Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED ALPHA-HELICAL PROTEIN - 32
BACKGROUND OF THE INVENTION
Proliferation, maintenance, survival and differentiation of cells of
multicellular organisms are controlled by hormones and polypeptide growth
factors.
These diffusable molecules allow cells to communicate with each other and act
in
concert to form cells and organs, and to repair and regenerate damaged tissue.
Examples of hormones and growth factors include the steroid hormones (e.g.
estrogen,
testosterone), parathyroid hormone, follicle stimulating hormone, the
interleukins,
platelet derived growth factor (PDGF), epidermal growth factor (EGF),
granulocyte-
macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and
calcitonin.
Hormones and growth factors influence cellular metabolism by binding
to proteins. Proteins may be integral membrane proteins that are linked to
signaling
pathways within the cell, such as second messenger systems. Other classes of
proteins
are soluble molecules, such as the transcription factors.
Of particular interest are cytokines, molecules that promote the
2 0 proliferation, maintenance, survival or differentiation of cells. Examples
of cytokines
include erythropoietin (EPO), which stimulates the development of red blood
cells;
thrombopoietin (TPO), which stimulates development of cells of the
megakaryocyte
lineage; and granmocyte-colony stimulating factor (G-CSF), which stimulates
development of neutrophils. These cytokines are useful in restoring normal
blood cell
2 5 levels in patients suffering from anemia or receiving chemotherapy for
cancer. The
demonstrated in vivo activities of these cytokines illustrates the enormous
clinical
potential of, and need for, other cytokines, cytokine agonists, and cytokine
antagonists.
Furthermore, the overexpression of cytokines generally results in unwanted
inflammation. Thus, there is a need to discover unknown cytokines so that
their
3 o antagonists can be administered to ameliorate inflammatory responses.
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2
DESCRIPTION OF THE INVENTION
The present invention addresses this need by providing novel
polypeptides and related compositions and methods. Within one aspect, the
present
invention provides an isolated polynucleotide encoding a mammalian cytokine
termed
'Secreted alpha helical protein-32', hereinafter referred to as "Zalpha32".
Zalpha32
defined by SEQ ID NOs 1 and 2 has four alpha helices A, B, C and D. Amino acid
residues 1-25 of SEQ ID NO: 2 define a signal sequence. Thus, the mature
sequence
extends from amino acid residue 26, a glutamine, to and including amino acid
residue
170, a phenylalanine. The mature sequence, which is also defined by SEQ ID NO:
3,
1 o has an unglycosylated molecular weight of about 16,578 Daltons (D). SEQ ID
NOs: 14
and 15 are mouse Zalpha32 cDNA and polypeptide. Mouse Zalpha32 polypeptide has
a
signal sequence comprised of amino acid residues 1 - 25 of SEQ ID NO: 15. The
mature sequence is comprised of the amino acid sequence of SEQ ID NO: 16. SEQ
ID
NOs: 17 and 18 show another variant of murine Zalpha32. The signal sequence of
SEQ
ID NO: 18 is comprised of amino acid residue 1 - 25.
Within a second aspect of the invention there is provided an expression
vector comprising (a) a transcription promoter; (b) a DNA segment encoding
Zalpha32
polypeptide, and (c) a transcription terminator, wherein the promoter, DNA
segment,
and terminator are operably linked.
2 0 Within a third aspect of the invention there is provided a cultured
eukaryotic cell into which has been introduced an expression vector as
disclosed above,
wherein said cell expresses a protein polypeptide encoded by the DNA segment.
Within a further aspect of the invention there is provided a chimeric
polypeptide consisting essentially of a first portion and a second portion
joined by a
2 5 peptide bond. The first portion of the chimeric polypeptide consists
essentially of (a) a
Zalpha32 polypeptide as shown in SEQ ID NOs: 3, 16 or 19 (b) allelic variants
of SEQ
ID NOs: 3, 16 or 1 ~~; and (c) protein polypeptides that are at least 80%
identical to (a)
or (b). The second portion of the chimeric polypeptide consists essentially of
another
polypeptide such as an affinity tag. Within one embodiment the affinity tag is
an
3 0 immunoglobulin Fc polypeptide. The invention also provides expression
vectors
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encoding the chimeric polypeptides and host cells transfected to produce the
chimeric
polypeptides.
Within an additional aspect of the invention there is provided an
antibody that specifically binds to a Zalpha32 polypeptide as disclosed above,
and also
an anti-idiotypic antibody that neutralizes the antibody to a Zalpha32
polypeptide.
An additional embodiment of the present invention relates to a peptide
or polypeptide that has the amino acid sequence of an epitope-bearing portion
of a
Zalpha32 polypeptide having an amino acid sequence described above. Peptides
or
polypeptides having the amino acid sequence of an epitope-bearing portion of a
l0 Zalpha32 polypeptide of the present invention include portions of such
polypeptides
with at least nine, preferably at least 15 and more preferably at least 30 to
50 amino
acids, although epitope-bearing polypeptides of any length up to and including
the
entire amino acid sequence of a polypeptide of the present invention described
above
are also included in the present invention. Also claimed are any of these
polypeptides
that are fused to another polypeptide or carrier molecule. Examples of said
epitope-
bearing polypeptides are the polypeptides of SEQ ID NOs: 26, 27, 28, 29, 30,
31, 32,
33 and 34.
Prior to setting forth the invention in detail, it may be helpful to the
understanding thereof to define the following terms:
2 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
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
2 5 histidine tract, protein A, Nilsson et al., EMBO J. 4:1075 ( 1985);
Nilsson et al.,
Methods Enzymol. 198:3 ( 1991 ), glutathione S transferase, Smith and Johnson,
Gene
67:31 (1988), Glu-Glu affinity tag, Grussenmeyer et al., Proc. Natl. Acad.
Sci. USA
82:7952-4 (1985), substance P, FIagTM peptide, Hopp et al., Biotechnology
6:1204-1210
(1988), streptavidin binding peptide, or other antigenic epitope or binding
domain. See,
3 0 in general, Ford et al., Protein Expression and Purification 2: 95-107
(1991). DNAs
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encoding affinity tags are available from commercial suppliers (e.g.,
Pharmacia
Biotech, Piscataway, NJ).
The term "allelic variant" is used herein to denote any of two or more
alternative forms of a gene occupying the same chromosomal locus. Allelic
variation
arises naturally through mutation, and may result in phenotypic polymorphism
within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or
may encode polypeptides having altered amino acid sequence. The term allelic
variant
is also used herein to denote a protein encoded by an allelic variant of a
gene.
The terms "amino-terminal" and "carboxyl-terminal" are used herein to
l0 denote positions within polypeptides. Where the context allows, these terms
are used
with reference to a particular sequence or portion of a polypeptide to denote
proximity
or relative position. For example, a certain sequence positioned carboxyl-
terminal to a
reference sequence within a polypeptide is located proximal to the carboxyl
terminus of
the reference sequence, but is not necessarily at the carboxyl terminus of the
complete
polypeptide.
"Angiogenic" denotes the ability of a compound to stimulate the
formation of new blood vessels from existing vessels, acting alone or in
concert with
one or more additional compounds. Angiogenic activity is measurable as
endothelial
cell activation, stimulation of protease secretion by endothelial cells,
endothelial cell
2 0 migration, capillary sprout formation, and endothelial cell proliferation.
The term "complement/anti-complement pair" denotes non-identical
moieties that form a non-covalently associated, stable pair under appropriate
conditions.
For instance, biotin and avidin (or streptavidin) are prototypical members of
a
complement/anti-complement pair. Other exemplary complement/anti-complement
2 5 pairs include receptor/ligand pairs, antibody/antigen (or hapten or
epitope) pairs,
sense/antisense polynucleotide pairs, and the like. Where subsequent
dissociation of
the complement/anti-complement pair is desirable, the complement/anti-
complement
pair preferably has a binding affinity of <109 M-1.
The term "complements of a polynucleotide molecule" is a
3 0 polynucleotide molecule having a complementary base sequence and reverse
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orientation as compared to a reference sequence. For example, the sequence 5'
ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
The term "contig" denotes a polynucleotide that has a contiguous stretch
of identical or complementary sequence to another polynucleotide. Contiguous
5 sequences are said to "overlap" a given stretch of polynucleotide sequence
either in
their entirety or along a partial stretch of the polynucleotide. For example,
representative contigs to the polynucleotide sequence 5'-ATGGCTTAGCTT-3' are
S'-
TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5'.
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,
etc. Expression vectors are generally derived from plasmid or viral DNA, or
may
2 0 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
genetically engineered protein production systems. Such isolated molecules are
those
2 5 that are separated from their natural environment and include cDNA and
genomic
clones. Isolated DNA molecules of the present invention are free of other
genes with
which they are ordinarily associated, but may include naturally occurring 5'
and 3'
untranslated regions such as promoters and terminators. The identification of
associated regions will be evident to one of ordinary skill in the art (see
for example,
3 0 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. In a preferred form, the isolated polypeptide is substantially
free of other
polypeptides, particularly other polypeptides of animal origin. It is
preferred to provide
the polypeptides in a highly purified form, i.e. greater than 95% pure, more
preferably
greater than 99% pure. When used in this context, the term "isolated" does not
exclude
the presence of the same polypeptide in alternative physical forms, such as
dimers or
alternatively glycosylated or derivatized forms.
The term "operably linked", when referring to DNA segments, indicates
1 o that the segments are arranged so that they function in concert for their
intended
purposes, e.g., transcription initiates in the promoter and proceeds through
the coding
segment to the terminator.
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.
"Paralogs" are distinct but structurally related proteins made by an
organism. Paralogs are believed to arise through gene duplication. For
example, a-
globin, b-globin, and myoglobin are paralogs of each other.
A "polynucleotide" is a single- or double-stranded polymer of
2 0 deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3'
end.
Polynucleotides include RNA and DNA, and may be isolated from natural sources,
synthesized in vitro, or prepared from a combination of natural and synthetic
molecules.
Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides
("nt"), or kilobases ("kb"). Where the context allows, the latter two terms
may describe
2 5 polynucleotides that are single-stranded or double-stranded. When the term
is applied
to double-stranded molecules it is used to denote overall length and will be
understood
to be equivalent to the term "base pairs". It will be recognized by those
skilled in the
art that the two strands of a double-stranded polynucleotide may differ
slightly in length
and that the ends thereof may be staggered as a result of enzymatic cleavage;
thus all
3 0 nucleotides within a double-stranded polynucleotide molecule may not be
paired. Such
unpaired ends will in general not exceed 20 nt in length.
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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 polymerase 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
l0 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.
The term "receptor" denotes a cell-associated protein that binds to a
bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on
the cell.
Membrane-bound receptors are characterized by a mufti-domain structure
comprising
an extracellular ligand-binding domain and an intracellular effector domain
that is
2 0 typically involved in signal transduction. Binding of ligand to receptor
results in a
conformational change in the receptor that causes an interaction between the
effector
domain and other molecules) in the cell. This interaction in turn leads to an
alteration
in the metabolism of the cell. Metabolic events that are linked to receptor-
ligand
interactions include gene transcription, phosphorylation, dephosphorylation,
increases
2 5 in cyclic AMP production, mobilization of cellular calcium, mobilization
of membrane
lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids. In
general, receptors can be membrane bound, cytosolic or nuclear; monomeric
(e.g.,
thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric
(e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF
3 0 receptor, erythropoietin receptor and IL-6 receptor).
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The term "secretory signal sequence" denotes a DNA sequence that
encodes a polypeptide (a "secretory peptide") that, as a component of a larger
polypeptide, directs the larger polypeptide through a secretory pathway of a
cell in
which it is synthesized. The larger polypeptide is commonly cleaved to remove
the
secretory peptide during transit through the secretory pathway.
The term "splice variant" is used herein to denote alternative forms of
RNA transcribed from a gene. Splice variation arises naturally through use of
alternative splicing sites within a transcribed RNA molecule, or less commonly
between separately transcribed RNA molecules, and may result in several mRNAs
l0 transcribed from the same gene. Splice variants may encode polypeptides
having
altered amino acid sequence. The term splice variant is also used herein to
denote a
protein encoded by a splice variant of an mRNA transcribed from a gene.
Molecular weights and lengths of polymers determined by imprecise
analytical methods (e.g., gel electrophoresis) will be understood to be
approximate
values. When such a value is expressed as "about" X or "approximately" X, the
stated
value of X will be understood to be accurate to X10%.
The present invention provides novel cytokine polypeptides/proteins.
The novel cytokine, termed "alpha helical protein-32" hereinafter referred to
as
"Zalpha32" was discovered and identified to be a cytokine by the presence of
2 0 polypeptide and polynucleotide features characteristic of four-helix-
bundle cytokines
(e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin and growth
hormone).
Analysis of the amino acid sequence shown in SEQ ID N0:2 indicates a signal
sequence which extends from the methionine at position 1 to and including
amino acid
residue 25. Thus the mature sequence extends from amino acid residue 26, a
glutamine,
2 5 to an including amino acid residue 170, a phenylalanine. The mature
Zalpha32
polypeptide is also represented by the amino acid sequence of SEQ ID N0:3
which has
an unglycosylated molecular weight of approximately 16,578 Daltons (D).
Further analysis of SEQ ID N0:2 indicates the presence of four
amphipathic, alpha-helical regions, namely helices A, B, C and D. Each helix
contains
3 0 an external region having amino acid residues, which are generally
hydrophilic, and an
internally located region which generally contains hydrophobic amino acid
residues.
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The amino acid residues that are positioned on the exterior of the helices are
considered
crucial for receptor binding and should not be changed to another amino acid
residue
except to one that is almost identical in charge. The amino acid residues that
are
positioned on the interior of the helix may be changed to any hydrophobic
amino acid
residue.
Helix A, SEQ ID NO: 4, contains at least amino acid residue 27, a
glutamine, to and including amino acid residue 41, a leucine of SEQ ID NO: 2.
Helix A
is also represented by SEQ ID NO: 4.
Helix B, SEQ ID NO: 5 contains at least amino acid residue 81, a
leucine, to and including amino acid residue 94, an aspartic acid of SEQ ID
N0:2.
Helix C, SEQ ID NO: 6, contains at least amino acid residue 97, a
leucine, to and including amino acid residue 11 l, a leucine of SEQ ID NO: 2.
Helix D, SEQ ID NO: 7, contains at least amino acid residue 139, a
valine, to and including amino acid residue 153, a tyrosine of SEQ ID NO: 2.
POLYNUCLEOTIDES:
The present invention also provides polynucleotide molecules, including
DNA and RNA molecules that encode the Zalpha32 polypeptides disclosed herein.
