12-20
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44
cell proliferation). See, in general, Ramakrishnan et al., Cancer Res. 56:1324-
1330,
1996.
Polypeptides and proteins of the present invention can be used to
identify and isolate receptors. Zalpha29 receptors may be involved in growth
regulation in the liver, blood vessel formation, and other developmental
processes.
For example, zalpha29 proteins and polypeptides can be immobilized on a
column,
and membrane preparations run over the column (as generally disclosed in
Immobilized Affini~ Li~and Techniques, Hermanson et al., eds., Academic Press,
San
Diego, CA, 1992, pp.195-202). Proteins and polypeptides can also be
radiolabeled
(Methods Enzymol., vol. 182, "Guide to Protein Purification", M. Deutscher,
ed.,
Academic Press, San Diego, 1990, 721-737) or photoaffinity labeled (Brunner et
al.,
Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol.
33:1167-1180, 1984) and used to tag specific cell-surface proteins. In a
similar
manner, radiolabeled zalpha29 proteins and polypeptides can be used to clone
the
cognate receptor in binding assays using cells transfected with an expression
cDNA
library.
The present invention also provides reagents for use in diagnostic
applications. For example, the zalpha29 gene, a probe comprising zalpha29 DNA
or
RNA, or a subsequence thereof can be used to determine the presence of
mutations at
or near the zalpha29 locus at chromosome 2p15. Detectable chromosomal
aberrations
at the zalpha29 gene locus include, but are not limited to, aneuploidy, gene
copy
number changes, insertions, deletions, restriction site changes, and
rearrangements.
These aberrations can occur within the coding sequence, within introns, or
within
flanking sequences, including upstream promoter and regulatory regions, and
may be
2 5 manifested as physical alterations within a coding sequence or changes in
gene
expression level. Analytical probes will generally be at least 20 nucleotides
in length,
although somewhat shorter probes (14-17 nucleotides) can be used. PCR primers
are
at least 5 nucleotides in length, often 15 or more nt, and frequently 20-30
nt. Short
polynucleotides can be used when a small region of the gene is targetted for
analysis.
3 0 For gross analysis of genes, a polynucleotide probe may comprise an entire
exon or
more. Probes will generally comprise a polynucleotide linked to a signal-
generating
CA 02377580 2001-12-20
WO 01/00831 PCT/US00/16736
moiety such as a radionucleotide. In general, these diagnostic methods
comprise the
steps of (a) obtaining a genetic sample from a patient; (b) incubating the
genetic
sample with a polynucleotide probe or primer as disclosed above, under
conditions
wherein the polynucleotide will hybridize to complementary polynucleotide
sequence,
5 to produce a first reaction product; and (c) comparing the first reaction
product to a
control reaction product. A difference between the first reaction product and
the
control reaction product is indicative of a genetic abnormality in the
patient. Genetic
samples for use within the present invention include genomic DNA, cDNA, and
RNA.
The polynucleotide probe or primer can be RNA or DNA, and will comprise a
portion
10 of SEQ >D NO:1, the complement of SEQ m NO:1, or an RNA equivalent thereof.
Suitable assay methods in this regard include molecular genetic techniques
known to
those in the art, such as restriction fragment length polymorphism (RFLP)
analysis,
short tandem repeat (STR) analysis employing PCR techniques, ligation chain
reaction
(Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection
15 assays, and other genetic linkage analysis techniques known in the art
(Sambrook et
al., ibid.; Ausubel et. al., ibid.; A.J. Marian, Chest 108:255-65, 1995).
Ribonuclease
protection assays (see, e.g., Ausubel et al., ibid., ch. 4) comprise the
hybridization of
an RNA probe to a patient RNA sample, after which the reaction product (RNA-
RNA
hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from
2 0 digestion. Within PCR assays, a patient genetic sample is incubated with a
pair of
polynucleotide primers, and the region between the primers is amplified and
recovered. Changes in size or amount of recovered product are indicative of
mutations in the patient. Another PCR-based technique that can be employed is
single
strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and
2 5 Applications 1:34-38, 1991 ).
The polypeptides, nucleic acids and/or antibodies of the present
invention may be used in diagnosis or treatment of disorders associated with
cell loss
or abnormal cell proliferation (including cancer). Labeled zalpha29
polypeptides may
be used for imaging tumors or other sites of abnormal cell proliferation.
3 0 Inhibitors of zalpha29 activity (zalpha29 antagonists) include anti-
zalpha29 antibodies and soluble zalpha29 receptors, as well as other peptidic
and non-
CA 02377580 2001-12-20
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46
peptidic agents (including ribozymes). Such antagonists can be used to block
the
effects of zalpha29 on cells or tissues. Of particular interest is the use of
antagonists
of zalpha29 activity in cancer therapy. As early detection methods improve it
becomes
possible to intervene at earlier times in tumor development, making it
feasible to use
inhibitors of growth factors to block cell proliferation, angiogenesis, and
other events
that lead to tumor development and metastasis. Inhibitors are also expected to
be
useful in adjunct therapy after surgery to prevent the growth of residual
cancer cells.
Inhibitors can also be used in combination with other cancer therapeutic
agents.
In addition to antibodies, zalpha29 inhibitors include small molecule
l0 inhibitors and inactive receptor-binding fragments of zalpha29
polypeptides.
Inhibitors are formulated for pharmaceutical use as generally disclosed above,
taking
into account the precise chemical and physical nature of the inhibitor and the
condition
to be treated. The relevant determinations are within the level of ordinary
skill in the
formulation art.
Polynucleotides encoding zalpha29 polypeptides are useful within gene
therapy applications where it is desired to increase or inhibit zalpha29
activity. If a
mammal has a mutated or absent zalpha29 gene, a zalpha29 gene can be
introduced
into the cells of the mammal. In one embodiment, a gene encoding a zalpha29
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,
2 5 without concern that the vector can infect other cells. Examples of
particular vectors
include, but are not limited to, a defective herpes simplex virus 1 (HSV1)
vector
(Kaplitt et al., Molec. Cell. Neurosci. 2:320-330, 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. 63:3822-3888, 1989).
Within
another embodiment, a zalpha29 gene can be introduced in a retroviral vector
as
CA 02377580 2001-12-20
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47
described, for example, by Anderson et al., U.S. Patent No. 5,399,346; Mann et
al.
Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S.
Patent
No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S.
Patent No.
5,124,263; Dougherty et al., WIPO Publication WO 95/07358; and Kuo et al.,
Blood
82:845, 1993. Alternatively, the vector can be introduced by liposome-mediated
transfection ("lipofection"). 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; 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, including molecular targeting of
liposomes to
specific cells. Directing transfection to particular cell types 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. Peptidic and non-peptidic molecules can be coupled to
liposomes chemically. Within another embodiment, cells are removed from the
body,
a vector is introduced into the cells as a naked DNA plasmid, and the
transformed
cells are re-implanted into the body as disclosed above.
Antisense methodology can be used to inhibit zalpha29 gene
transcription in a patient as generally disclosed above.
2 0 Zalpha29 polypeptides and anti-zalpha29 antibodies can 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 may be used to identify or treat tissues
or organs
that express a corresponding anti-complementary molecule (receptor or antigen,
2 5 respectively, for instance). More specifically, zalpha29 polypeptides or
anti-zalpha29
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.
Suitable detectable molecules can be directly or indirectly attached to
3 0 the polypeptide or antibody, and include radionuclides, enzymes,
substrates, cofactors,
inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles,
and the
CA 02377580 2001-12-20
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48
like. Suitable cytotoxic molecules can be directly or indirectly attached to
the
polypeptide or antibody, and include bacterial or plant toxins (for instance,
diphtheria
toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well as
therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90.
These can
be either directly attached to the polypeptide or antibody, or indirectly
attached
according to known methods, such as through a chelating moiety. Polypeptides
or
antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For
indirect
attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic
molecule
may 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.
Polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion
proteins may be used for targeted cell or tissue inhibition or ablation, such
as in cancer
therapy. Of particular interest in this regard are conjugates of a zalpha29
polypeptide
and a cytotoxin, which can be used to target the cytotoxin to a tumor or other
tissue
that is undergoing undesired angiogenesis or neovascularization. Target cells
(i.e.,
those displaying the zalpha29 receptor) bind the zalpha29-toxin conjugate,
which is
then internalized, killing the cell. The effects of receptor-specific cell
killing (target
ablation) are revealed by changes in whole animal physiology or through
histological
2 0 examination. Thus, ligand-dependent, receptor-directed cyotoxicity can be
used to
enhance understanding of the physiological significance of a protein ligand.
One such
toxin is saporin. Mammalian cells have no receptor for saporin, which is non-
toxic
when it remains extracellular.
In another embodiment, zalpha29-cytokine fusion proteins or
2 5 antibody/fragment-cytokine fusion proteins may be used for enhancing in
vitro
cytotoxicity (for instance, that mediated by monoclonal antibodies against
tumor
targets) and for enhancing in vivo killing of target tissues (for example,
blood and
bone marrow cancers). See, generally, Hornick et al., Blood 89:4437-4447,
1997). In
general, cytokines are toxic if administered systemically. The described
fusion
3 0 proteins enable targeting of a cytokine to a desired site of action, such
as a cell having
binding sites for zalpha29, thereby providing an elevated local concentration
of
CA 02377580 2001-12-20
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49
cytokine. Suitable cytokines for this purpose include, for example,
interleukin-2 and
granulocyte-macrophage colony-stimulating factor (GM-CSF). Such fusion
proteins
may be used to cause cytokine-induced killing of tumors and other tissues
undergoing
angiogenesis or neovascularization.
The bioactive polypeptide or antibody conjugates described herein can
be delivered intravenously, intra-arterially or intraductally, or may be
introduced
locally at the intended site of action.
The invention is further illustrated by the following non-limiting
examples.
Examples
Example 1
Zalpha29 Northern blot analysis was performed using commercially
prepared blots of human RNA (Human Multiple Tissue Northern Blots I, II, and
III;
Human Fetal Multiple Tissue Northern Blot II; and Human RNA Master Blot;
Clontech Laboratories, Inc., Palo Alto, CA).
The zalpha29 hybridization probe was generated as a gel purified PCR
amplification product. The amplification product was made using
oligonucleotides
ZC21,720 (SEQ ID N0:8) and ZC21,721 (SEQ ID N0:9) as PCR primers and a
2 o cloned zalpha29 cDNA (see SEQ ID NO:1 ) as template. The PCR amplification
was
performed as follows: 1 p,1 of zalpha29 cDNA (~2 ng) and 40 pmoles each of
oligonucleotide primers ZC21,720 and ZC21,721 were added to a reaction mixture
containing commercially available reagents (AdvantageTM KlenTaq Polymerase
Kit,
Clontech Laboratories, Inc.) following the manufacturer's recommended
protocol.
2 5 The reaction was run as follows: 94°C for 30 seconds, 25 cycles of
94°C for 5
seconds, 55°C for 5 seconds, and 68°C for 1 minute, followed by
68°C for 3 minutes
and a hold at 4°C. The 422 by PCR amplified fragment was gel purified
and
recovered using silica gel particles (QIAEX~ II gel extraction kit; Qiagen,
Valencia,
CA) according to the manufacturer's recommended protocol.
3 0 The probe was a radioactively labeled using a commercially available
kit (RediprimeTM II random-prime labeling system; Amersham Corp., Arlington
CA 02377580 2001-12-20
WO 01/00831 PCT/US00/16736
Heights, IL) according to the manufacturer's protocol. The probe was purified
using a
a commercially available push column (NucTrap~ column; Stratagene, La Jolla,
CA;
see U.S. Patent No. 5,336,412). A hybridization solution (ExpressHybTM
Hybridization Solution; Clontech Laboratories, Inc.) solution was used for the
5 prehybridization and hybridization solutions for the Northern blots.
Hybridization
took place overnight at 65°C. Following hybridization, the blots were
washed in 2X
SSC, 0.1 % SDS at room temperature, followed by a wash in O.1X SSC and 0.1 %
SDS
at 50°C. The blots were exposed to film (BIOMAX, Eastman Kodak, New
Haven,
CT).
10 An overnight exposure showed an approximately 870 base band in
every lane on all of the blots. Every RNA sample on the RNA Master Blot was
positive while the negative controls were negative.
The positive tissues on the Northerns included heart, ovary, fetal lung,
brain, small intestine, fetal liver, placenta, colon (mucosal lining), fetal
kidney, lung,
15 peripheral blood leukocyte, liver, stomach, skeletal muscle, thyroid,
kidney, spinal
cord, pancreas, lymph node, spleen, trachea, thymus, adrenal gland, prostate,
bone
marrow, testis, fetal brain.
Positive tissues on the RNA Master Blot that were not also on the
Northerns included amygdala, aorta, caudate nucleus, bladder, cerebellum,
uterus,
2 0 cerebral cortex, pituitary gland, frontal lobe, salivary gland,
hippocampus, mammary
gland, medulla oblongata, appendix, occipital lobe, trachea, putamen, fetal
heart,
substantia nigra, fetal spleen, thalamus, fetal thymus, and subthalamic
nucleus.
The Northern blots were reprobed for human transferrin receptor. The
resulting signal generated from the transferrin receptor probe was used to
normalize
2 5 the zalpha29 signal. The tissues with the greatest ratio of zalpha29
signal to
transferrin receptor signal were heart, liver, and testis.
Example 2
Zalpha29 was mapped to chromosome 2 using the commercially
3 o available version of the Stanford G3 Radiation Hybrid Mapping Panel
(Research
Genetics, Inc., Huntsville, AL). This panel contains PCRable DNAs from each of
83
CA 02377580 2001-12-20
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51
radiation hybrid clones of the whole human genome, plus two control DNAs (the
RM
donor and the A3 recipient). A publicly available WWW server (http://shgc-
www.stanford.edu) allows chromosomal localization of markers.