Those skilled in the art will readily recognize that, in view of the
degeneracy of the
2 0 genetic code, considerable sequence variation is possible among these
polynucleotide
molecules.
Polynucleotides, generally a cDNA sequence, of the present invention
encode the described polypeptides herein. A cDNA sequence that encodes a
polypeptide of the present invention is comprised of a series of codons, each
amino acid
2 5 residue of the polypeptide being encoded by a codon and each codon being
comprised
of three nucleotides. The amino acid residues are encoded by their respective
codons as
follows.
Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;
Cysteine (Cys) is encoded by TGC or TGT;
3 0 Aspartic acid (Asp) is encoded by GAC or GAT;
Glutamic acid (Glu) is encoded by GAA or GAG;
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Phenylalanine (Phe) is encoded by TTC or TTT;
Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;
Histidine (His) is encoded by CAC or CAT;
Isoleucine (Ile) is encoded by ATA, ATC or ATT;
5 Lysine (Lys) is encoded by AAA, or AAG;
Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;
Methionine (Met) is encoded by ATG;
Asparagine (Asn) is encoded by AAC or AAT;
Proline (Pro) is encoded by CCA, CCC, CCG or CCT;
10 Glutamine (Gln) is encoded by CAA or CAG;
Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT;
Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT;
Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;
Valine (Val) is encoded by GTA, GTC, GTG or GTT;
Tryptophan (Trp) is encoded by TGG; and
Tyrosine (Tyr) is encoded by TAC or TAT.
It is to be recognized that according to the present invention, when a
polynucleotide is claimed as described herein, it is understood that what is
claimed are
2 0 both the sense strand, the anti-sense strand, and the DNA as double-
stranded having
both the sense and anti-sense strand annealed together by their respective
hydrogen
bonds. Also claimed is the messenger RNA (mRNA) that encodes the polypeptides
of
the president invention, and which mRNA is encoded by the cDNA described
herein.
Messenger RNA (mRNA) will encode a polypeptide using the same codons as those
2 5 defined herein, with the exception that each thymine nucleotide (T) is
replaced by a
uracil nucleotide (U).
One of ordinary skill in the art will also appreciate that different species
can exhibit "preferential codon usage." In general, see, Grantham, et al.,
Nuc. Acids
Res. 8:1893-1912 (1980); Haas, et al. Curr. Biol. 6:315-324 (1996); Wain-
Hobson, et
3 0 al., Gene 13:355-364 (1981); Grosjean and Fiers, Gene 18:199-209 (1982);
Holm, Nuc.
Acids Res. 14:3075-3087 (1986); Ikemura, J. Mol. Biol. 158:573-597 (1982). As
used
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herein, the term "preferential codon usage" or "preferential codons" is a term
of art
referring to protein translation codons that are most frequently used in cells
of a certain
species, thus favoring one or a few representatives of the possible codons
encoding
each amino acid. For example, the amino acid Threonine (Thr) may be encoded by
ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses or
bacteria, different
Thr codons may be preferential. Preferential codons for a particular species
can be
introduced into the polynucleotides of the present invention by a variety of
methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA
can, for example, enhance production of the protein by making protein
translation more
efficient within a particular cell type or species. Sequences containing
preferential
codons can be tested and optimized for expression in various species, and
tested for
functionality as disclosed herein.
Within preferred 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
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
2 0 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
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
2 5 amounts of Zalpha32 RNA. Such tissues and cells are identified by Northern
blotting,
Thomas, Proc. Natl. Acad. Sci. USA 77:5201 (1980) and are discussed below.
Total
RNA can be prepared using guanidine HCI 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.
3 0 Acad. Sci. USA 69:1408-1412 (1972). Complementary DNA (cDNA) is prepared
from
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poly(A)+ RNA using known methods. In the alternative, genomic DNA can be
isolated. Polynucleotides encoding Zalpha32 polypeptides are then identified
and
isolated by, for example, hybridization or PCR.
A full-length clone encoding Zalpha32 can be obtained by conventional
cloning procedures. Complementary DNA (cDNA) clones are preferred, although
for
some applications (e.g., expression in transgenic animals) it may be
preferable to use a
genomic clone, or to modify a cDNA clone to include at least one genomic
intron.
Methods for preparing cDNA and genomic clones are well known and within the
level
of ordinary skill in the art, and include the use of the sequence disclosed
herein, or parts
l0 thereof, for probing or priming a library. Expression libraries can be
probed with
antibodies to Zalpha32, receptor fragments, or other specific binding
partners.
The polynucleotides of the present invention can also be synthesized
using DNA synthesizers. Currently the method of choice is the phosphoramidite
method. If chemically synthesized double stranded DNA is required for an
application
such as the synthesis of a gene or a gene fragment, then each complementary
strand is
made separately. The production of short genes (60 to 80 bp) is technically
straightforward and can be accomplished by synthesizing the complementary
strands
and then annealing them. For the production of longer genes (>300 bp),
however,
special strategies must be invoked, because the coupling efficiency of each
cycle during
2 0 chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic
genes
(double-stranded) are assembled in modular form from single-stranded fragments
that
are from 20 to 100 nucleotides in length.See Glick and Pasternak, Molecular
Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press,
Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-356 (1984)
and
Climie et al., Proc. Natl. Acad. Sci. USA 87:633-637 (1990).
The present invention further provides counterpart polypeptides and
polynucleotides from other species (orthologs). These species include, but are
not
limited to mammalian, avian, amphibian, reptile, fish, insect and other
vertebrate and
invertebrate species. Of particular interest are Zalpha32 polypeptides from
other
3 0 mammalian species, including murine, porcine, ovine, bovine, canine,
feline, equine,
and other primate polypeptides. Orthologs of human Zalpha32 can be cloned
using
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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 Zalpha32 as disclosed
herein.
Suitable sources of mRNA can be identified by probing Northern blots with
probes
designed from the sequences disclosed herein. A library is then prepared from
mRNA
of a positive tissue or cell line. A Zalpha32-encoding cDNA can then be
isolated by a
variety of methods, such as by probing with a complete or partial human cDNA
or with
one or more sets of degenerate probes based on the disclosed sequences. A cDNA
can
also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S.
Patent No.
4,683,202), using primers designed from the representative human Zalpha32
sequence
disclosed herein. Within an additional method, the cDNA library can be used to
transform or transfect host cells, and expression of the cDNA of interest can
be detected
with an antibody to Zalpha32 polypeptide. Similar techniques can also be
applied to
the isolation of genomic clones.
Those skilled in the art will recognize that the sequence disclosed in
SEQ ID NO: 1 represents a single allele of human Zalpha32 and that allelic
variation
and alternative splicing are expected to occur. Allelic variants of this
sequence can be
cloned by probing cDNA or genomic libraries from different individuals
according to
standard procedures. Allelic variants of the DNA sequence shown in SEQ ID
NO:1,
2 0 including those containing silent mutations and those in which mutations
result in
amino acid sequence changes, are within the scope of the present invention, as
are
proteins which are allelic variants of SEQ ID N0:2. cDNAs generated from
alternatively spliced mRNAs, which retain the properties of the Zalpha32
polypeptide
are included within the scope of the present invention, as are polypeptides
encoded by
2 5 such cDNAs and mRNAs. Allelic variants and splice variants of these
sequences can
be cloned by probing cDNA or genomic libraries from different individuals or
tissues
according to standard procedures known in the art.
The present invention also provides isolated Zalpha32 polypeptides that
are substantially homologous to the polypeptides of SEQ ID N0:2 and their
orthologs.
3 0 The term "substantially homologous" is used herein to denote polypeptides
having
50%, preferably 60%, more preferably at least 80%, sequence identity to the
sequences
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WO 00/71717 PCT/US00/14563
14
shown in SEQ ID N0:2 or their orthologs. Such polypeptides will more
preferably be
at least 90% identical, and most preferably 95% or more identical to SEQ ID
N0:2 or
its orthologs.) Percent sequence identity is determined by conventional
methods. See,
for example, Altschul et al., Bull. Math. Bio. 48: 603-616 (1986) and Henikoff
and
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992). Briefly, two amino
acid
sequences are aligned to optimize the alignment scores using a gap opening
penalty of
10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and
Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the
standard one-
letter codes).
to
The percent identity is then calculated as:
Total number of identical matches
x 100
[length of the longer sequence plus the
nm:~ber of gaps introduced into the longer
sequence in order to align the two sequences]
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
N
M
n
~c7N N
O
~ M N
N
i
i
I~~ .~~t M
N
i i i
i
l0 ~ N N ~ M
~
i i ~ i
tnO N
i i ~ i
tl~~ M ~ O ~ M N
i i ~ ~ i N
i
i
J ~ N N O M N ~ N
n n v i n n
'-" ~ N M ~ O M N ~ M ~
n n n n n M
n
N M M ~ N .-rN ~ N N N
i i i i i i i i i M
U' l0 N W t N M M N O N N M
i n n n n i i n n M
n
n
w ICJN O M M ~ N M ~ O ~ M N
n n n n n n n n N
n
n
O W -(7N N O M N ~ O M ~ O ~ N ~
n n n v n n ~ N
n
n
U 01M ~ M M ~ .-iM .-iN M ~ ~ N N
~
n i n n n n n n n ~ n n n n
n
D LO M o N .~ ,~M ~t~ M M ~ O ~ V- M
n n n n n n n n n n n M
n
n
Z l0 ~ M O O O ~ M M O N M N ~ O ~ N
i i i ~ i i i M
i
i
LC)O N M ~ O N O M N N ~ M N ~ ~ M N
n v n n n n i i n n n M
n
n
Q '~t.-~N N O ~ .-iO N ~ ~ ~ ~ N ~ ~ O M N
i i i i i i i i i i i i i O
i
Q L z D U O' UJU' Z ~ J ~G ~ L~ d.V7 H 3
Lf1 O L(1 O
N
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16
Those skilled in the art appreciate that there are many established
algorithms to align two amino acid sequences. The "FASTA" similarity search
algorithm of Pearson and Lipman is a suitable protein alignment method for
examining
the level of identity shared by an amino acid sequence and the amino acid
sequence of a
putative variant. The FASTA algorithm is described by Pearson and Lipman,
Proc.
Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990).
Briefly, FASTA first characterizes sequence similarity by identifying regions
shared by
the query sequence (e.g., SEQ ID N0:2) and a test sequence that have either
the highest
density of identities (if the ktup variable is 1 ) or pairs of identities (if
ktup=2), without
considering conservative amino acid substitutions, insertions or deletions.
The ten
regions with the highest density of identities are then rescored by comparing
the
similarity of all paired amino acids using an amino acid substitution matrix,
and the
ends of the regions are "trimmed" to include only those residues that
contribute to the
highest score. If there are several regions with scores greater than the
"cutoff' value
(calculated by a predetermined formula based upon the length of the sequence
and the
ktup value), then the trimmed initial regions are examined to determine
whether the
regions can be joined to form an approximate alignment with gaps. Finally, the
highest
scoring regions of the two amino acid sequences are aligned using a
modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444
(1970); Sellers, SIAMJ. Appl. Math. 26:787 (1974), which allows for amino acid
insertions and deletions. Illustrative parameters for FASTA analysis are:
ktup=1, gap
opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62.
These parameters can be introduced into a FASTA program by modifying the
scoring
matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol.
2 5 183:63 ( 1990).
FASTA can also be used to determine the sequence identity of nucleic
acid molecules using a ratio as disclosed above. For nucleotide sequence
comparisons,
the ktup value can range between one to six, preferably from four to six.
The present invention includes nucleic acid molecules that encode a
3 o polypeptide having one or more conservative amino acid changes, compared
with the
amino acid sequence of SEQ ID N0:3. The BLOSUM62 table is an amino acid
CA 02374520 2001-11-23
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17
substitution matrix derived from about 2,000 local multiple alignments of
protein
sequence segments, representing highly conserved regions of more than 500
groups of
related proteins [Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915
(1992)].
Accordingly, the BLOSUM62 substitution frequencies can be used to define
conservative amino acid substitutions that may be introduced into the amino
acid
sequences of the present invention. As used herein, the language "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 0,1,2, or 3. Preferred conservative amino
acid
substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1,2
or 3),
while more preferred conservative substitutions are characterized by a
BLOSUM62
value of at least 2 (e.g., 2 or 3). Accordingly the present invention claims
those
polypeptides which are at least 90%, preferably 95% and most preferably 99%
identical
to SEQ ID N0:3 and which are able to stimulate antibody production in a
mammal, and
said antibodies are able to bind the native sequence of SEQ ID N0:3.
Variant Zalpha32 polypeptides or substantially homologous Zalpha32
polypeptides are characterized as having one or more amino acid substitutions,
deletions or additions. These changes are preferably of a minor nature, that
is
conservative amino acid substitutions (see Table 2) and other substitutions
that do not
2 0 significantly affect the folding or activity of the polypeptide; small
deletions, typically
of one to about 30 amino acids; and small amino- or carboxyl-terminal
extensions, such
as an amino-terminal methionine residue, a small linker peptide of up to about
20-25
residues, or an affinity tag. The present invention thus includes polypeptides
of from
to 30 amino acid residues that comprise a sequence that is at least 90%,
preferably at
2 5 least 95%, and more preferably 99% or more identical to the corresponding
region of
SEQ ID N0:4. Polypeptides comprising affinity tags can further comprise a
proteolytic
cleavage site between the Zalpha32 polypeptide and the affinity tag. Preferred
such
sites include thrombin cleavage sites and factor Xa cleavage sites.
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18
Conservative amino acid substitutions
Basic: arginine
lysine
histidine
Acidic: glutamic acid
aspartic acid
Polar: glutamine
Asparagine
Hydrophobic: leucine
isoleucine
valine
Aromatic: phenylalanine
2 0 tryptophan
tyrosine
Small: glycine
alanine
2 5 serine
threonine
methionine
The present invention further provides a variety of other polypeptide
3 0 fusions [and related multimeric proteins comprising one or more
polypeptide fusions].
For example, a Zalpha32 polypeptide can be prepared as a fusion to a
dimerizing
CA 02374520 2001-11-23
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19
protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Preferred
dimerizing proteins in this regard include immunoglobulin constant region
domains.
Immunoglobulin-Zalpha32 polypeptide fusions can be expressed in genetically
engineered cells [to produce a variety of multimeric Zalpha32 analogs].