For the mapping of Zalpha29 with the Stanford G3 RH Panel, 20-~.l
reaction mixtures were set up in a PCRable 96-well microtiter plate
(Stratagene, La
Jolla, CA) and used in a thermal cycler (RoboCycler~ Gradient 96; Stratagene).
Each
of the 85 PCR reactions consisted of 2 ~,1 buffer (10X KlenTaq PCR reaction
buffer;
(Clontech Laboratories, Inc., Palo Alto, CA), 1.6 ~,1 dNTPs mix (2.5 mM each,
Perkin-
Elmer, Foster City, CA), 1 ~1 sense primer ZC22,737 (SEQ ID NO:10), 1 ~l
antisense
primer ZC22,738 (SEQ ID NO:11), 2 ~l of a density increasing agent and
tracking
dye (RediLoad, Research Genetics, Inc., Huntsville, AL), 0.4 ~ 1 of a
commercially
available DNA polymerase/antibody mix (SOX AdvantageTM KlenTaq Polymerase
Mix; Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid
clone or
control and x ~1 ddH20 for a total volume of 20 ~1. The mixtures were overlaid
with
an equal amount of mineral oil and sealed. The PCR cycler conditions were as
follows: an initial 5-minute denaturation at 94°C, 35 cycles of a 45-
second
denaturation at 94°C, 45 seconds annealing at 64°C and 75
seconds extension at 72°C;
followed by a final extension of 7 minutes at 72°C. The reactions were
separated by
electrophoresis on a 2% agarose gel (obained from Life Technologies,
Gaithersburg,
2 0 MD).
The results showed linkage of Zalpha29 to the chromosome 2
framework marker SHGC-30949 with a LOD score of >11 and at a distance of 0
cR_10000 from the marker. The use of surrounding genes that have been
physically
mapped positions Zalpha29 in the 2p16-p15 region on chromosome 2.
Example 3
The protein coding region of mouse zalpha29 was amplified by PCR
using primers that added FseI and AscI restriction sties at the 5' and 3'
termini
respectively. PCR primers ZC23019 (SEQ ID N0:12) and ZC23018 (SEQ ID N0:13)
3 0 were used with a template plasmid (pT7T3D-Pac) containing the full-length
murine
zalpha29 cDNA in a PCR reaction as follows: one cycle at 95°C for 5
minutes;
CA 02377580 2001-12-20
WO 01/00831 PCT/US00/16736
52
followed by 15 cycles at 95°C for 0.5 min., 58°C for 0.5 min.,
and 72°C for 0.5 min.;
followed by 72°C for 7 min.; followed by a 4°C soak. The PCR
reaction product was
loaded onto a 1.2°l0 (low melt) agarose (SeaPlaque~ GTG; FMC Corp.,
Rockland,
ME) gel in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA). The zalpha29 PCR
product was excised from the gel. The gel slice was melted at 65°, and
the DNA was
extracted twice with phenol and precipitated with ethanol. The PCR product was
then
digested with FseI + AscI, phenol/chloroform extracted, EtOH precipitated, and
rehydrated in 20,1 TE (Tris/EDTA pH 8). The 567-by zalpha29 fragment was then
ligated into the FseI-AscI sites of a modified pAdTrack CMV (He et al., Proc.
Natl.
Acad. Sci. USA 95:2509-2514, 1998). This construct also contained the green
fluorescent protein (GFP) marker gene. The CMV promoter driving GFP expression
was replaced with the SV40 promoter and the SV40 polyadenylation signal was
replaced with the human growth hormone polyadenylation signal. In addition,
the
native polylinker was replaced with FseI, EcoRV, and AscI sites. This modified
form
of pAdTrack CMV was named pZyTrack. Ligation was performed using a DNA
ligation and screening kit (Fast-LinkT"~; Epicentre Technologies, Madison,
WI).
Clones containing the zalpha29 cDNA were identified by standard mini-prep
procedures. To linearize the plasmid, approximately 5 ~,g of the pZyTrack
zalpha29
plasmid was digested with PmeI. Approximately 1 ~g of the linearized plasmid
was
cotransformed with 200ng of supercoiled pAdEasy (He et al., ibid.) into BJ5183
cells.
The co-transformation was done using an electroporator (Gene Pulser~; Bio-Rad
Laboratories, Inc., Hercules, CA) at 2.5 kV, 200 ohms, and 25 ~Fa. The entire
co-
transformation mixture was plated on 4 LB plates containing 25 ~g/ml
kanamycin.
The smallest colonies were picked and expanded in LB/kanamycin, and
recombinant
2 5 adenovirus DNA was identified by standard DNA miniprep procedures.
Digestion of
the recombinant adenovirus DNA with FseI + AscI confirmed the presence of the
zalpha29 sequence. The recombinant adenovirus miniprep DNA was transformed
into
E. coli host cells (DH10BTM; Life Technologies, Gaithersburg, MD), and DNA was
prepared using a commercially available plasmid isolation kit (QIAGEN~ Plasmid
3 0 Maxi Kit; Qiagen, Inc., Valencia, CA) as directed by the supplier.
CA 02377580 2001-12-20
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53
Approximately 5 pg of recombinant adenoviral DNA was digested with
PacI enzyme (New England Biolabs) for 3 hours at 37°C in a reaction
volume of 100
~.l containing 20-30U of PacI. The digested DNA was extracted twice with an
equal
volume of phenol/chloroform and precipitated with ethanol. The DNA pellet was
resuspended in 5 ~l distilled water. A T25 flask of QBI-293A cells (Quantum
Biotechnologies, Inc. Montreal, Canada), inoculated the day before and grown
to 60-
70% confluence, was transfected with the PacI-digested DNA. The PacI-digested
DNA was diluted to a total volume of 50 ~l with sterile HBS (150 mM NaCI, 20
mM
HEPES). In a separate tube, 25 p,1 of lmg/ml N-[1-(2,3-Dioleoyloxy)propyl]-
N,N,N-
trimethyl-ammonium salts (DOTAP) (Boehringer Mannheim, Indianapolis, IN) was
diluted to a total volume of 100 p,1 with HBS. The DNA was added to the DOTAP,
mixed gently by pipeting up and down, and left at room temperature for 15
minutes.
The media was removed from the 293A cells, and the cells were washed with 5 ml
serum-free MEMalpha containing 1 mM sodium pyruvate, 0.1 mM MEM non-
essential amino acids, and 25 mM HEPES buffer (media components obtained from
Life Technologies, Gaithersburg, MD). 5 ml of serum-free MEM was added to the
293A cells and held at 37°C. The DNA/lipid mixture was added drop-wise
to the T25
flask of 293A cells, mixed gently and incubated at 37°C for 4 hours.
After 4 hours the
media containing the DNA/lipid mixture was aspirated off and replaced with 5
ml
2 0 complete MEM containing 5% fetal bovine serum. The transfected cells were
monitored for GFP expression and formation of foci (viral plaques).