Auxiliary
domains can be fused to Zalpha32 polypeptides to target them to specific
cells, tissues,
or macromolecules (e.g., collagen). For example, a Zalpha32 polypeptide or
protein
could be targeted to a predetermined cell type by fusing a Zalpha32
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
l0 Zalpha32 polypeptide can be fused to two or more moieties, such as an
affinity tag for
purification and a targeting domain. Polypeptide fusions can also comprise one
or
more cleavage sites, particularly between domains. See, Tuan et al.,
Connective Tissue
Research 34:1-9 (1996).
The proteins of the present invention can also comprise non-naturally
occurring amino acid residues. Non-naturally occurring amino acids include,
without
limitation, traps-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
traps-4-
hydroxyproline, N methylglycine, allo-threonine, methylthreonine,
hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine,
pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-
methylproline,
2 0 3,3-dimethylproline, tent-leucine, norvaline, 2-azaphenylalanine, 3-
azaphenylalanine, 4-
azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the
art for
incorporating non-naturally occurring amino acid residues into proteins. For
example,
an in vitro system can be employed wherein nonsense mutations are suppressed
using
chemically aminoacylated suppressor tRNAs.
2 5 Methods for synthesizing amino acids and aminoacylating tRNA are
known in the art. Transcription and translation of plasmids containing
nonsense
mutations is carried out in a cell-free system comprising an E coli S30
extract and
commercially available enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722
3 0 (1991); Ellman et al., Methods Enzymol. 202:301 (1991; Chung et al.,
Science 259:806-
809 (1993); and Chung et al., Proc. Natl. Acad Sci. USA 90:10145-1019 (1993).
In a
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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 amino acid that is to be replaced (e.g.,
phenylalanine) and in
5 the presence of the desired non-naturally occurring amino acids) (e.g., 2-
azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-
fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the protein in
place of its
natural counterpart. See, Koide et al., Biochem. 33:7470-7476 (1994).
Naturally
occurring amino acid residues can be converted to non-naturally occurring
species by in
10 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).
A limited number of non-conservative amino acids, amino acids that are
not encoded by the genetic code, non-naturally occurring amino acids, and
unnatural
15 amino acids may b:, substituted for Zalpha32 amino acid residues.
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 88:4498-502 (1991). In the
latter
2 0 technique, single alanine mutations are introduced at every residue in the
molecule, and
the resultant mutant molecules are tested for biological activity as disclosed
below to
identify amino acid residues that are critical to the activity of the
molecule. See also,
Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites of ligand-receptor
interaction
can also be determined by physical analysis of structure, as determined by
such
2 5 techniques as nuclear magnetic resonance, crystallography, electron
diffraction or
photoaffinity labeling, in conjunction with mutation of putative contact site
amino
acids. See, for example, de Vos et al., Science 255:306-312 (1992); Smith et
al., J.
Mol. Biol. 224:899-904 (1992); Wlodaver et al., FEBS Lett. 309:59-64 (1992).
Multiple amino acid substitutions can be made and tested using known
3 0 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
CA 02374520 2001-11-23
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21
86:2152-2156 (1989). Briefly, these authors disclose methods for
simultaneously
randomizing two or more positions in a polypeptide, selecting for functional
polypeptide, and then sequencing the mutagenized polypeptides to determine the
spectrum of allowable substitutions at each position. Other methods that can
be used
include phage display, e.g., Lowman et al., Biochem. 30:10832-10837 (1991);
Ladner
et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and
region-
directed mutagenesis, Derbyshire et al., Gene 46:145 (1986); Ner et al., DNA
7:127
( 1988).
Variants of the disclosed Zalpha32 DNA and polypeptide sequences can
be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-
391,
(1994), Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751 (1994) and WIPO
Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro
homologous recombination by random fragmentation of a parent DNA followed by
reassembly using PCR, resulting in randomly introduced point mutations. This
technique can be modified by using a family of parent DNAs, such as allelic
variants or
DNAs from different species, to introduce additional variability into the
process
Selection or screening for the desired activity, followed by additional
iterations of
mutagenesis and assay provides for rapid "evolution" of sequences by selecting
for
desirable mutations while simultaneously selecting against detrimental
changes.
2 o Mutagenesis methods as disclosed herein can be combined with high-
throughput, automated screening methods to detect activity of cloned,
mutagenized
polypeptides in host cells. Mutagenized DNA molecules that encode active
polypeptides can be recovered from the host cells and rapidly sequenced using
modern
equipment. These methods allow the rapid determination of the importance of
2 5 individual amino acid residues in a polypeptide of interest, and can be
applied to
polypeptides of unknown structure.
Using the methods discussed herein, one of ordinary skill in the art can
identify and/or prepare a variety of polypeptide fragments or variants of SEQ
ID
NOs:2, 4 or 6 or that retain the properties of the wild-type Zalpha32 protein.
For any
3 0 Zalpha32 polypeptide, including variants and fusion proteins, one of
ordinary skill in
CA 02374520 2001-11-23
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22
the art can readily generate a fully degenerate polynucleotide sequence
encoding that
variant using the information set forth in Tables 1 and 2 above.
PROTEIN PRODUCTION
The Zalpha32 polypeptides of the present invention, including full-
length polypeptides, biologically active fragments, and fusion polypeptides,
can be
produced in genetically engineered host cells according to conventional
techniques.
Suitable host cells are those cell types that can be transformed or
transfected with
l0 exogenous DNA and grown in culture, and include bacteria, fungal cells, and
cultured
higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of
multicellular
organisms, are preferred. Techniques for manipulating cloned DNA molecules and
introducing exogenous DNA into a variety of host cells are disclosed by
Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd ed., (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989), and Ausubel et al., eds.,
Current
Protocols in Molecular Biology (John Wiley and Sons, Inc., NY, 1987).
In general, a DNA sequence encoding a Zalpha32 polypeptide is
operably linked to other genetic elements required for its expression,
generally
including a transcription promoter and terminator, within an expression
vector. The
2 0 vector will also commonly contain one or more selectable markers and one
or more
origins of replication, although those skilled in the art will recognize that
within certain
systems selectable markers may be provided on separate vectors, and
replication of the
exogenous DNA may be provided by integration into the host cell genome.
Selection
of promoters, terminators, selectable markers, vectors and other elements is a
matter of
2 5 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 Zalpha32 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
3 0 be that of Zalpha32, or may be derived from another secreted protein
(e.g., t-PA) or
synthesized de novo. The secretory signal sequence is operably linked to the
Zalpha32
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
23
DNA sequence, i.e., the two sequences are joined in the correct reading frame
and
positioned to direct the newly synthesized polypeptide into the secretory
pathway of the
host cell. Secretory signal sequences are commonly positioned 5' to the DNA
sequence
encoding the polypeptide of interest, although certain secretory signal
sequences may
be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et
al., U.S.
Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
Alternatively, the secretory signal sequence contained in the
polypeptides of the present invention is used to direct other polypeptides
into the
secretory pathway. The present invention provides for such fusion
polypeptides. The
secretory signal sequence contained in the fusion polypeptides of the present
invention
is preferably fused amino-terminally to an additional peptide to direct the
additional
peptide into the secretory pathway. Such constructs have numerous applications
known
in the art. For example, these novel secretory signal sequence fusion
constructs can
direct the secretion of an active component of a normally non-secreted
protein, such as
a receptor. Such fusions may be used in vivo or in vitro to direct peptides
through the
secretory pathway.
Cultured mammalian cells are suitable hosts within the present
invention. Methods for introducing exogenous DNA into mammalian host cells
include
calcium phosphate-mediated transfection, Wigler et al., Cell 14:725 (1978);
Corsaro
2 0 and Pearson, Somatic Cell Genetics 7:603 ( 1981 ); Graham and Van der Eb,
l~irology
52:456 (1973), electroporation, Neumann et al., EMBO J. 1:841-845 (1982), DEAE-
dextran mediated transfection (Ausubel et al., ibid., and liposome-mediated
transfection, Hawley-Nelson et al., Focus 15:73 (1993); Ciccarone et al.,
Focus 15:80
(1993), and viral vectors, Miller and Rosman, BioTechniques 7:980(1989); Wang
and
Finer, Nature Med 2:714 (1996). 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
3 0 (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL
1573; Graham et al., J. Gen. Virol. 36:59 (1977) and Chinese hamster ovary
(e.g. CHO-
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24
K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in
the art
and available from public depositories such as the American Type Culture
Collection,
Rockville, Maryland. In general, strong transcription promoters are preferred,
such as
promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
Other suitable promoters include those from metallothionein genes (U.S. Patent
Nos.
4,579,821 and 4,601,978) and the adenovirus major late promoter.
Drug selection is generally used to select for cultured mammalian cells
into which foreign DNA has been inserted. Such cells are commonly referred to
as
"transfectants". Cells that have been cultured in the presence of the
selective agent and
are able to pass the gene of interest to their progeny are referred to as
"stable
transfectants." A preferred selectable marker is a gene encoding resistance to
the
antibiotic neomycin. Selection is carried out in the presence of a neomycin-
type drug,
such as G-418 or the like. Selection systems can also be used to increase the
expression
level of the gene of interest, a process referred to as "amplification."
Amplification is
carried out by culturing transfectants in the presence of a low level of the
selective
agent and then increasing the amount of selective agent to select for cells
that produce
high levels of the products of the introduced genes. A preferred amplifiable
selectable
marker is dihydrofolate reductase, which confers resistance to methotrexate.
Other
drug resistance genes (e.g. hygromycin resistance, mufti-drug resistance,
puromycin
2 0 acetyltransferase) can also be used. Alternative markers that introduce an
altered
phenotype, such as green fluorescent protein, or cell surface proteins such as
CD4,
CDB, Class I MHC, placental alkaline phosphatase may be used to sort
transfected cells
from untransfected cells by such means as FACS sorting or magnetic bead
separation
technology.
2 5 Other higher eukaryotic cells can also be used as hosts, including plant
cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a
vector for
expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci.
(Bangalore) 11:47 (1987). Transformation of insect cells and production of
foreign
polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222
and
3 0 WIPO publication WO 94/06463. Insect cells can be infected with
recombinant
baculovirus, commonly derived from Autographa californica nuclear polyhedrosis
virus
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
(AcNPV). DNA encoding the Zalpha32 polypeptide is inserted into the
baculoviral
genome in place of the AcNPV polyhedrin gene coding sequence by one of two
methods. The first is the traditional method of homologous DNA recombination
between wild-type AcNPV and a transfer vector containing the Zalpha32 flanked
by
5 AcNPV sequences. Suitable insect cells, e.g. SF9 cells, are infected with
wild-type
AcNPV and transfected with a transfer vector comprising a Zalpha32
polynucleotide
operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking
sequences. See, King, L.A. and Possee, R.D., The Baculovirus Expression
System: A
Laboratory Guide, (Chapman & Hall, London); O'Reilly, D.R. et al., Baculovirus
10 Expression Vectors: A Laboratory Manual (Oxford University Press, New York,
New
York, 1994); and, Richardson, C. D., Ed., Baculovirus Expression Protocols.
Methods
in Molecular Biology, (Humana Press, Totowa, NJ 1995). Natural recombination
within an insect cell will result in a recombinant baculovirus which contains
Zalpha32
driven by the polyhedrin promoter. Recombinant viral stocks are made by
methods
15 commonly used in the art.
The second method of making recombinant baculovirus utilizes a
transposon-based system described by Luckow, V.A, et al., J Virol 67:4566
(1993).
This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD).
This
system utilizes a transfer vector, pFastBac 1 T"" (Life Technologies)
containing a Tn7
2 0 transposon to move the DNA encoding the Zalpha32 polypeptide into a
baculovirus
genome maintained in E. coli as a large plasmid called a "bacmid." The
pFastBaclT""
transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression
of the
gene of interest, in this case Zalpha32. However, pFastBaclT"" can be modified
to a
considerable degree. The polyhedrin promoter can be removed and substituted
with the
2 5 baculovirus basic protein promoter (also known as Pcor, p6.9 or MP
promoter) which is
expressed earlier in the baculovirus infection, and has been shown to be
advantageous
for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D.,
JGen Virol
71:971 (1990); Bonning, B.C. et al., JGen Virol 75:1551 (1994); and,
Chazenbalk,
G.D., and Rapoport, B., JBiol Chem 270:1543 (1995). In such transfer vector
3 0 constructs, a short or long version of the basic protein promoter can be
used. Moreover,
transfer vectors can be constructed which replace the native Zalpha32
secretory signal
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
26
sequences with secretory signal sequences derived from insect proteins. For
example, a
secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee
Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San
Diego, CA)
can be used in constructs to replace the native Zalpha32 secretory signal
sequence. In
addition, transfer vectors can include an in-frame fusion with DNA encoding an
epitope
tag at the C- or N-terminus of the expressed Zalpha32 polypeptide, for
example, a Glu-
Glu epitope tag, Grussenmeyer, T. et al., Proc Natl Acad Sci. 82:7952 (1985).
Using a
technique known in the art, a transfer vector containing Zalpha32 is
transformed into E.
coli, and screened for bacmids which contain an interrupted lacZ gene
indicative of
recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus
genome is isolated, using common techniques, and used to transfect Spodoptera
frugiperda cells, e.g. Sf~7 cells. Recombinant virus that expresses Zalpha32
is
subsequently produced. Recombinant viral stocks are made by methods commonly
used the art.
The recombinant virus is used to infect host cells, typically a cell line
derived from the fall army worm, Spodoptera frugiperda. See, in general, Glick
and
Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant
DNA (ASM Press, Washington, D.C., 1994). Another suitable cell line is the
High
FiveOT"" cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent
#5,300,435).
2 0 Commercially available serum-free media are used to grow and maintain the
cells.
Suitable media are Sf~00 IIT"~ (Life Technologies) or ESF 921 T"" (Expression
Systems)
for the Sf~ cells; and Ex-ce11O405T"" (JRH Biosciences, Lenexa, KS) or Express
FiveOT"" (Life Technologies) for the T. ni cells. 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
2 5 which time a recombinant viral stock is added at a multiplicity of
infection (MOI) of
0.1 to 10, more typically near 3. The recombinant virus-infected cells
typically produce
the recombinant Zalpha32 polypeptide at 12-72 hours post-infection and secrete
it with
varying efficiency into the medium. The culture is usually harvested 48 hours
post-
infection. Centrifugation is used to separate the cells from the medium
(supernatant).
3 0 The supernatant containing the Zalpha32 polypeptide is filtered through
micropore
filters, usually 0.45 qm pore size. Procedures used are generally described in
available
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
27
laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et
al., ibid.;
Richardson, C. D., ibid. ). Subsequent purification of the Zalpha32
polypeptide from
the supernatant can be achieved using methods described herein.
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;
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.
Transformed cells are selected by phenotype determined by the selectable
marker,
commonly drug resistance or the ability to grow in the absence of a particular
nutrient
(e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae
is the
POTI 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
2 0 4,661,454. Transformation systems for other yeasts, including Hansenula
polymorpha,
Schizosaccharomyces pombe, Kluyveromyces lactic, Kluyveromyces fragilis,
Ustilago
maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida
maltosa are known in the art. See, for example, Gleeson et al., J. Gen.
Microbiol.
132:3459 (1986) and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be
2 5 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.
The use of Pichia methanolica as host for the production of recombinant
3 0 proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO
98/02536, and WO 98/02565. DNA molecules for use in transforming P.
methanolica
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28
will commonly be prepared as double-stranded, circular plasmids, which are
preferably
linearized prior to transformation. For polypeptide production in P.
methanolica, it is
preferred that the promoter and terminator in the plasmid be that of a P.
methanolica
gene, such as a P. methanolica alcohol utilization gene (AUGI or AUG2). Other
useful
promoters include those of the dihydroxyacetone synthase (DHAS), formate
dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of
the DNA
into the host chromosome, it is preferred to have the entire expression
segment of the
plasmid flanked at both ends by host DNA sequences. A preferred selectable
marker
for use in Pichia m~thanolica is a P. methanolica ADE2 gene, which encodes
phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21 ), which allows
ade2 host cells to grow in the absence of adenine. For large-scale, industrial
processes
where it is desirable to minimize the use of methanol, it is preferred to use
host cells in
which both methanol utilization genes (AUGI and AUG2) are deleted. For
production
of secreted proteins, host cells deficient in vacuolar protease genes (PEP4
and PRBI )
are preferred. Electroporation is used to facilitate the introduction of a
plasmid
containing DNA encoding a polypeptide of interest into P. methanolica cells.
It is
preferred to transform P. methanolica cells by electroporation using an
exponentially
decaying, pulsed electric field having a field strength of from 2.5 to 4.5
kV/cm,
preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40
milliseconds, most
2 0 preferably about 20 milliseconds.
Prokaryotic host cells, including strains of the bacteria Escherichia coli,
Bacillus and other genera are also useful host cells within the present
invention.
Techniques for transforming these hosts and expressing foreign DNA sequences
cloned
therein are well known in the art, see, e.g., Sambrook et al., ibid.). When
expressing a
2 5 Zalpha32 polypeptide in bacteria such as E. coli, the polypeptide may be
retained in the
cytoplasm, typically as insoluble granules, or may be directed to the
periplasmic space
by a bacterial secretion sequence. In the former case, the cells are lysed,
and the
granules are recovered and denatured using, for example, guanidine
isothiocyanate or
urea. The denatured polypeptide can then be refolded and dimerized by diluting
the
3 0 denaturant, such as by dialysis against a solution of urea and a
combination of reduced
and oxidized glutathione, followed by dialysis against a buffered saline
solution. In the
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29
latter case, the poll. peptide 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
and
minerals. Media may also contain such components as growth factors or serum,
as
required. The growth medium will generally select for cells containing the
exogenously added DNA by, for example, drug selection or deficiency in an
essential
nutrient which is complemented by the selectable marker carried on the
expression
vector or co-transfected into the host cell. P. methanolica cells are cultured
in a
medium comprising adequate sources of carbon, nitrogen and trace nutrients at
a
temperature of about 25°C to 35°C. Liquid cultures are provided
with sufficient
aeration by conventional means, such as shaking of small flasks or sparging of
fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-
glucose,
2% BactoTM Peptone (Difco Laboratories, Detroit, MI), 1 % BactoTM yeast
extract (Difco
2 0 Laboratories), 0.004% adenine and 0.006% L-leucine).
Another embodiment of the present invention provides for a peptide or
polypeptide comprising an epitope-bearing portion of a Zalpha32 polypeptide of
the
invention. The epitope of the this polypeptide portion is an immunogenic or
antigenic
epitope of a polypeptide of the invention. A region of a protein to which an
antibody
2 5 can bind is defined as an "antigenic epitope". See for instance, Geysen,
H.M. et al.,
Proc. Natl. Acad Sw. USA 81: 3998-4002 (1984). As to the selection of peptides
or
polypeptides bearing an antigenic epitope (i. e., that contain a region of a
protein
molecule to which an antibody can bind), it is well known in the art that
relatively short
synthetic peptides that mimic part of a protein sequence are routinely capable
of
3 o eliciting an antiserum that reacts with the partially mimicked protein.
See Sutcliffe, J.G.
et al. Science 219:660-666 (1983). Peptides capable of eliciting protein-
reactive sera
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are frequently represented in the primary sequence of a protein, can be
characterized by
a set of simple chemical rules, and are confined neither to immunodominant
regions of
intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl
terminals.
Peptides that are extremely hydrophobic and those of six or fewer residues
generally are
5 ineffective at inducing antibodies that bind to the mimicked protein; longer
soluble
peptides, especially those containing proline residues, usually are effective.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore useful to raise antibodies, including monoclonal antibodies, that
bind
specifically to a polypeptide of the invention. Antigenic epitope-bearing
peptides and
10 polypeptides of the present invention contain a sequence of at least nine,
preferably
between 15 to about 30 amino acids contained within the amino acid sequence of
a
polypeptide of the invention. However, peptides or polypeptides comprising a
laxger
portion of an amino acid sequence of the invention, containing from 30 to 50
amino
acids, or any length up to and including the entire amino acid sequence of a
polypeptide
15 of the invention, also are useful for inducing antibodies that react with
the protein.
Preferably, the amino acid sequence of the epitope-bearing peptide is selected
to
provide substantial solubility in aqueous solvents (i.e., the sequence
includes relatively
hydrophilic residues and hydrophobic residues are preferably avoided); and
sequences
containing proline residues are particularly preferred. All of the
polypeptides shown in
2 o the sequence listing contain antigenic epitopes to be used according to
the present
invention, however, specifically designed antigenic epitopes include the
peptides
defined by SEQ ID NOs: 26-34. The present invention also provides polypeptide
fragments or peptides comprising an epitope-bearing portion of a Zalpha32
polypeptide
described herein. Such fragments or peptides may comprise an "immunogenic
2 5 epitope," which is a part of a protein that elicits an antibody response
when the entire
protein is used as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods [see, for example, Geysen et al., supra. See
also U.S.
Patent No. 4,708,781 (1987) further describes how to identify a peptide
bearing an
immunogenic epitope of a desired protein.
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31
Protein Isolation
It is preferred to purify the polypeptides of the present invention to
>80% purity, more preferably to >_90% purity, even more preferably >_95%
purity, and
particularly preferred is a pharmaceutically pure state, that is greater than
99.9% pure
with respect to contaminating macromolecules, particularly other proteins and
nucleic
acids, and free of infectious and pyrogenic agents. Preferably, a purified
polypeptide is
substantially free of other polypeptides, particularly other polypeptides of
animal
origin.
Expressed recombinant Zalpha32 polypeptides (or chimeric Zalpha32
polypeptides) can be purified using fractionation and/or conventional
purification
methods and media. Ammonium sulfate precipitation and acid or chaotrope
extraction
may be used for fractionation of samples. Exemplary purification steps may
include
hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid
chromatography. Suitable chromatographic media include derivatized dextrans,
agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI,
DEAE, QAE
and Q derivatives are preferred. Exemplary chromatographic media include those
media
derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF
(Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-
Sepharose
2 0 (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71
(Toso
Haas) and the like. Suitable solid supports include glass beads, silica-based
resins,
cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene
beads, cross-
linked polyacrylamide resins and the like that are insoluble under the
conditions in
which they are to be used. These supports may be modified with reactive groups
that
2 5 allow attachment of proteins by amino groups, carboxyl groups, sulfliydryl
groups,
hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries
include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide
activation, sulfhydryl activation, hydrazide activation, and carboxyl and
amino
derivatives for carbodiimide coupling chemistries. These and other solid media
are
3 0 well known and widely used in the art, and are available from commercial
suppliers.
Methods for binding receptor polypeptides to support media are well known in
the art.
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32
Selection of a particular method is a matter of routine design and is
determined in part
by the properties of the chosen support. See, for example, Amity
Chromatography:
Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988).
The polypeptides of the present invention can be isolated by exploitation
of their properties. For example, immobilized metal ion adsorption (IMAC)
chromatography can be used to purify histidine-rich proteins, including those
comprising polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions to
form a chelate, Sulkowski, Trends in Biochem. 3:1 (1985). Histidine-rich
proteins will
be adsorbed to this matrix with differing affinities, depending upon the metal
ion used,
and will be eluted by competitive elution, lowering the pH, or use of strong
chelating
agents. Other methods of purification include purification of glycosylated
proteins by
lectin affinity chromatography and ion exchange chromatography. Methods in
Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.),page
529-539
(Acad. Press, San Diego, 1990). Within additional embodiments of the
invention, a
fusion of the polypeptide of interest and an affinity tag (e.g., maltose-
binding protein,
an immunoglobulin domain) may be constructed to facilitate purification.
Moreover, using methods described in the art, polypeptide fusions, or
hybrid Zalpha32 proteins, are constructed using regions or domains of the
inventive
Zalpha32, Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin.
Biology,
2 0 5:511 ( 1994). These methods allow the determination of the biological
importance of
larger domains or regions in a polypeptide of interest. Such hybrids may alter
reaction
kinetics, binding, constrict or expand the substrate specificity, or alter
tissue and
cellular localization of a polypeptide, and can be applied to polypeptides of
unknown
structure.
2 5 Fusion proteins can be prepared by methods known to those skilled in
the art by preparing each component of the fusion protein and chemically
conjugating
them. Alternatively, a polynucleotide encoding both components of the fusion
protein
in the proper reading frame can be generated using known techniques and
expressed by
the methods described herein. For example, part or all of a domains)
conferring a
3 o biological function may be swapped between Zalpha32 of the present
invention with
the functionally equivalent domains) from another family member. Such domains
CA 02374520 2001-11-23
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33
include, but are not limited to, the secretory signal sequence, conserved, and
significant
domains or regions in this family. Such fusion proteins would be expected to
have a
biological functional profile that is the same or similar to polypeptides of
the present
invention or other known family proteins, depending on the fusion constructed.
Moreover, such fusion proteins may exhibit other properties as disclosed
herein.
Zalpha32 polypeptides or fragments thereof may also be prepared
through chemical synthesis. Zalpha32 polypeptides may be monomers or
multimers;
glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may
not
include an initial methionine amino acid residue.
Chemical Synthesis of Polypeptides
Polypeptides, especially polypeptides of the present invention can also
be synthesized by exclusive solid phase synthesis, partial solid phase
methods,
fragment condensation or classical solution synthesis. The polypeptides are
preferably
prepared by solid phase peptide synthesis, for example as described by
Merrifield, J.
Am. Chem. Soc. 85:2149 (1963).
ASSAYS
2 0 The activity of molecules of the present invention can be measured using
a variety of assays. Of particular interest are changes in steroidogenesis,
spermatogenesis, in the testis, LH and FSH production and GnRH in the
hypothalamus.
Such assays are well known in the art.
Proteins of the present invention are useful for increasing sperm
2 5 production. Zalpha32 can be measured in vitro using cultured cells or in
vivo by
administering molecules of the claimed invention to the appropriate animal
model. For
instance, Zalpha32 transfected (or co-transfected) expression host cells may
be
embedded in an alginate environment and injected (implanted) into recipient
animals.
Alginate-poly-L-lysine microencapsulation, permselective membrane
encapsulation and
3 0 diffusion chambers have been described as a means to entrap transfected
mammalian
cells or primary mammalian cells. These types of non-immunogenic
"encapsulations"
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34
or microenvironments permit the transfer of nutrients into the
microenvironment, and
also permit the diffusion of proteins and other macromolecules secreted or
released by
the captured cells across the environmental barrier to the recipient animal.
Most
importantly, the capsules or microenvironments mask and shield the foreign,
embedded
cells from the recipient animal's immune response. Such microenvironments can
extend the life of the injected cells from a few hours or days (naked cells)
to several
weeks (embedded cells).
Alginate threads provide a simple and quick means for generating
embedded cells. The materials needed to generate the alginate threads are
readily
l0 available and relatively inexpensive. Once made, the alginate threads are
relatively
strong and durable, both in vitro and, based on data obtained using the
threads, in vivo.
The alginate threads are easily manipulable and the methodology is scalable
for
preparation of numerous threads. In an exemplary procedure, 3% alginate is
prepared
in sterile H20, and sterile filtered. Just prior to preparation of alginate
threads, the
alginate solution is again filtered. An approximately 50% cell suspension
(containing
about S x 105 to about 5 x 10~ cells/ml) is mixed with the 3% alginate
solution. One
ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered
CaCl2
solution over a time period of ~15 min, forming a "thread". The extruded
thread is then
transferred into a solution of 50 mM CaCl2, and then into a solution of 25 mM
CaCl2.
2 0 The thread is then rinsed with deionized water before coating the thread
by incubating
in a 0.01 % solution of poly-L-lysine. Finally, the thread is rinsed with
Lactated
Ringer's Solution and drawn from solution into a syringe barrel (without
needle
attached). A large bore needle is then attached to the syringe, and the thread
is
intraperitoneally injected into a recipient in a minimal volume of the
Lactated Ringer's
Solution.
An alternative in vivo approach for assaying proteins of the present
invention involves viral delivery systems. Exemplary viruses for this purpose
include
adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV).
Adenovirus, a double-stranded DNA virus, is currently the best studied gene
transfer
3 0 vector for delivery of heterologous nucleic acid (for a review, see T.C.
Becker et al.,
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Meth. Cell Biol. 43:161 (1994); and J.T. Douglas and D.T. Curiel, Science &
Medicine
4:44 (1997). The adenovirus system offers several advantages: adenovirus can
(i)
accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii)
infect a
broad range of mammalian cell types; and (iv) be used with a large number of
available
5 vectors containing different promoters. Also, 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-
l0 transfected plasmid. In an exemplary system, the essential E1 gene has been
deleted
from the viral vector, and the virus will not replicate unless the E1 gene is
provided by
the host cell (the human 293 cell line is exemplary). When intravenously
administered
to intact animals, adenovirus primarily targets the liver. If the adenoviral
delivery
system has an El gene deletion, the virus cannot replicate in the host cells.
However,
15 the host's tissue (e.g., liver) will express and process (and, if a
secretory 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.
The adenovirus system can also be used for protein production in vitro.