Seven days after transfection of 293A cells with the recombinant
adenoviral DNA, the cells expressed GFP and started to form foci. The crude
viral
lysate was collected with a cell scraper and transferred to a 50-ml conical
tube. To
2 5 release most of the virus particles from the cells, three freeze/thaw
cycles were done in
a dry ice/ethanol bath and a 37° waterbath.
The crude lysate was amplified (primary ( 1 °) amplification) to
obtain a
working "stock" of zalpha29 recombinant adenovirus (rAdV) lysate. Ten 10-cm
plates of nearly confluent (80-90%) 293A cells were set up 20 hours in
advance. 200
3 0 ml of crude rAdV lysate was added to each 10-cm plate, and the plates were
monitored
for 48 to 72 hours for CPE (cytopathic effect) under the white light
microscope and
CA 02377580 2001-12-20
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54
expression of GFP under the fluorescent microscope. When all of the 293A cells
showed CPE, the 1 ° stock lysate was collected, and freeze/thaw cycles
were performed
as above.
For secondary (2°) amplification, 20 15-cm tissue culture dishes
of
293A cells were prepared so that the cells were 80-90% confluent. All but 20
ml of
5% MEM media was removed, and each dish was inoculated with 300-500 ml
1°
amplified rAdv lysate. After 48 hours the cells were lysed from virus
production.
This lysate was collected into 250-ml polypropylene centrifuge bottles.
To purify the rAdV, NP-40 detergent was added to a final concentration
of 0.5% to the bottles of crude lysate to lyse all cells. Bottles were placed
on a
rotating platform for 10 minutes, agitating as fast as possible without the
bottles
falling over. The debris was pelleted by centrifugation at 20,000 X G for 15
minutes.
The supernatants were transferred to 250-ml polycarbonate centrifuge bottles,
and 0.5
volume of 20% PEG-8000/2.5 M NaCI solution was added. The bottles were shaken
overnight on ice. The bottles were centrifuged at 20,000 X G for 15 minutes,
and
supernatants were discarded into a bleach solution. Using a sterile cell
scraper, the
precipitate from 2 bottles was resuspended in 2.5 ml PBS. The virus solution
was
placed in 2-ml microcentrifuge tubes and centrifuged at 14,000 X G for 10
minutes to
remove any additional cell debris. The supernatant from the 2-ml
microcentrifuge
2 0 tubes was transferred to a 15-ml polypropylene snapcap tube and adjusted
to a density
of 1.34 g/ml with CsCI. The volume of the virus solution was estimated, and
0.55
g/ml of CsCI was added. The CsCI was dissolved, and 1 ml of this solution
weighed
1.34 g. The solution was transferred to polycarbonate thick-walled centrifuge
tubes
(3.2m1; Beckman #362305) and spun at 348,000 X G for 3-4 hours at 25°C
in a
2 5 Beckman Optima TLX micro-ultracentrifuge with a TLA-100.4 rotor. The virus
formed a white band. Using wide-bore pipette tips, the virus band was
collected.
The virus preparation was desalted by gel filtration using commercially
available columns and cross-linked dextran media (PD-10 columns prepacked with
Sephadex~ G-25M; Pharmacia, Piscataway, NJ). The column was equilibrated with
3 0 20 ml of PBS. The virus was loaded and allow it to run into the column. 5
ml of PBS
was added to the column, and fractions of 8-10 drops were collected. The
optical
CA 02377580 2001-12-20
WO 01/00831 PCT/US00/16736
densities of 1:50 dilutions of each fraction was determined at 260 nm on a
spectrophotometer. A clear absorbance peak was present between fractions 7-12.
These fractions were pooled, and the optical density (OD) of a 1:25 dilution
determined. Virus concentration was determined by the formula: (OD at
5 260nm)(25)(1.1 x 1012) = virions/ml. The OD of a 1:25 dilution of the
zalpha29
rAdV was 0.059, giving a virus concentration of 3.3 X IOIZ virions/ml.
To store the virus, glycerol was added to the purified virus to a final
concentration of 15%, mixed gently but effectively, and stored in aliquots at -
80°C.
A protocol developed by Quantum Biotechnologies, Inc. (Montreal,
l0 Canada) was followed to measure recombinant virus infectivity. Briefly, two
96-well
tissue culture plates were seeded with 1X104 293A cells per well in MEM
containing
2% fetal bovine serum for each recombinant virus to be assayed. After 24
hours, 10-
fold dilutions of each virus from 1X10-2 to 1X10-'4 were made in MEM
containing 2%
fetal bovine serum. 100 ~l of each dilution was placed in each of 20 wells.
After 5
15 days at 37°C, wells were read either positive or negative for CPE,
and a value for
plaque forming units/ml (PFU) was calculated.
TCmS° formulation used was as per Quantum Biotechnologies, Inc.,
above. The titer (T) was determined from a plate where virus used was diluted
from
10-'' to 10-14, and read 5 days after the infection. At each dilution a ratio
(R) of
2 o positive wells for CPE per the total number of wells was determined.
To calculate titer of the undiluted virus sample: the factor, "F" -
1+d(S-0.5); where "S" is the sum of the ratios (R); and "d" is LoglO of the
dilution
series, for example, "d" is equal to 1 for a ten-fold dilution series. The
titer of the
undiluted sample is T = 101+~ = TCmS°/ml. To convert TCmsdml to pfu/ml,
0.7 is
2 5 subtracted from the exponent in the calculation for titer (T).
The zalpha29 adenovirus had a titer of 1.3 X 10'° pfu/ml.
From the foregoing, it will be appreciated that, although specific
embodiments of the invention have been described herein for purposes of
illustration,
3 0 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.
CA 02377580 2001-12-20
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1
SEQUENCE LISTING
<110> ZymoGenetics, Inc.