2 0 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
2 5 without significant cell division. Alternatively, adenovirus vector
infected 293 S cells
can be grown in suspension culture at relatively high cell density to produce
significant
amounts of protein (see Gamier et al., Cytotechnol. 15:145 (1994). With either
protocol, an expressed, secreted heterologous protein can be repeatedly
isolated from
the cell culture supernatant. Within the infected 293S cell production
protocol, non-
3 0 secreted proteins may also be effectively obtained.
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36
Antagonists
Antagonists are also useful as research reagents for characterizing sites
of ligand-receptor interaction. Also as a treatment for prostate cancer.
Inhibitors of
Zalpha32 activity (Zalpha32 antagonists) include anti-Zalpha32 antibodies and
soluble
Zalpha32 receptors, as well as other peptidic and non-peptidic agents
(including
ribozymes).
Zalpha32 can also be used to identify inhibitors (antagonists) of its
activity. Test compounds are added to the assays disclosed herein to identify
compounds that inhibit the activity of Zalpha32. In addition to those assays
disclosed
herein, samples can be tested for inhibition of Zalpha32 activity within a
variety of
assays designed to measure receptor binding or the stimulation/inhibition of
Zalpha32-
dependent cellular responses. For example, Zalpha32-responsive cell lines can
be
transfected with a reporter gene construct that is responsive to a Zalpha32-
stimulated
cellular pathway. Reporter gene constructs of this type are known in the art,
and will
generally comprise a Zalpha32-DNA response element operably linked to a gene
encoding a protein which can be assayed, such as luciferase. DNA response
elements
can include, but are not limited to, cyclic AMP response elements (CRE),
hormone
response elements (HRE) insulin response element (IRE), Nasrin et al., Proc.
Natl.
2 0 Acad Sci. USA 87:5273 ( 1990) and serum response elements (SRE) (Shaw et
al. Cell
56: 563 (1989). Cyclic AMP response elements are reviewed in Roestler et al.,
J. Biol.
Chem. 263 (19):9063 (1988) and Habener, Molec. Endocrinol. 4 (8):1087 (1990).
Hormone response elements are reviewed in Beato, Cell 56:335 (1989). Candidate
compounds, solutions, mixtures or extracts are tested for the ability to
inhibit the
2 5 activity of Zalpha32 on the target cells as evidenced by a decrease in
Zalpha32
stimulation of reporter gene expression. Assays of this type will detect
compounds that
directly block Zalpha32 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
Zalpha32
30 binding to receptor using Zalpha32 tagged with a detectable label
(e.g.,'ZSI, biotin,
horseradish peroxidase, FITC, and the like). Within assays of this type, the
ability of a
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37
test sample to inhibit the binding of labeled Zalpha32 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.
A Zalpha32 polypeptide can be expressed as a fusion with an
immunoglobulin heavy chain constant region, typically an Fc fragment, which
contains
two constant region domains and lacks the variable region. Methods for
preparing such
fusions are disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Such
fusions are
typically secreted as multimeric molecules wherein the Fc portions are
disulfide bonded
to each other and two non-Ig polypeptides are arrayed in closed proximity to
each
other. Fusions of this type can be used to affinity purify the ligand. For use
in assays,
the chimeras are bound to a support via the Fc region and used in an ELISA
format.
A Zalpha32 ligand-binding polypeptide can also be used for purification
of ligand. The polypeptide is immobilized on a solid support, such as beads of
agarose,
cross-linked agarose, glass, cellulosic resins, silica-based resins,
polystyrene, cross-
linked polyacrylamide, or like materials that are stable under the conditions
of use.
Methods for linking polypeptides to solid supports are known in the art, and
include
amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation,
epoxide activation, sulfhydryl activation, and hydrazide activation. The
resulting
medium will generally be configured in the form of a column, and fluids
containing
2 0 ligand are passed through the column one or more times to allow ligand to
bind to the
receptor polypeptide. The ligand is then eluted using changes in salt
concentration,
chaotropic agents (guanidine HCl), or pH to disrupt ligand-receptor binding.
An assay system that uses a ligand-binding receptor (or an antibody, one
member of a complement/ anti-complement pair) or a binding fragment thereof,
and a
2 5 commercially available biosensor instrument (BIAcore, Pharmacia Biosensor,
Piscataway, NJ) may be advantageously employed. Such receptor, antibody,
member
of a complement/anti-complement pair or fragment is immobilized onto the
surface of a
receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol.
Methods
145:229 (1991) and Cunningham and Wells, J. Mol. Biol. 234:554 (1993). A
receptor,
3 0 antibody, member or fragment is covalently attached, using amine or
sulfhydryl
chemistry, to dextran fibers that are attached to gold film within the flow
cell. A test
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38
sample is passed through the cell. If a ligand, epitope, or opposite member of
the
complement/anti-complement pair is present in the sample, it will bind to the
immobilized receptor, antibody or member, respectively, causing a change in
the
refractive index of the medium, which is detected as a change in surface
plasmon
resonance of the gold film. This system allows the determination of on- and
off rates,
from which binding affinity can be calculated, and assessment of stoichiometry
of
binding.
Ligand-binding receptor polypeptides can also be used within other
assay systems known in the art. Such systems include Scatchard analysis for
determination of binding affinity, Scatchard, Ann. NYAcad. Sci. 51: 660 (1949)
and
calorimetric assays, Cunningham et al., Science 253:545 (1991); Cunningham et
al.,
Science 245:821 (1991).
Zalpha32 polypeptides can also be used to prepare antibodies that
specifically bind to Zalpha32 epitopes, peptides or polypeptides. The Zalpha32
polypeptide or a fragment thereof serves as an antigen (immunogen) to
inoculate an
animal and elicit an immune response. Suitable antigens would be the Zalpha32
polypeptides encoded by SEQ ID NOs:2-24. Antibodies generated from this immune
response can be isolated and purified as described herein. Methods for
preparing and
isolating polyclonal and monoclonal antibodies are well known in the art. See,
for
2 o example, Current Protocols in Immunology, Cooligan, et al. (eds.),
National Institutes
of Health, (John Wiley and Sons, Inc., 1995); Sambrook et al., Molecular
Cloning: A
Laboratory Manual, Second Edition (Cold Spring Harbor, NY, 1989); and Hurrell,
J.
G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC
Press, Inc., Boca Raton, FL, 1982).
2 5 As would be evident to one of ordinary skill in the art, polyclonal
antibodies can be generated from inoculating a variety of warm-blooded animals
such
as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a
Zalpha32
polypeptide or a fragment thereof. The immunogenicity of a Zalpha32
polypeptide
may be increased through the use of an adjuvant, such as alum (aluminum
hydroxide)
3 0 or Freund's complete or incomplete adjuvant. Polypeptides useful for
immunization
also include fusion polypeptides, such as fusions of Zalpha32 or a portion
thereof with
CA 02374520 2001-11-23
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39
an immunoglobulin polypeptide or with maltose binding protein. The polypeptide
immunogen may be a full-length molecule or a portion thereof. If the
polypeptide
portion is "hapten-like", such portion may be advantageously joined or linked
to a
macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum
albumin (BSA) or tetanus toxoid) for immunization.
As used herein, the term "antibodies" includes polyclonal antibodies,
affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-
binding
fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically
engineered intact
antibodies or fragments, such as chimeric antibodies, Fv fragments, single
chain
antibodies and the like, as well as synthetic antigen-binding peptides and
polypeptides,
are also included. Non-human antibodies may be humanized by grafting non-human
CDRs onto human framework and constant regions, or by incorporating the entire
non-
human variable domains (optionally "cloaking" them with a human-like surface
by
replacement of exposed residues, wherein the result is a "veneered" antibody).
In some
instances, humanized antibodies may retain non-human residues within the human
variable region framework domains to enhance proper binding characteristics.
Through
humanizing antibodies, biological half life may be increased, and the
potential for
adverse immune reactions upon administration to humans is reduced.
Alternative techniques for generating or selecting antibodies useful
2 0 herein include in vitro exposure of lymphocytes to Zalpha32 protein or
peptide, and
selection of antibody display libraries in phage or similar vectors (for
instance, through
use of immobilized or labeled Zalpha32 protein or peptide). Genes encoding
polypeptides having potential Zalpha32 polypeptide-binding domains can be
obtained
by screening random peptide libraries displayed on phage (phage display) or on
2 5 bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides
can be
obtained in a number of ways, such as through random mutagenesis and random
polynucleotide synthesis. These random peptide display libraries can be used
to screen
for peptides which interact with a known target which can be a protein or
polypeptide,
such as a ligand or receptor, a biological or synthetic macromolecule, or
organic or
3 o inorganic substances. Techniques for creating and screening such random
peptide
display libraries are known in the art (Ladner et al., US Patent NO.
5,223,409; Ladner
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et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and
Ladner et
al., US Patent NO. 5,571,698) and random peptide display libraries and kits
for
screening such libraries are available commercially, for instance from
Clontech (Palo
Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc.
(Beverly, MA)
5 and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide
display
libraries can be screened using the Zalpha32 sequences disclosed herein to
identify
proteins that bind to Zalpha32. These "binding proteins" which interact with
Zalpha32
polypeptides can be used for tagging cells; for isolating homolog polypeptides
by
affinity purification: they can be directly or indirectly conjugated to drugs,
toxins,
1 o radionuclides and the like. These binding proteins can also be used in
analytical
methods such as for screening expression libraries and neutralizing activity.
The
binding proteins can also be used for diagnostic assays for determining
circulating
levels of polypeptides; for detecting or quantitating soluble polypeptides as
marker of
underlying pathology or disease. These binding proteins can also act as
Zalpha32
15 "antagonists" to block Zalpha32 binding and signal transduction in vitro
and in vivo.
Antibodies are determined to be specifically binding if: 1 ) they exhibit a
threshold level of binding activity, and 2) they do not significantly cross-
react with
related polypeptide molecules. First, antibodies herein specifically bind if
they bind to
a Zalpha32 polypeptide, peptide or epitope with a binding affinity (Ka) of 106
M 1 or
2 0 greater, preferably 107 M 1 or greater, more preferably 108 M 1 or
greater, and most
preferably 109 M 1 or greater. The binding affinity of an antibody can be
readily
determined by one of ordinary skill in the art, for example, by Scatchard
analysis.
Second, antibodies are determined to specifically bind if they do not
significantly cross-react with related polypeptides. Antibodies do not
significantly
25 cross-react with related polypeptide molecules, for example, if they detect
Zalpha32 but
not known related polypeptides using a standard Western blot analysis (Ausubel
et al.,
ibid.). Examples of known related polypeptides are orthologs, proteins from
the same
species that are members of a protein family (e.g. IL-16), Zalpha32
polypeptides, and
non-human Zalpha32. Moreover, antibodies may be "screened against" known
related
3 0 polypeptides to isolate a population that specifically binds to the
inventive
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41
polypeptides. For example, antibodies raised to Zalpha32 are adsorbed to
related
polypeptides adhered to insoluble matrix; antibodies specific to Zalpha32 will
flow
through the matrix under the proper buffer conditions. Such screening allows
isolation
of polyclonal and monoclonal antibodies non-crossreactive to closely related
polypeptides, Antibodies: A Laboratory Manual, Harlow and Lane (eds.) (Cold
Spring
Harbor Laboratory Press, 1988); Current Protocols in Immunology, Cooligan, et
al.
(eds.), National Institutes of Health (John Wiley and Sons, Inc., 1995).
Screening and
isolation of specific antibodies is well known in the art. See, Fundamental
Immunology, Paul (eds.) (Raven Press, 1993); Getzoff et al., Adv. in Immunol.
43: 1-98
(1988); Monoclonal Antibodies: Principles and Practice, Goding, J.W. (eds.),
(Academic Press Ltd., 1996); Benjamin et al., Ann. Rev. Immunol. 2: 67-101
(1984).
A variety of assays known to those skilled in the art can be utilized to
detect antibodies that specifically bind to Zalpha32 proteins or peptides.
Exemplary
assays are described in detail in Antibodies: A Laboratory Manual, Harlow and
Lane
(Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of
such
assays include: concurrent immunoelectrophoresis, radioimmunoassay,
radioimmuno-
precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western
blot
assay, inhibition or competition assay, and sandwich assay. In addition,
antibodies can
be screened for binding to wild type versus mutant Zalpha32 protein or
polypeptide.
2 0 Antibodies to Zalpha32 may be used for tagging cells that express
Zalpha32; for isolating Zalpha32 by affinity purification; for diagnostic
assays for
determining circulating levels of Zalpha32 polypeptides; for detecting or
quantitating
soluble Zalpha32 as marker of underlying pathology or disease; in analytical
methods
employing FACS; for screening expression libraries; for generating anti-
idiotypic
2 5 antibodies; and as neutralizing antibodies or as antagonists to block
Zalpha32 in vitro
and in vivo. Suitable direct tags or labels include radionuclides, enzymes,
substrates,
cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic
particles and the like; indirect tags or labels may feature use of biotin-
avidin or other
complement/anti-complement pairs as intermediates. Antibodies herein may also
be
3 o directly or indirectly conjugated to drugs, toxins, radionuclides and the
like, and these
conjugates used for in vivo diagnostic or therapeutic applications. Moreover,
antibodies
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42
to Zalpha32 or fragments thereof may be used in vitro to detect denatured
Zalpha32 or
fragments thereof in assays, for example, Western Blots or other assays known
in the
art.
BIOACTIVE CONJUGATES:
Antibodies or polypeptides herein can also be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like, and these conjugates
used for in
vivo diagnostic or therapeutic applications. For instance, polypeptides or
antibodies of
the present invention can be used to identify or treat tissues or organs that
express a
corresponding anti-complementary molecule (receptor or antigen, respectively,
for
instance). More specifically, Zalpha32 polypeptides or anti-Zalpha32
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 may be directly or indirectly attached to
the polypeptide or antibody, and include radionuclides, enzymes, substrates,
cofactors,
inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles
and the
like. Suitable cytotoxic molecules may be directly or indirectly attached to
the
polypeptide or antibody, and include bacterial or plant toxins (for instance,
diphtheria
2 o toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as
therapeutic
radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly
attached
to the polypeptide or antibody, or indirectly attached through means of a
chelating
moiety, for instance). Polypeptides or antibodies may also be conjugated to
cytotoxic
drugs, such as adriamycin. For indirect attachment of a detectable or
cytotoxic
2 5 molecule, the detectable or cytotoxic molecule can be conjugated with a
member of a
complementary/ anticomplementary pair, where the other member is bound to the
polypeptide or antibody portion. For these purposes, biotin/streptavidin is an
exemplary complementary/ anticomplementary pair.
In another embodiment, polypeptide-toxin fusion proteins or antibody-
3 0 toxin fusion proteins can be used for targeted cell or tissue inhibition
or ablation (for
instance, to treat cancer cells or tissues). Alternatively, if the polypeptide
has multiple
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43
functional domains (i.e., an activation domain or a ligand binding domain,
plus a
targeting domain), a fusion protein including only the targeting domain may be
suitable
for directing a detectable molecule, a cytotoxic molecule or a complementary
molecule
to a cell or tissue type of interest. In instances where the domain only
fusion protein
includes a complementary molecule, the anti-complementary molecule can be
conjugated to a detectable or cytotoxic molecule. Such domain-complementary
molecule fusion proteins thus represent a generic targeting vehicle for
cell/tissue-
specific delivery of generic anti-complementary-detectable/ cytotoxic molecule
conjugates.
In another embodiment, Zalpha32-cytokine fusion proteins or antibody-
cytokine fusion proteins can be used for enhancing in vivo killing of target
tissues (for
example, blood and bone marrow cancers), if the Zalpha32 polypeptide or anti-
Zalpha32 antibody targets the hyperproliferative blood or bone marrow cell.
See,
generally, Hornick et al., Blood 89:4437 (1997). They described fusion
proteins enable
targeting of a cytokine to a desired site of action, thereby providing an
elevated local
concentration of cytokine. Suitable Zalpha32 polypeptides or anti-Zalpha32
antibodies
target an undesirable cell or tissue (i. e., a tumor or a leukemia), and the
fused cytokine
mediated improved target cell lysis by effector cells. Suitable cytokines for
this purpose
include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM-
2 o CSF), for instance.
In yet another embodiment, if the Zalpha32 polypeptide or anti-
Zalpha32 antibody targets vascular cells or tissues, such polypeptide or
antibody may
be conjugated with a radionuclide, and particularly with a beta-emitting
radionuclide, to
reduce restenosis. Such therapeutic approach poses less danger to clinicians
who
2 5 administer the radioactive therapy. For instance, iridium-192 impregnated
ribbons
placed into stented vessels of patients until the required radiation dose was
delivered
showed decreased tissue growth in the vessel and greater luminal diameter than
the
control group, which received placebo ribbons. Further, revascularisation and
stmt
thrombosis were significantly lower in the treatment group. Similar results
are
3 0 predicted with targeting of a bioactive conjugate containing a
radionuclide, as described
herein.
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44
The bioactive polypeptide or antibody conjugates described herein can
be delivered intravenously, intraarterially or intraductally, or may be
introduced locally
at the intended site of action.
USES OF POLYNUCLEOTIDE/POLYPEPTIDE:
Molecules of the present invention can be used to identify and isolate
receptors involved in spermatogenesis, steroidogenesis, testicular
differentiation and
regulatory control of the hypothalamic-pituitary-gonadal axis. For example,
proteins
1 o and peptides of the present invention can be immobilized on a column and
membrane
preparations run over the column, Immobilized Amity Ligand Techniques,
Hermanson
et al., eds., pp.195-202 (Academic Press, San Diego, CA, 1992,). Proteins and
peptides
can also be radiolabeled, Methods in Enzymol., vol. 182, "Guide to Protein
Purification", M. Deutscher, ed., pp 721-737 (Acad. Press, San Diego, 1990) or
photoaffinity labeled, Brunner et al., Ann. Rev. Biochem. 62:483-514 (1993)
and Fedan
et al., Biochem. Pharmacol. 33:1167 (1984) and specific cell-surface proteins
can be
identified.
The molecules of the present invention will be useful for testing
disorders of the reproductive system and immunological systems.
GENE THERAPY:
Polynucleotides encoding Zalpha32 polypeptides are useful within gene
therapy applications where it is desired to increase or inhibit Zalpha32
activity. If a
mammal has a mutated or absent Zalpha32 gene, the Zalpha32 gene can be
introduced
2 5 into the cells of the mammal. In one embodiment, a gene encoding a
Zalpha32
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
3 o 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,
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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 (1991); an attenuated adenovirus
vector,
such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest.
90:626
5 (1992); and a defective adeno-associated virus vector, Samulski et al., J.
Virol. 61:3096
(1987); Samulski et al., J. Virol. 63:3822 (1989).
In another embodiment, a Zalpha32 gene can be introduced in a
retroviral vector, e.g., as described in 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.
10 Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120 (1988); Temin et
al., U.S.
Patent No. 5,124,263; International Patent Publication No. WO 95/07358,
published
March 16, 1995 by Dougherty et al.; and Kuo et al., Blood 82:845 (1993).
Alternatively, the vector can be introduced by lipofection in vivo using
liposomes.
Synthetic cationic lipids can be used to prepare liposomes for in vivo
transfection of a
15 gene encoding a marker, Felgner et al., Proc. Natl. Acad Sci. USA 84:7413
(1987);
Mackey et al., Proc. Natl. Acad Sci. USA 85:8027 (1988). The use of
lipofection to
introduce exogenous genes into specific organs in vivo has certain practical
advantages.
Molecular targeting of liposomes to specific cells represents one area of
benefit. More
particularly, directing transfection to particular cells represents one area
of benefit. For
2 0 instance, directing transfection to particular cell types would be
particularly
advantageous in a tissue with cellular heterogeneity, such as the pancreas,
liver, kidney,
and brain. Lipids may be chemically coupled to other molecules for the purpose
of
targeting. Targeted peptides (e.g., hormones or neurotransmitters), proteins
such as
antibodies, or non-peptide molecules can be coupled to liposomes chemically.
2 5 It is possible to remove the target cells from the body; to introduce the
vector as a naked DNA plasmid; and then to re-implant the transformed cells
into the
body. Naked DNA vectors for gene therapy can be introduced into the desired
host
cells by methods known in the art, e.g., transfection, electroporation,
microinjection,
transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use
of a gene
3 0 gun or use of a DNa vector transporter. See, e.g., Wu et al., J. Biol.
Chem. 267:963
(1992); Wu et al., J. Biol. Chem. 263:14621-4, 1988.
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46
Antisense methodology can be used to inhibit Zalpha32 gene
transcription, such as to inhibit cell proliferation in vivo. Polynucleotides
that are
complementary to a segment of a Zalpha32-encoding polynucleotide (e.g., a
polynucleotide as set froth in SEQ ID NO:1 ) are designed to bind to Zalpha32-
encoding
mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides
are
used to inhibit expression of Zalpha32 polypeptide-encoding genes in cell
culture or in
a subj ect.
The present invention also provides reagents that will find use in
diagnostic applications. For example, the Zalpha32 gene, a probe comprising
Zalpha32
1 o DNA or RNA or a subsequence thereof can be used to determine if the
Zalpha32 gene
is present on chromosome 19p13.2-19p13.1 or if a mutation has occurred.
Detectable
chromosomal aberrations at the Zalpha32 gene locus include, but are not
limited to,
aneuploidy, gene copy number changes, insertions, deletions, restriction site
changes
and rearrangements. Such aberrations can be detected using polynucleotides of
the
present invention by employing molecular genetic techniques, such as
restriction
fragment length polymorphism (RFLP) analysis, short tandem repeat (STR)
analysis
employing PCR techniques, and other genetic linkage analysis techniques known
in the
art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255
(1995).
Transgenic mice, engineered to express the Zalpha32 gene, and mice
2 o that exhibit a complete absence of Zalpha32 gene function, referred to as
"knockout
mice", Snouwaert et al., Science 257:1083 (1992), may also be generated,
Lowell et al.,
Nature 366:740-42 (1993). These mice may be employed to study the Zalpha32
gene
and the protein encoded thereby in an in vivo system.
2 5 CHROMOSOMAL LOCALIZATION:
Radiation hybrid mapping is a somatic cell genetic technique developed
for constructing high-resolution, contiguous maps of mammalian chromosomes
(Cox et
al., Science 250:245 (1990). Partial or full knowledge of a gene's sequence
allows one
to design PCR primers suitable for use with chromosomal radiation hybrid
mapping
3 o panels. Radiation hybrid mapping panels are commercially available which
cover the
entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH
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47
Panel (Research Genetics, Inc., Huntsville, AL). These panels enable rapid,
PCR-based
chromosomal localizations and ordering of genes, sequence-tagged sites (STSs),
and
other nonpolymorphic and polymorphic markers within a region of interest. This
includes establishing directly proportional physical distances between newly
discovered
genes of interest and previously mapped markers. The precise knowledge of a
gene's
position can be useful for a number of purposes, including: 1 ) determining if
a sequence
is part of an existing contig and obtaining additional surrounding genetic
sequences in
various forms, such as YACs, BACs or cDNA clones; 2) providing a possible
candidate gene for an inheritable disease which shows linkage to the same
l0 chromosomal region; and 3) cross-referencing model organisms, such as
mouse, which
may aid in determining what function a particular gene might have. Zalpha32
has been
mapped to chromosome 19p13.2-19p13.1.
Sequence tagged sites (STSs) can also be used independently for
chromosomal localization. An STS is a DNA sequence that is unique in the human
genome and can be used as a reference point for a particular chromosome or
region of a
chromosome. An STS is defined by a pair of oligonucleotide primers that are
used in a
polymerase chain reaction to specifically detect this site in the presence of
all other
genomic sequences. Since STSs are based solely on DNA sequence they can be
completely described within an electronic database, for example, Database of
Sequence
2 0 Tagged Sites (dbSTS), GenBank, (National Center for Biological
Information, National
Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be
searched
with a gene sequence of interest for the mapping data contained within these
short
genomic landmark STS sequences.
For pharmaceutical use, the proteins of the present invention are
2 5 formulated for parenteral, particularly intravenous or subcutaneous,
delivery according
to conventional methods. Intravenous administration will be by bolus injection
or
infusion over a typical period of one to several hours. In general,
pharmaceutical
formulations will include a Zalpha32 protein in combination with a
pharmaceutically
acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or
the like.
3 0 Formulations may further include one or more excipients, preservatives,
solubilizers,
buffering agents, albumin to prevent protein loss on vial surfaces, etc.
Methods of
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48
formulation are well known in the art and are disclosed, for example, in
Remington:
The Science and Practice of Pharmacy, Gennaro, ed.,(Mack Publishing Co.,
Easton,
PA, 19th ed., 1995). Therapeutic doses will generally be in the range of 0.1
to 100
~g/kg of patient weight per day, preferably 0.5-20 mg/kg per day, with the
exact dose
determined by the clinician according to accepted standards, taking into
account the
nature and severity of the condition to be treated, patient traits, etc.
Determination of
dose is within the level of ordinary skill in the art. The proteins may be
administered
for acute treatment, over one week or less, often over a period of one to
three days or
may be used in chronic treatment, over several months or years.
Tissue Expression and Use
Zalpha32 represents a novel polypeptide with a putative signal peptide
leader sequence and alpha helical structure. It is expressed primarily in the
thymus,
testis, fetal liver and fetal kidney. Therefore this gene may encode a
secreted
polypeptide with secondary structure indicating it is a member of the four-
helix bundle
cytokine family.
Most four-helix bundle cytokines as well as other proteins produced by
activated T lymphocytes play an important biological role in cell
differentiation,
2 o activation, recruitment and homeostasis of cells throughout the body and
are involved
in inflammation in one form or another. Thus, antagonists to Zalpha32 can be
used to
reduce inflammation.
ED UCA TIONAL KIT UTILITY OF ZALPHA32 POL YPEPTIDES,
POLYNUCLEOTIDES AND ANTIBODIES.
Polynucleotides and polypeptides of the present invention will additionally
find
use as educational tools as a laboratory practicum kits for courses related to
genetics
and molecular biology, protein chemistry and antibody production and analysis.
Due to
its unique polynucleotide and polypeptide sequence molecules of Zalpha32 can
be used
3 0 as standards or as "unknowns" for testing purposes. For example, Zalpha32
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49
polynucleotides can be used as an aid, such as, for example, to teach a
student how to
prepare expression constructs for bacterial, viral, and/or mammalian
expression,
including fusion constructs, wherein Zalpha32 is the gene to be expressed; for
determining the restriction endonuclease cleavage sites of the
polynucleotides; for
determining mRNA and DNA localization of Zalpha32 polynucleotides in tissues
(i.e.,
by Northern and Southern blotting as well as polymerise chain reaction); and
for
identifying related polynucleotides and polypeptides by nucleic acid
hybridization.
Zalpha32 polypeptides can be used educationally as an aid to teach
preparation of antibodies; identifying proteins by Western blotting; protein
purification;
1 o determining the weight of expressed Zalpha32 polypeptides as a ratio to
total protein
expressed; identifying peptide cleavage sites; coupling amino and carboxyl
terminal
tags; amino acid sequence analysis, as well as, but not limited to monitoring
biological
activities of both the native and tagged protein (i.e., receptor binding,
signal
transduction, proliferation, and differentiation) in vitro and in vivo.
Zalpha32
polypeptides can also be used to teach analytical skills such as mass
spectrometry,
circular dichroism to determine conformation, in particular the locations of
the disulfide
bonds, x-ray crystallography to determine the three-dimensional structure in
atomic
detail, nuclear magnetic resonance spectroscopy to reveal the structure of
proteins in
solution. For example, a kit containing the Zalpha32 can be given to the
student to
2 0 analyze. Since the amino acid sequence would be known by the professor,
the protein
can be given to the student as a test to determine the skills or develop the
skills of the
student, the teacher would then know whether or not the student has correctly
analyzed
the polypeptide. Since every polypeptide is unique, the educational utility of
Zalpha32
would be unique unto itself.
2 5 The antibodies which bind specifically to Zalpha32 can be used as a
teaching aid to instruct students how to prepare affinity chromatography
columns to
purify Zalpha32, cloning and sequencing the polynucleotide that encodes an
antibody
and thus as a practicum for teaching a student how to design humanized
antibodies. The
Zalpha32 gene, polypeptide or antibody would then be packaged by reagent
companies
3 0 and sold to universities so that the students gain skill in art of
molecular biology. Since
Zalpha32 is actually expressed in the body, the antibodies to Zalpha32 can be
used to
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teach the students tissue localization using labeled antibodies. Because each
gene and
protein is unique, each gene and protein creates unique challenges and
learning
experiences for students in a lab practicum. Because the Zalpha32 gene and
polypeptide are actually present in the body they provide for real-life
experiences that
5 mere hypothetical sequences are unable to provide. Such educational kits
containing the
Zalpha32 gene, polypeptide or antibody are considered within the scope of the
present
invention.