<120> HELICAL POLYPEPTIDE ZALPHA29
<130> 99-28PC
<150> US 09/343,163
<151> 1999-06-28
<160> 16
<170> FastSEQ for Windows Version 3.0
<210>1
<211>813
<212>DNA
<213>Homo Sapiens
<220>
<221> CDS
<222> (21)...(593)
<400> 1
ggctcgagcc ttcgcagagc atg gcg gcg ggc gag ctt gag ggt ggc aaa ccc 53
Met Ala Ala Gly Glu Leu Glu Gly Gly Lys Pro
1 5 10
ctg agc ggg ctg ctg aat gcg ctg gcc cag gac act ttc cac ggg tac 101
Leu Ser Gly Leu Leu Asn Ala Leu Ala Gln Asp Thr Phe His Gly Tyr
15 20 25
ccc ggc atc aca gag gag ctg cta cgg agc cag cta tat cca gag gtg 149
Pro Gly Ile Thr Glu Glu Leu Leu Arg Ser Gln Leu Tyr Pro Glu Val
30 35 40
cca ccc gag gag ttc cgc ccc ttt ctg gca aag atg agg ggg att ctt 197
Pro Pro Glu Glu Phe Arg Pro Phe Leu Ala Lys Met Arg Gly Ile Leu
45 50 55
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2
aag tct att gcg tct gca gac atg gat ttc aac cag ctg gag gca ttc 245
Lys Ser Ile Ala Ser Ala Asp Met Asp Phe Asn Gln Leu Glu Ala Phe
60 65 70 75
ttg act get caa acc aaa aag caa ggt ggg atc aca tct gac caa get 293
Leu Thr Ala Gln Thr Lys Lys Gln Gly Gly Ile Thr Ser Asp Gln Ala
80 85 90
get gtc att tcc aaa ttc tgg aag agc cac aag aca aaa atc cgt gag 341
Ala Val Ile Ser Lys Phe Trp Lys Ser His Lys Thr Lys Ile Arg Glu
95 100 105
agc ctc atg aac cag agc cgc tgg aat agc ggg ctt cgg ggc ctg agc 389
Ser Leu Met Asn Gln Ser Arg Trp Asn Ser Gly Leu Arg Gly Leu Ser
110 115 120
tgg aga gtt gat ggc aag tct cag tca agg cac tca get caa ata cac 437
Trp Arg Val Asp Gly Lys Ser Gln Ser Arg His Ser Ala Gln Ile His
125 130 135
aca cct gtt gcc att ata gag ctg gaa tta ggc aaa tat gga cag gaa 485
Thr Pro Val Ala Ile Ile Glu Leu Glu Leu Gly Lys Tyr Gly Gln Glu
140 145 150 155
tct gaa ttt ctg tgt ttg gaa ttt gat gag gtc aaa gtc aac caa att 533
Ser Glu Phe Leu Cys Leu Glu Phe Asp Glu Val Lys Val Asn Gln Ile
160 165 170
ctg aag acg ctg tca gag gta gaa gaa agt atc agc aca ctg atc agc 581
Leu Lys Thr Leu Ser Glu Val Glu Glu Ser Ile Ser Thr Leu Ile Ser
175 180 185
cag cct aac tga agatgatgta tgaaggagtt ggagttgttg aaaccaaggt 633
Gln Pro Asn
190
gtccatgatc cctccccact gaccttttct aagaaaattc ttgtgcccgc attggtatta 693
aatcctcgca ttcagtcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 753
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 813
<210> 2
<211> 190
<212> PRT
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3
<213> Homo Sapiens
<400> 2
Met Ala Ala Gly Glu Leu Glu Gly Gly Lys Pro Leu Ser Gly Leu Leu
1 5 10 15
Asn Ala Leu Ala Gln Asp Thr Phe His Gly Tyr Pro Gly Ile Thr Glu
20 25 30
Glu Leu Leu Arg Ser Gln Leu Tyr Pro Glu Val Pro Pro Glu Glu Phe
35 40 45
Arg Pro Phe Leu Ala Lys Met Arg Gly Ile Leu Lys Ser Ile Ala Ser
50 55 60
Ala Asp Met Asp Phe Asn Gln Leu Glu Ala Phe Leu Thr Ala Gln Thr
65 70 75 80
Lys Lys Gln Gly Gly Ile Thr Ser Asp Gln Ala Ala Val Ile Ser Lys
85 90 95
Phe Trp Lys Ser His Lys Thr Lys Ile Arg Glu Ser Leu Met Asn Gln
100 105 110
Ser Arg Trp Asn Ser Gly Leu Arg Gly Leu Ser Trp Arg Val Asp Gly
115 120 125
Lys Ser Gln Ser Arg His Ser Ala Gln Ile His Thr Pro Val Ala Ile
130 135 140
Ile Glu Leu Glu Leu Gly Lys Tyr Gly Gln Glu Ser Glu Phe Leu Cys
145 150 155 160
Leu Glu Phe Asp Glu Val Lys Val Asn Gln Ile Leu Lys Thr Leu Ser
165 170 175
Glu Val Glu Glu Ser Ile Ser Thr Leu Ile Ser Gln Pro Asn
180 185 190
<210>3
<211>805
<212>DNA
<213>Mus musculus
<220>
<221> CDS
<222> (23)...(589)
<400> 3
ggatcttggg ccctccttag cc atg gcg ggc gat ctg gag ggt ggc aag tcc 52
Met Ala Gly Asp Leu Glu Gly Gly Lys Ser
1 5 10
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4
ctg agc ggg ctg ctg agc ggc cta gcg cag aac gcc ttt cac gga cac 100
Leu Ser Gly Leu Leu Ser Gly Leu Ala Gln Asn Ala Phe His Gly His
15 20 25
tcg ggt gtc acg gag gag ctg ctg cac agc caa ctc tat ccg gaa gtg 148
Ser Gly Ual Thr Glu Glu Leu Leu His Ser Gln Leu Tyr Pro Glu Val
30 35 40
cca ccg gag gag ttc cgc ccc ttc ctg gcg aag atg aga gga ctt ctc 196
Pro Pro Glu Glu Phe Arg Pro Phe Leu Ala Lys Met Arg Gly Leu Leu
45 50 55
aag tct att gca tct gca gac atg gat ttc aac cag tta gag gca ttc 244
Lys Ser Ile Ala Ser Ala Asp Met Asp Phe Asn Gln Leu Glu Ala Phe
60 65 70
ctg act get caa acc aaa aag caa ggt ggc atc acc agt gag caa get 292
Leu Thr Ala Gln Thr Lys Lys Gln Gly Gly Ile Thr Ser Glu Gln Ala
75 80 85 90
gca gtc atc tcc aag ttt tgg aag agc cac aag ata aaa atc cga gag 340
Ala Val Ile Ser Lys Phe Trp Lys Ser His Lys Ile Lys Ile Arg Glu
95 100 105
agt ctc atg aag cag agc cgc tgg gac aac ggc ctt cgg ggc ctg agc 388
Ser Leu Met Lys Gln Ser Arg Trp Asp Asn Gly Leu Arg Gly Leu Ser
110 115 120
tgg aga gtc gat ggc aag tct cag tca cgg cac tca act cag ata cac 436
Trp Arg Val Asp Gly Lys Ser Gln Ser Arg His Ser Thr Gln Ile His
125 130 135
agc cct gtt gcc ata ata gag ctg gaa ttt gga aaa aat gga cag gaa 484
Ser Pro Val Ala Ile Ile Glu Leu Glu Phe Gly Lys Asn Gly Gln Glu
140 145 150
tct gaa ttt ttg tgt ctg gaa ttt gat gaa gtt aaa gtc aag caa atc 532