10 The invention is further illustrated by the following non-limiting
examples.
Example 1
Cloning of Zalpha32
Zalpha32 was discovered by using SEQ ID NO: 7 as a probe in a spleen
cDNA library. cDNAs from human hematopoietic cell lines, K562 (ATCC #CCL243),
Daudi (ATCC #CCL213, HL-60(ATCC CCL240), MOLT-4 (ATCC #CRL1582) and
Raji ATCC #CCL86 were synthesized in separate reactions and size fractionated
in the
2 0 following manner. RNA extracted from each one of the cell lines was
reversed
transcribed. The resulting cDNA library was subjected to large scale
sequencing to
identify novel express sequence tags (ESTs). The EST defined by SEQ ID NO: 13
was
discovered and the cloned sequence resulting in Zalphal3 gene and protein of
SEQ ID
NOs: 1 and 2.
Example 2
Using the human zalpha32 cDNA sequence a mouse expressed sequence
tag (EST) database was searched and two ESTs were delivered, EST664085, SEQ ID
NO: 20, and EST629520, SEQ ID NO: 21, from the Washington University, IMAGE
3 0 consortium, St. Louis Missouri. The clone corresponding to SEQ ID NO: 20
was full-
length, SEQ ID NO: 14, with a 3'end splicing different from the clone
corresponding to
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51
SEQ ID NO: 21, which was missing 5'end start. A full-length sequence was
constructed
annealing the 5'end of SEQ ID NO: 14 with SEQ ID NO: 21 to produce SEQ ID NO:
17.
Example 3
Alpha32m cloning for Baculovirus expression
The full length zAlpha32mu underwent the PCR using primers which added a
5' BamHI RES and a 3' XbaI RES. The PCR product was digested with BamHI and
1 o XbaI then purified using Qiagen's PCR purification kit. The cut product
was ligated
into pZBV32L, heat shocked into pZBV32L and plated on an Amp resistant plate.
Five
colonies were selected and mini-preps were done. Colonies were screened via
restriction enzyme digestion. Two of the colonies were transformed into
DHlOBac
cells and also submitted for sequencing. The protein sequence was found to be
correct
for both clones and one was selected. Recombinant Bacmid was isolated from the
DHlOBac cells and transfected into Sf~ cells. Virus was produced from the
initial
transfection and was amplified using standard methods. An infection was done
and
protein was detected via western blot in the conditioned media. Work on
protein is
currently on hold.
From the foregoing, it will be appreciated that, although specific embodiments
of the invention have been described herein for purposes of illustration,
various
modifications may be made without deviating from the spirit and scope of the
invention. Accordingly, the invention is not limited except as by the appended
claims.
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1
SEQUENCE LISTING
<110> ZymoGenetics, Inc.
<120> Secreted Alpha Helical Protein - 32
<130> 99-40
<160> 34
<170> FastSEQ for Windows Version 3.0
<210>1
<211>731
<212>DNA
<213>Homo sapiens
<220>
<221> CDS
<222> (24)...(533)
<400> 1
gcgggttgga gcctggcgta gtc atg gcc gcc ttc cgc gac ata gag gag gtg 53
Met Ala Ala Phe Arg Asp Ile Glu Glu Val
1 5 10
agc cag ggg ctg ctc agc ctg ctg ggc gcc aac cgc gcg gag gcg cag 101
Ser Gln Gly Leu Leu Ser Leu Leu Gly Ala Asn Arg Ala Glu Ala Gln
15 20 25
cag cga cgg ctg ctg ggg cgc cac gag cag gtg gtg gag cgg ctg ctg 149
Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val Val Glu Arg Leu Leu
30 35 40
gaa acg caa gac ggt gcc gag aag cag ctg cga gag atc ctc acc atg 197
Glu Thr Gln Asp Gly Ala Glu Lys Gln Leu Arg Glu Ile Leu Thr Met
45 50 55
gag aag gaa gtg gcc cag agc ctt ctc aat gcg aag gag cag gtg cac 245
Glu Lys Glu Val Ala Gln Ser Leu Leu Asn Ala Lys Glu Gln Val His
60 65 70
cag gga ggc gtg gag ctg cag cag ctg gaa get ggg ctt cag gag get 293
Gln Gly Gly Val Glu Leu Gln Gln Leu Glu Ala Gly Leu Gln Glu Ala
75 80 85 90
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
2
ggg gag gag gac acc cgt ctg aag gcc agc ctc ctt cag ctc acc aga 341
Gly Glu Glu Asp Thr Arg Leu Lys Ala Ser Leu Leu Gln Leu Thr Arg
95 100 105
gag ctg gaa gag ctc aag gag att gag gcg gat ctg gag cga cag gag 389
Glu Leu Glu Glu Leu Lys Glu Ile Glu Ala Asp Leu Glu Arg Gln Glu
110 115 120
aag gag gtc gac gag gac acg aca gtc aca atc ccc tcg gcc gtg tac 437
Lys Glu Val Asp Glu Asp Thr Thr Val Thr Ile Pro Ser Ala Val Tyr
125 130 135
gtg get caa ctt tac cac caa gtt agt aaa att gag tgg gat tat gag 485
Val Ala Gln Leu Tyr His Gln Val Ser Lys Ile Glu Trp Asp Tyr Glu
140 145 150
tgt gag cca ggg atg gtc aaa ggc agt atc ctt ttt ggg gag cca ttt 533
Cys Glu Pro Gly Met Val Lys Gly Ser Ile Leu Phe Gly Glu Pro Phe
155 160 165 170
taacccttgt gcactgtagg tagggacata aaatggtgca tagcaggacc ctgtaaaaat 593
tagccgggtg tggtggcgtg catctgttgt cccagctacc tgggaggctg aggtgggagg 653
atcacttgag gccaggagtt tgagaccagc ctgggtatca gtgagacccc acgtctataa 713
taaatatagt aaagtata 731
<210>2
<211>170
<212>PRT
<213>Homo Sapiens
<400> 2
Met Ala Ala Phe Arg Asp Ile Glu Glu Val Ser Gln Gly Leu Leu Ser
1 5 10 15
Leu Leu Gly Ala Asn Arg Ala Glu Ala Gln Gln Arg Arg Leu Leu Gly
20 25 30
Arg His Glu Gln Ual Val Glu Arg Leu Leu Glu Thr Gln Asp Gly Ala
35 40 45
Glu Lys Gln Leu Arg Glu Ile Leu Thr Met Glu Lys Glu Val Ala Gln
50 55 60
Ser Leu Leu Asn Ala Lys Glu Gln Val His Gln Gly Gly Val Glu Leu
65 70 75 80
Gln Gln Leu Glu Ala Gly Leu Gln Glu Ala Gly Glu Glu Asp Thr Arg
85 90 95
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
3
Leu Lys Ala Ser Leu Leu Gln Leu Thr Arg Glu Leu Glu Glu Leu Lys
100 105 110
Glu Ile Glu Ala Asp Leu Glu Arg Gln Glu Lys Glu Val Asp Glu Asp
115 120 125
Thr Thr Val Thr Ile Pro Ser Ala Val Tyr Val Ala Gln Leu Tyr His
130 135 140
Gln Val Ser Lys Ile Glu Trp Asp Tyr Glu Cys Glu Pro Gly Met Val
145 150 155 160
Lys Gly Ser Ile Leu Phe Gly Glu Pro Phe
165 170
<210>3
<211>145
<212>PRT
<213>Homo sapiens
<400> 3
Gln Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val Val Glu Arg Leu
1 5 10 15
Leu Glu Thr Gln Asp Gly Ala Glu Lys Gln Leu Arg Glu Ile Leu Thr
20 25 30
Met Glu Lys Glu Val Ala Gln Ser Leu Leu Asn Ala Lys Glu Gln Val
35 40 45
His Gln Gly Gly Val Glu Leu Gln Gln Leu Glu Ala Gly Leu Gln Glu
50 55 60
Ala Gly Glu Glu Asp Thr Arg Leu Lys Ala Ser Leu Leu Gln Leu Thr
65 70 75 80
Arg Glu Leu Glu Glu Leu Lys Glu Ile Glu Ala Asp Leu Glu Arg Gln
85 90 95
Glu Lys Glu Val Asp Glu Asp Thr Thr Val Thr Ile Pro Ser Ala Val
100 105 110
Tyr Val Ala Gln Leu Tyr His Gln Val Ser Lys Ile Glu Trp Asp Tyr
115 120 125
Glu Cys Glu Pro Gly Met Val Lys Gly Ser Ile Leu Phe Gly Glu Pro
130 135 140
Phe
145
<210>4
<211>15
<212>PRT
<213>Homo sapiens
<400> 4
Gln Arg Arg Leu Leu Gly Arg His Glu Gln Ual Val Glu Arg Leu
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
4
1 5 10 15
<210> 5
<211> 15
<212> PRT
<213> Homo Sapiens
<400> 5
Leu Gln Gln Leu Glu Ala Gly Leu Gln Glu Ala Gly Glu Glu Asp
1 5 10 15
<210>6
<211>15
<212>PRT
<213>Homo sapiens
<400> 6
Leu Lys Ala Ser Leu Leu Gln Leu Thr Arg Glu Leu Glu Glu Leu
1 5 10 15
<210>7
<211>15
<212>PRT
<213>Homo Sapiens
<400> 7
Val Ala Gln Leu Tyr His Gln Val Ser Lys Ile Glu Trp Asp Tyr
1 5 10 15
<210>8
<211>13
<212>PRT
<213>Homo Sapiens
<400> 8
Ala Gln Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val
1 5 10
<210>9
<211>16
<212>PRT
<213>Homo sapiens
<400> 9
Glu Arg Leu Leu Glu Thr Gln Asp Gly Ala Glu Lys Gln Leu Arg Glu
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
1 5 10 15
<210> 10
<211> 26
<212> PRT
<213> Homo sapiens
<400> 10
Thr Arg Glu Leu Glu Glu Leu Lys Glu Ile Glu Ala Asp Leu Glu Arg
1 5 10 15
Gln Glu Lys Glu Val Asp Glu Asp Thr Thr
20 25
<210>11
<211>31
<212>PRT
<213>Homo Sapiens
<400> 11
Thr Arg Glu Leu Glu Glu Leu Lys Glu Ile Glu Ala Asp Leu Glu Arg
1 5 10 15
Gln Glu Lys Glu Val Asp Glu Asp Thr Thr Val Thr Ile Pro Ser
20 25 30
<210>12
<211>15
<212>PRT
<213>Homo sapiens
<400> 12
Ser Lys Ile Glu Trp Asp Tyr Glu Cys Glu Pro Gly Met Val Lys
1 5 10 15
<210>13
<211>592
<212>DNA
<213>Homo sapiens
<400> 13
gcacgagggc gggttggagc ctggcgtagt catggccgcc ttccgcgaca tagaggaggt 60
gagccagggg ctgctcagcc tgctgggcgc caaccgcgcg gaggcgcagc agcgacggct 120
gctggggcgc cacgagcagg tggtggagcg gctgctggaa acgcaagacg gtgccgagaa 180
gcagctgcga gagatcctca ccatggagaa ggaagtggcc cagagccttc tcaatgcgaa 240
ggagcaggtg caccagggag gcgtggagct gcagcagctg gaagctgggc ttcaggaggc 300
tggggaggag gacacccgtc tgaaggccag cctccttcag ctcaccagag agctggaaga 360
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
6
gctcaaggag attgaggcgg atctggagcg acaggagaag gaggtcgacg aggacacgac 420
agtcacaatc ccctcggccg tgtacgtggc tcaactatac caccaagtta gtaaaattga 480
gtgggattat gagtgtgagc cagggatggt caaaggcagt atcctttttg gggagccatt 540
ttaacccttg tgcactgtag gtagggacat aaaatggtgc atagcaggac cc 592
<210>14
<211>777
<212>DNA
<213>Mus musculus
<220>
<221> CDS
<222> (19)...(615)
<400> 14
gaattcggca cgagggtc atg gcg get ttc cgc gac atg gtg gag gtg agc 51
Met Ala Ala Phe Arg Asp Met Val Glu Val Ser
1 5 10
aac tgg cta ctg agc ctg ctg ggg gcc aac cgc gcc gag gcg cag cag 99
Asn Trp Leu Leu Ser Leu Leu Gly Ala Asn Arg Ala Glu Ala Gln Gln
15 20 25
cgg cgg ctg ctc ggg agc tac gag cag atg atg gag cgg ctg ctg gag 147
Arg Arg Leu Leu Gly Ser Tyr Glu Gln Met Met Glu Arg Leu Leu Glu
30 35 40
atg cag gac ggc gcc tac cgg cag ctt cgg gag act ctg get gtg gag 195
Met Gln Asp Gly Ala Tyr Arg Gln Leu Arg Glu Thr Leu Ala Val Glu
45 50 55
gag gaa gtg get cag agc ctt ctt gaa ctg aaa gaa tgt acg cgc cag 243
Glu Glu Val Ala Gln Ser Leu Leu Glu Leu Lys Glu Cys Thr Arg Gln
60 65 70 75
ggg gac acc gag ctg cag cag ctg gag gtg gag ctc cag agg acc agc 291
Gly Asp Thr Glu Leu Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser
80 85 90
aag gag gac acc tgt gtg cag get agg cta cgt cag ctc atc aca gag 339
Lys Glu Asp Thr Cys Val Gln Ala Arg Leu Arg Gln Leu Ile Thr Glu
95 100 105
ctg cag gag ctc agg gag atg gag gaa gag ctc cag cgc cag gag agg 387
Leu Gln Glu Leu Arg Glu Met Glu Glu Glu Leu Gln Arg Gln Glu Arg
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
7
110 115 120