Ser Glu Phe Leu Cys Leu Glu Phe Asp Glu Val Lys Val Lys Gln Ile
155 160 165 170
ctg aag aag ctg tca gag gta gaa gag agt atc aac agg ctg atg cag 580
Leu Lys Lys Leu Ser Glu Val Glu Glu Ser Ile Asn Arg Leu Met Gln
175 180 185
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gca gcc taa ctgaagagag tatcaatagg ctgatgcagg cagcctaact 629
A1 a A1 a
gaaggctgga ggaaggggcg tttgaagtga agctgctcac agactttctc cactgaccct 689
ttgaaagtcc tgtttgccca ctggtgttac caaaagacat tgtatacatg catgaaagtc 749
ttcaagaata aataaaaata tattttaaaa agtgggtaaa aaagagaaac ctctca 805
<210>4
<211>188
<212>PRT
<213>Mus musculus
<400> 4
Met Ala Gly Asp Leu Glu Gly Gly Lys Ser Leu Ser Gly Leu Leu Ser
1 5 10 15
Gly Leu Ala Gln Asn Ala Phe His Gly His Ser Gly Val Thr Glu Glu
20 25 30
Leu Leu His Ser Gln Leu Tyr Pro Glu Val Pro Pro Glu Glu Phe Arg
35 40 45
Pro Phe Leu Ala Lys Met Arg Gly Leu Leu Lys Ser Ile Ala Ser Ala
50 55 60
Asp Met Asp Phe Asn Gln Leu Glu Ala Phe Leu Thr Ala Gln Thr Lys
65 70 75 80
Lys Gln Gly Gly Ile Thr Ser Glu Gln Ala Ala Val Ile Ser Lys Phe
85 90 95
Trp Lys Ser His Lys Ile Lys Ile Arg Glu Ser Leu Met Lys Gln Ser
100 105 110
Arg Trp Asp Asn Gly Leu Arg Gly Leu Ser Trp Arg Val Asp Gly Lys
115 120 125
Ser Gln Ser Arg His Ser Thr Gln Ile His Ser Pro Val Ala Ile Ile
130 135 140
Glu Leu Glu Phe Gly Lys Asn Gly Gln Glu Ser Glu Phe Leu Cys Leu
145 150 155 160
Glu Phe Asp Glu Val Lys Val Lys Gln Ile Leu Lys Lys Leu Ser Glu
165 170 175
Val Glu Glu Ser Ile Asn Arg Leu Met Gln Ala Ala
180 185
<210> 5
<211> 6
<212> PRT
CA 02377580 2001-12-20
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6
<213> Artificial Sequence
<220>
<223> peptide tag
<400> 5
Glu Tyr Met Pro Met Glu
1 5
<210> 6
<211> 190
<212> PRT
<213> Artificial Sequence
<220>
<223> variant polypeptides
<221> VARIANT
<222> (48)...(105)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly or Ala
<221> VARIANT
<222> (108)...(108)
<223> Xaa is Leu. Ile, Val, Met, Phe, Trp, Gly, Ala or
Ser
<221> VARIANT
<222> (109)...(109)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly or Ala
<221> VARIANT
<222> (112)...(112)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly, Ala or
Gln
<221> VARIANT
<222> (115)...(115)
<223> Xaa is Leu, Ile. Val, Met. Phe, Trp, Gly or Ala
<221> VARIANT
<222> (116)...(116)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly, Ala, Asn
or Asp
CA 02377580 2001-12-20
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7
<221> VARIANT
<222> (119)...(138)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly or Ala
<221> VARIANT
<222> (141)...(141)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly, Ala or
Pro
<221> VARIANT
<222> (142)...(145)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly or Ala
<221> VARIANT
<222> (148)...(148)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly, Ala or
Glu
<221> VARIANT
<222> (149)...(149)
<223> Xaa is Leu, Ile, Ual, Met, Phe, Trp, Gly or Ala
<221> VARIANT
<222> (152)...(152)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly, Ala, Asn
or Tyr
<221> VARIANT
<222> (171)...(171)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly or Ala
<221> VARIANT
<222> (174)...(174)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly, Ala, Lys
or Thr
<221> VARIANT
<222> (175)...(178)
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly or Ala
<221> VARIANT
<222> (181)...(181)
CA 02377580 2001-12-20
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8
<223> Xaa is Leu, Ile, Val, Met, Phe, Trp, Gly, Ala or
Ser
<221> VARIANT
<222> (182)...(185)
<223> Xaa is Leu, Ile, Val, Met. Phe. Trp, Gly or Ala
<400> 6
Met Ala Ala Gly Glu Leu Glu Gly Gly Lys Pro Leu Ser Gly Leu Leu
1 5 10 15
Asn Ala Leu Ala Gln Asp Thr Phe His Gly Tyr Pro Gly Ile Thr Glu
20 25 30
Glu Leu Leu Arg Ser Gln Leu Tyr Pro Glu Val Pro Pro Glu Glu Xaa
35 40 45
Arg Pro Xaa Xaa Ala Lys Xaa Arg Gly Xaa Xaa Lys Ser Xaa Ala Ser
50 55 60
Ala Asp Met Asp Phe Asn Gln Leu Glu Ala Phe Leu Thr Ala Gln Thr
65 70 75 80
Lys Lys Gln Gly Gly Ile Thr Ser Asp Gln Ala Ala Val Ile Ser Lys
85 90 95
Phe Trp Lys Ser His Lys Thr Lys Xaa Arg Glu Xaa Xaa Met Asn Xaa
100 105 110
Ser Arg Xaa Xaa Ser Gly Xaa Arg Gly Leu Ser Trp Arg Val Asp Gly
115 120 125
Lys Ser Gln Ser Arg His Ser Ala Gln Xaa His Thr Xaa Xaa Ala Ile
130 135 140
Xaa Glu Leu Xaa Xaa Gly Lys Xaa Gly Gln Glu Ser Glu Phe Leu Cys
145 150 155 160
Leu Glu Phe Asp Glu Val Lys Val Asn Gln Xaa Leu Lys Xaa Xaa Ser
165 170 175
Glu Xaa Glu Glu Xaa Xaa Ser Thr Xaa Ile Ser Gln Pro Asn
180 185 190
<210> 7
<211> 570
<212> DNA
<213> Artificial Sequence
<220>
<223> degenerate sequence
<221> misc_feature
<222> (1). .(570)
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WO 01/00831 PCT/US00/16736
9
<223> n = A.T,C or G
<400> 7
atggcngcnggngarytngarggnggnaarccnytnwsnggnytnytnaaygcnytngcn 60
cargayacnttycayggntayccnggnathacngargarytnytnmgnwsncarytntay 120
ccngargtnccnccngargarttymgnccnttyytngcnaaratgmgnggnathytnaar 180
wsnathgcnwsngcngayatggayttyaaycarytngargcnttyytnacngcncaracn 240
aaraarcarggnggnathacnwsngaycargcngcngtnathwsnaarttytggaarwsn 300
cayaaracnaarathmgngarwsnytnatgaaycarwsnmgntggaaywsnggnytnmgn 360
ggnytnwsntggmgngtngayggnaarwsncarwsnmgncaywsngcncarathcayacn 420
ccngtngcnathathgarytngarytnggnaartayggncargarwsngarttyytntgy 480
ytngarttygaygargtnaargtnaaycarathytnaaracnytnwsngargtngargar 540
wsnathwsnacnytnathwsncarccnaay 570
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC21.720
<400> 8
ggtaccccgg catcacag 18
<210> 9
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC21,721
<400> 9
gacctcatca aattccaaac aca 23
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC22.737
CA 02377580 2001-12-20
WO 01/00831 PCT/US00/16736
<400> 10
gctggcccag gacacttt 1g
<210> 11
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC22,738
<400> 11
gaatccccct catctttg 1g
<210> 12
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC23019
<400> 12
cacacaggcc ggccaccatg gcgggcgatc tggagggtgg 40
<210> 13
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> oligonucleotide primer ZC23018
<400> 13
cacacaggcg cgcctttagg ctgcctgcat cagcctgttg 40
<210>14
<211>6644
<212>DNA
<213>Homo Sapiens
<400> 14
CA 02377580 2001-12-20
WO 01/00831 PCT/US00/16736
11
tcaactttgcgctttagggcttacgcacggacgccagggtgcctggggggtgagatttga60
caaaatcgtattgaactcctggcttcaagtgatcctcctgcgtcagccttccaaaatgct120
ggggttacaggcatgagccaccatgcctggcctggcaggtctcattttttagaagtttct180
gctcttgttcagataatgtaaactccaagacttatttttctatctgttgagtctcaattg240
ccttcagcacaaagtaacctataggccaaagtggcaaagttggggctggtatattctgat300
acttttcagtagttaggaaaatacagagaaaagaacattgacttagtgactgtcatcaac360
agagggacattaattagccacatcatttaacatgcctggggcttaatttaattatctaga420
aaacaaggcaattggattatatgctctgtaaggattcatactgctctaaattacttgatt480
attgttctgaaggattttgaatgtagttgctcctagatacataaaatttatccttgtctc540
tcaagataatgcagatgtagaatttgtctagtgtgcatgaaattcctgctgccataactc600
agagatcttatgactgggcaacatactgatcctccccctacccccatctctacaaaaaaa660
aaaaaaaattttttttttttagttaaccggggcagggttggcaaccctgtagtccaagca720
actcaggaggctgaggtgggaggatcactttagcccaggagtttgaggctacagtgagct780
atgatcatgccactgcactccagcctggaaacagagggggaaaaaaatcttattttttga840
tatctaccatctacctaacctaggattgattgaccaatcctaacagtgattgaagggaac900
gtatttaagggagctgtgaggagggttgcttgcagcaagtggagggaccccagaaatgtt960
tgttgttatttcaagatctggttatctttcagttatctggctcatgtttcccaagcaact1020
tgtcacaatttctggcataaccactaaatccaagttagctcacttcccagactaactcag1080
agtccatcagagtcagtcaaaattctcctttcatttttgtaatccaaggtgctgtggaga1140
cagtatggacttcggcgtcatccagagctgagagttcccacagtctgattctgcctttat1200
atggtatttatttttgatacaggttaacctctttgtgcctcagggtcctcatttttaaaa1260
cagcactactcaagttcttaccttaaaaattattagaggatccaatgagatgacaaagag1320
gaagagcttctcatatgcctcctagtgagaacatctgacatttgtacgcctcttatttat1380
caatatgaaacaccatcataaaagcacttttttttttttttgagatggactctcactctc1440
tcccccaggctggagtgcagtggcccgatctcggctcactgcaagctccgactttcgggt1500
tcacgccattctcctgcctcagcctcctgagtagctgggactcacaggtgcccgcaactg1560
cgcctggctaattttttatatttttagtagagacggggtttcacccgtgttagccaggat1620
ggtctcgatctcctgaccttgtgatctgcccgcctcggcctcccaaagtgctgggattac1680
aggtgtgagccactgcgcctggcataaaagcacttttatttagggcattatgtggatatg1740
ctttgctgggtaaaatatcacttcgatattaaggtctgagctgggcttgctggattggga1800
ccctgggtattttgagtttggtcatgccagatggccttggctatcttgtggtttccctct1860
aaaatatccttttatgttttccaggaatctgaatttctgtgtttggaatttgatgaggtc1920
aaagtcaaccaaattctgaagacgctgtcagaggtagaagaaagtatcagcacactgatc1980
agccagcctaactgaagatgatgtatgaaggagttggagttgttgaaaccaaggtgtcca2040
tgatccctccccactgaccttttctaagaaaattcttgtgcccgcattggtattaaatcc2100
tcgcattcagtcttcctgcctctacttgctcagatttctttttttctagctttcatttag2160
tcttacatttgttccagtgcagaggttctcacccttcagtgtgcataaatgttataaggg2220
gtacttgtaaaagcattcacttttttgttgttattattaaattcggagtgttgctctgtt2280
gcccaggctggagtgcagtggtgcagtcatggctcactgcagcctcaagctcctggactc2340
aagcgagcctcccacctcagcctctcaagtagctgggactacaggtgcatgccaccacac2400
tcaggtaatttttgtattttttgtagagatggggtttcaccatgttgcccaggctggtct2460
ggaaatcctgggctcaagtgatcctcccaccctggcttcccaaagcccaaagtgctggga2520
ttacaggcgtgagaaaagcatttacatttaaaaaaaaaaaaaaaaaaaaaaagtaggctt2580
CA 02377580 2001-12-20
WO 01/00831 PCT/US00/16736
12
ccagggctctatccccagagacttggattcaataggattagggtgagagagatcagcaat 2640
ggaaatccttgatatagtggtttgtcctgggtggggttttaagaatttatacatgataaa 2700
tcatgtaggaattatttttaaagtagaaaaaaaagcttttatcatgcaaatatagggctg 2760
accaaagtgctatgtacttagctgaggcataggagcacctacctaacctagaaaagatgt 2820
acctgaccctagttaaaacctgagctctttctgaaactgatttgggcatttggattagtt 2880
ctgcttaaatctggggcatctggtttgatctgaactactgagagactcaggcttttctgg 2940
aacctagaactaaattggcctcacaacaaagggactcccttcacttgcctcaagtcagga 3000
tcatgggaaggggcagatgtctgctgagactgatgtgaggtcttttacctcagaaaattt 3060
tacctgagtcattaaaataaaacccctttcaaaaaatttctttaagaaaaactagtgtaa 3120
taaaaagtaggtcctattagagaacttaccaaacaccaagaacattctaacggcggaggc 3180
tgtcaactagcatattgaggctatggtccttgttgaacaggttttgtcatctgatactga 3240
taatatttagaaatctaagatgcctttggagtataatatgttcaaaaaatgtggttatct 3300
tggtctgtgattacagcatatgtccatgctaaggagtttgtttcaggacaggaataagtc 3360
ctcttctgttaagcagtttctcctaaatcagtttggagacatttcaggagcttttctaac 3420
acccaagctgaaattattggcttcttctctgattaaaaccatcccagcagttagcaaaca 3480
ataaccagaaggttttcaatgtagcccctgtgcacccttcagaaaacatcttgaaacagt 3540
actgtaaatagattcaagaaaggaatgtggtttggaaaaaaaaaaaactattttaaactt 3600
gccttctgttcccagggctgctgtcatgtaatctaggagaattttgataaggtctcctgc 3660
tgtaaaatggagcaatagaatatctcatcagataatcggattccagatgtccttggaagg 3720
aataactagagctatcaccttagtattgactcatatatcccatggaagtctgtggaagtg 3780
tgaaggaacagcacatgggccacaaggagaggaaatcattgtcatagtctgaaactctgt 3840
ttaggtcatcccatgaaagtaatagctacaagagtggccatgggcttttaaaattgtatt 3900
cccaaaccctaggtcattggaagactacagttaatgtatactgagttttcaagaattaaa 3960
aagaaaaccaaaaactggttgttgcaggtggtccccacatttaacactaagcacttctga 4020
atgcaagttgtttctaacagggtatattttatatttactgatgatttttaattttttatt 4080
atcaaaggtatatatgtttattatagtctattatgaaaatacagaaacatactaagaaca 4140
gttaatgacccatcaatctaatgtacagaaagaaggctagaactaggaaaagagttgact 4200
ttccttgaataaattcccagaagtggagtacacagttttatttatttatttagagacaga 4260
gtttcactctgtcacccatgctggaatgcagtggcgcaatctcagctcactgcaacctcc 4320
gcctcccgggttcaagtgattctcctgtctcagcctcctgagtagctgggactacaggca 4380
cctgccaccacgtccggctaatttttttttgtatttttagtagagacagggtttcactat 4440
gttggccaggctggtctcgaactcctgacctccagtgattgacccgcctcagcctcccaa 4500
agtgctgggattacaggcgtgagccactgcacccggcctagtacacagtttttaactttg 4560
ataaacattgccaaattcctctccaggaaggctgtattaatttgtattccctctgagaaa 4620
gtataagactaaattaccccctctcttgcctaattggctatcatcattttttgtattttc 4680
tgggagtaagttcttagaaagttttgtaagggacacttacattaaaccaggacatctccc 4740
tggtaacaataaaagcatggagaaaggaccagggaaggagaaaacaggtataaagttccc 4800
agagaccccactaggttttctacctgtgcgatcctagattaaaaccacttgttttgattt 4860
caggaaattagggacaaaataaaaatctcagcctgaactggaccttgtagaaattatccc 4920
tgcttgagcaataagcactctaaattcagtctgtttagaaagattcctgcccgttagcca 4980
ggtgtggtagcacaggcctcaagtccaagctgctcaggaggctgaggaaggaggatgcct 5040
tgagcccaggagtttggggcttcaggcaacaacagcaagagcccatctctaaaaaagaaa 5100
gaaaaggagagagagagagagagagagagagatgagagagagagaaagatgagagaagaa 5160
CA 02377580 2001-12-20
WO 01/00831 PCT/US00/16736
13
aagaaaaaaacagtccagccaagctaaaagttagctttcagaataaagtcagaaaataac 5220
tccagattttggtagcgttgtgttgatacgaagcaaaagatttggccttattcttaggtc 5280
aggctttccttggaagctctagttcttctcagctgtaacagcaaaagcctaaattccatt 5340
atagactctttatttcctttatataacctctcttcccccagtcttattttaataatgatt 5400
caaaaagagttccagcattaaaaaaaagtagtttaactcttcacccccaaatgcaagaag 5460
gtggtgaaaagcagaggatgatgttgagtatcttaaatagctgacatcatgtcaaactat 5520
taattgttgaagttatttttttacacctgagtgaacatttagaaaataatataaatagaa 5580
attaaagggaaataaatgctaaaccgatgttagaaaatactgttttctgaagtgtacagt 5640
aagtatctttttgtatgtttttttttctttttaatttatttattgaaatggagtctcact 5700
ctgtcacccaggctggagtgcagtggcgcgatcttggctcactgcaacctccgccctttg 5760
agttcaagcgattctcctgcctcagcctcctgagtacctgggatcacaggcacctgccac 5820
cgcacccagctaattttttttttacttttagtagagacggagtttcaccatcttggccag 5880
gctagtcttgaactcctgacctcatgatccatccgcctcggcctcccaaagtgctgggat 5940
tacaggtgtgagccaccatgcccagccttttatttatttatttatttttgagaaggagtc 6000
tcactctgtcgcccagggtggagtgcagtggtgcaatctctgctcactgcaacctctgcc 6060
tcccaggttcaagcgattctcctgtgtcagcctcccgagtagctgggattacaggcatgc 6120
gccaccgcacccagctaatttttatatttttagtagagacgtggtttcaccatgttggcc 6180
aggctggtctcaaactcctgaccttcggtgatccacccacctcggcctcccaaagtgctg 6240
ggatgacaggcatgagccgctgcacccagcctcaaagtgtatagtaaatatctaaacaaa 6300
tgaaagggacaagatatagaaggaatcttaggatcagctgagagataattgaatactttc 6360
ctaaaagaacacaatactggaagggatggggctttgtgggacaattgctattttgaattc 6420
ttaggtgtccaactttacaaccaaggtttacaaatattttaaatggtgatttagtcagca 6480
gaagggaagactcaaatagaacataattagcttaagcttacctctagttgtagagtatac 6540
aggttttgacctcaaaatttgaaaaatcgcaatttttatctaagtgcaatcaagttttcc 6600
ttatttggggatggccataattgtctctcatggcatctttgtaa 6644
<210>15
<211>560
<212>DNA
<213>Mus musculus
<400>
15
accatgggtggcaagtccctgagcgggctgctgagcggcctagcgcagaacgcctttcac 60
ggacactcgggtgtcacggaggagctgctgcacagccaactctatccggaagtgccaccg 120
gaggagttccgccccttcctggcgaagatgagaggacttctcaaggtacggtggttccgc 180
cgagcagccctgccctctcgcagcctcaggcccgccccagcctcgggtgctgctgtcttt 240
gggcgctcagggacccttctgagccgtggaggtcggtctgttgcggccttgttttaggga 300
cacataacggtgaaaacattggatttttttttctctccctcaagactttctgtgtctgta 360
gtatagataagtttcgagtttttttcgcctcggactttgatgttgcaccgggcgttgtag 420
tgcactcctttaatctgtgcacttggagaggcagaggctggcagagagttgtgtgagttc 480
gaggccagcctgttgcacagagttccggggcagtcagggcaatgtggtgagacccttgtt 540
CA 02377580 2001-12-20
WO 01/00831 PCT/US00/16736
14
taaagagagc gagagcgtgc 560
<210>16
<211>295
<212>DNA
<213>Mus musculus
<400> 16
ggtcctacagacccacagcttccaggatctccatgacacagggcaacagcaggctatccg 60
agaggagccctggtgaaactaagttcaatcaanatatgttctgtagctaggcagctagct 120
ttgtctagttatctaccaagttcaaatatattgctttttcttttatctttatagtctatt 180
gcatctgcagacatggatttcaaccagttagaggcattcctgactgctcaaaccaaaaag 240
caaggtggcatcaccagtgagcaagctgcagtcatctccaagttttggaagagcc 295