gat gta gat gag gac aac acc gtc acc atc ccc tct gca gtg tat gtg 435
Asp Val Asp Glu Asp Asn Thr Val Thr Ile Pro Ser Ala Val Tyr Val
125 130 135
get cat ctc tat cac caa att agt aaa ata cag tgg gat tat gaa tgc 483
Ala His Leu Tyr His Gln Ile Ser Lys Ile Gln Trp Asp Tyr Glu Cys
140 145 150 155
gag cca ggg atg atc aag ggc aga gga ccg aaa aca ctt tcc ttt cat 531
Glu Pro Gly Met Ile Lys Gly Arg Gly Pro Lys Thr Leu Ser Phe His
160 165 170
ctc gtc ctc agt cca cca cgg ccc cac agt ggc cca gcc cat cca ctt 579
Leu Val Leu Ser Pro Pro Arg Pro His Ser Gly Pro Ala His Pro Leu
175 180 185
gga cag tgc aca get ctc gcc gaa gtt cat cag tga ctacctctgg 625
Gly Gln Cys Thr Ala Leu Ala Glu Val His Gln
190 195
agcctggtgg acaccacgtg ggagccagag ccttgacctc ataccttgca cagaactggg 685
gttgagggag ccaaggaggg gatcactcta aaattaaatg tcgtgtatgt gaaaaaaaaa 745
aaaaaaaaaa aaaaaaattt ccgcggccgc as 777
<210>15
<211>198
<212>PRT
<213>Mus musculus
<400> 15
MetAla AlaPhe ArgAspMet ValGluVal SerAsn TrpLeu LeuSer
1 5 10 15
LeuLeu GlyAla AsnArgAla GluAlaGln GlnArg ArgLeu LeuGly
20 25 30
SerTyr GluGln MetMetGlu ArgLeuLeu GluMet GlnAsp GlyAla
35 40 45
TyrArg GlnLeu ArgGluThr LeuAlaVal GluGlu GluVal AlaGln
50 55 60
SerLeu LeuGlu LeuLysGlu CysThrArg GlnGly AspThr GluLeu
65 70 75 80
Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser Lys Glu Asp Thr Cys
85 90 95
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
8
Val Gln Ala Arg Leu Arg Gln Leu Ile Thr Glu Leu Gln Glu Leu Arg
100 105 110
Glu Met Glu Glu Glu Leu Gln Arg Gln Glu Arg Asp Val Asp Glu Asp
115 120 125
Asn Thr Val Thr Ile Pro Ser Ala Val Tyr Val Ala His Leu Tyr His
130 135 140
Gln Ile Ser Lys Ile Gln Trp Asp Tyr Glu Cys Glu Pro Gly Met Ile
145 150 155 160
Lys Gly Arg Gly Pro Lys Thr Leu Ser Phe His Leu Val Leu Ser Pro
165 170 175
Pro Arg Pro His Ser Gly Pro Ala His Pro Leu Gly Gln Cys Thr Ala
180 185 190
Leu Ala Glu Val His Gln
195
<210>16
<211>173
<212>PRT
<213>Mus musculus
<400> 16
GlnGlnArg ArgLeu LeuGlySer TyrGlu GlnMetMet GluArg Leu
1 5 10 15
LeuGluMet GlnAsp GlyAlaTyr ArgGln LeuArgGlu ThrLeu Ala
20 25 30
ValGluGlu GluVal AlaGlnSer LeuLeu GluLeuLys GluCys Thr
35 40 45
ArgGlnGly AspThr GluLeuGln GlnLeu GluValGlu LeuGln Arg
50 55 60
ThrSerLys GluAsp ThrCysVal GlnAla ArgLeuArg GlnLeu Ile
65 70 75 80
ThrGluLeu GlnGlu LeuArgGlu MetGlu GluGluLeu GlnArg Gln
85 90 95
GluArgAsp ValAsp GluAspAsn ThrVal ThrIlePro SerAla Val
100 105 110
TyrValAla HisLeu TyrHisGln IleSer LysIleGln TrpAsp Tyr
115 120 125
GluCysGlu ProGly MetIleLys GlyArg GlyProLys ThrLeu Ser
130 135 140
PheHisLeu ValLeu SerProPro ArgPro HisSerGly ProAla His
145 150 155 160
ProLeuGly GlnCys ThrAlaLeu AlaGlu ValHisGln
165 170
<210> 17
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
9
<211> 1445
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (19)...(624)
<400> 17
gaattcggca cgagggtc atg gcg get ttc cgc gac atg gtg gag gtg agc 51
Met Ala Ala Phe Arg Asp Met Val Glu Val Ser
1 5 10
aac tgg cta ctg agc ctg ctg ggg gcc aac cgc gcc gag gcg cag cag 99
Asn Trp Leu Leu Ser Leu Leu Gly Ala Asn Arg Ala Glu Ala Gln Gln
15 20 25
cgg cgg ctg ctc ggg agc tac gag cag atg atg gag cgg ctg ctg gag 147
Arg Arg Leu Leu Gly Ser Tyr Glu Gln Met Met Glu Arg Leu Leu Glu
30 35 40
atg cag gac ggc gcc tac cgg cag ctt cgg gag act ctg get gtg gag 195
Met Gln Asp Gly Ala Tyr Arg Gln Leu Arg Glu Thr Leu Ala Val Glu
45 50 55
gag gaa gtg get cag agc ctt ctt gaa ctg aaa gaa tgt acg cgc cag 243
Glu Glu Val Ala Gln Ser Leu Leu Glu Leu Lys Glu Cys Thr Arg Gln
60 65 70 75
ggg gac acc gag ctg cag cag ctg gag gtg gag ctc cag agg acc agc 291
Gly Asp Thr Glu Leu Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser
80 85 90
aag gag gac acc tgt gtg cag get agg cta cgt cag ctc atc aca gag 339
Lys Glu Asp Thr Cys Val Gln Ala Arg Leu Arg Gln Leu Ile Thr Glu
95 100 105
ctg cag gag ctc agg gag atg gag gaa gag ctc cag cgc cag gag agg 387
Leu Gln Glu Leu Arg Glu Met Glu Glu Glu Leu Gln Arg Gln Glu Arg
110 115 120
gat gta gat gag gac aac acc gtc acc atc ccc tct gca gtg tat gtg 435
Asp Val Asp Glu Asp Asn Thr Val Thr Ile Pro Ser Ala Val Tyr Val
125 130 135
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
get cat ctc tat cac caa att agt aaa ata cag tgg gat tat gaa tgc 483
Ala His Leu Tyr His Gln Ile Ser Lys Ile Gln Trp Asp Tyr Glu Cys
140 145 150 155
gag cca ggg atg atc aag ggc atc cac cac ggc ccc aca gtg gcc cag 531
Glu Pro Gly Met Ile Lys Gly Ile His His Gly Pro Thr Val Ala Gln
160 165 170
ccc atc cac ttg gac agt gca cag ctc tcg ccg aag ttc atc agt gac 579
Pro Ile His Leu Asp Ser Ala Gln Leu Ser Pro Lys Phe Ile Ser Asp
175 180 185
tac ctc tgg agc ctg gtg gac acc acg tgg gag cca gag cct tga 624
Tyr Leu Trp Ser Leu Val Asp Thr Thr Trp Glu Pro Glu Pro
190 195 200
cctcataccttgcacagaactggggttgagggagccaaggaggggatcactctaaaatta684
aatgtctgtatgtgagtgcgttcattgatttatctacttgctttgagacagcatggagtc744
caggctggcctgcagcttcttttttatttgtaattacatttactgtatgaatgttttgtc804
tgcatgtgtgtctgttagctgtgtattccaggagaggttagagagggcttcagaccccct864
gaaactggagttatgggtggttctgagctgccatgtggctactgggaatcgaacctgtat924
tctatagaagagcagccagtgctcttaattgttgagctgtctctccatccccttaattac984
aattttaaaaaatgtgtgcctagccgggcgtggtggcgcacgcctttaatcccagcactt1044
gggaggcagaggcaggcggatttctgagttcgaggccagcctggtctacagagtgagttc1104
caggacagccagggctatacagagaaaccctgtcttgaaaaaacaaaaaaaaaaaaaaaa1164
caaacaaacaaaaaaacaaaaacaaaaatgtgtgcagttggggctggagagatggctcag1224
tggttaagagcacactgattgctcttccagaggttctgggttcaattcccatctgtaatg1284
ggatccgatgccctcttctggtgtgtctgaagacagccacagtgtactcacatacattaa1344
ataaatactcttttttaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa1404
aaaaaaaaaaaaaaaaaaaaaaaaaaaatttccgcggccgc 1445
<210>18
<211>201
<212>PRT
<213>Mus musculus
<400> 18
Met Ala Ala Phe Arg Asp Met Val Glu Val Ser Asn Trp Leu Leu Ser
1 5 10 15
Leu Leu Gly Ala Asn Arg Ala Glu Ala Gln Gln Arg Arg Leu Leu Gly
25 30
Ser Tyr Glu Gln Met Met Glu Arg Leu Leu Glu Met Gln Asp Gly Ala
35 40 45
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
11
Tyr Arg Gln Leu Arg Glu Thr Leu Ala Val Glu Glu Glu Val Ala Gln
50 55 60
Ser Leu Leu Glu Leu Lys Glu Cys Thr Arg Gln Gly Asp Thr Glu Leu
65 70 75 80
Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser Lys Glu Asp Thr Cys
85 90 95
Val Gln Ala Arg Leu Arg Gln Leu Ile Thr Glu Leu Gln Glu Leu Arg
100 105 110
Glu Met Glu Glu Glu Leu Gln Arg Gln Glu Arg Asp Val Asp Glu Asp
115 120 125
Asn Thr Val Thr Ile Pro Ser Ala Val Tyr Val Ala His Leu Tyr His
130 135 140
Gln Ile Ser Lys Ile Gln Trp Asp Tyr Glu Cys Glu Pro Gly Met Ile
145 150 155 160
Lys Gly Ile His His Gly Pro Thr Val Ala Gln Pro Ile His Leu Asp
165 170 175
Ser Ala Gln Leu Ser Pro Lys Phe Ile Ser Asp Tyr Leu Trp Ser Leu
180 185 190
Val Asp Thr Thr Trp Glu Pro Glu Pro
195 200
<210>19
<211>176
<212>PRT
<213>Mus musculus
<400> 19
Gln GlnArgArg LeuLeuGly SerTyrGlu GlnMet MetGluArg Leu
1 5 10 15
Leu GluMetGln AspGlyAla TyrArgGln LeuArg GluThrLeu Ala
20 25 30
Val GluGluGlu ValAlaGln SerLeuLeu GluLeu LysGluCys Thr
35 40 45
Arg GlnGlyAsp ThrGluLeu GlnGlnLeu GluVal GluLeuGln Arg
50 55 60
Thr SerLysGlu AspThrCys ValGlnAla ArgLeu ArgGlnLeu Ile
65 70 75 80
Thr GluLeuGln GluLeuArg GluMetGlu GluGlu LeuGlnArg Gln
85 90 95
Glu ArgAspVal AspGluAsp AsnThrVal ThrIle ProSerAla Val
100 105 110
Tyr ValAlaHis LeuTyrHis GlnIleSer LysIle GlnTrpAsp Tyr
115 120 125
Glu CysGluPro GlyMetIle LysGlyIle HisHis GlyProThr Val
130 135 140
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
12
Ala Gln Pro Ile His Leu Asp Ser Ala Gln Leu Ser Pro Lys Phe Ile
145 150 155 160
Ser Asp Tyr Leu Trp Ser Leu Val Asp Thr Thr Trp Glu Pro Glu Pro
165 170 175
<210>20
<211>352
<212>DNA
<213>Mus musculus
<400>
20
gtcatggcggctttcccggacatggtggaggtgagcaactggctactgagcctgctgggg 60
gccaaccgcgccgagcgagcagcgcggcatgctcagggagctacgagcagatgatggagc 120
ggctgctggagatgcaggacggcgcctaccaggcagcttcgggagactctggctgtggag 180
gaggaagtggctcagagccttcttgaactgaaagaatgtacgcgccagggggacaccgag 240
ctgcagcagctggaggtggagctccagaggaccagcaaggaggacacctgtgtgcaggct 300
aggctacgtcagctcatcacagagctgcaggagctcagggagatggaggaag 352
<210>21
<211>455
<212>DNA
<213>Mus musculus
<400> 21
tggtggaggtgagcaactggctactgagcctgctgggggccaaccgcgccgaggcggcag 60
cggggctgctcgggagctacgagcagatgatggagcggctgctggagatgcaggacggcg 120
cctaccggcagcttcgggagactctggctgtggaggaggaagtggctcagagccttcttg 180
aactgaaagaatgtacgcgccagggggacaccgagctgcagcagctggaggtggagctcc 240
agaggaccagcaaggaggacacctgtgtgcaggctaggctacgtcagctcatcacagagc 300
tgcaggagctcagggagatggaggaagagctccagcgccaggagagggatgtagatgagg 360
acaacaccgtcaccatcccctctgcagtgtatgtggctcatctctatcaccaaattagta 420
aaatacagtgggattatgaatgcgagccagggatg 455
<210>22
<211>15
<212>PRT
<213>Mus musculus
<400> 22
Gln Arg Arg Leu Leu Gly Ser Tyr Glu Gln Met Met Glu Arg Leu
1 5 10 15
<210> 23
<211> 15
<212> PRT
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
13
<213> Mus musculus
<400> 23
Leu Gln Gln Leu Glu Val Glu Leu Gln Arg Thr Ser Lys Glu Asp
1 5 10 15
<210>24
<211>15
<212>PRT
<213>Mus musculus
<400> 24
Val Gln Ala Arg Leu Arg Gln Leu Ile Thr Glu Leu Gln Glu Leu
1 5 10 15
<210>25
<211>15
<212>PRT
<213>Mus musculus
<400> 25
Val Ala His Leu Tyr His Gln Ile Ser Lys Ile Gln Trp Asp Tyr
1 5 10 15
<210>26
<211>68
<212>PRT
<213>Homo Sapiens
<400> 26
GlnArg ArgLeu LeuGlyArg HisGluGln ValVal GluArgLeu Leu
1 5 10 15
GluThr GlnAsp GlyAlaGlu LysGlnLeu ArgGlu IleLeuThr Met
20 25 30
GluLys GluVal AlaGlnSer LeuLeuAsn AlaLys GluGlnVal His
35 40 45
GlnGly GlyVal GluLeuGln GlnLeuGlu AlaGly LeuGlnGlu Ala
50 55 60
GlyGlu GluAsp
65
<210>27
<211>85
<212>PRT
<213>Homo sapiens
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
14
<400> 27
GlnArg ArgLeuLeu GlyArg HisGluGln ValValGlu ArgLeuLeu
1 5 10 15
GluThr GlnAspGly AlaGlu LysGlnLeu ArgGluIle LeuThrMet
20 25 30
GluLys GluValAla GlnSer LeuLeuAsn AlaLysGlu GlnValHis
35 40 45
GlnGly GlyValGlu LeuGln GlnLeuGlu AlaGlyLeu GlnGluAla
50 55 60
GlyGlu GluAspThr ArgLeu LysAlaSer LeuLeuGln LeuThrArg
65 70 75 80
GluLeu GluGluLeu
85
<210>28
<211>127
<212>PRT
<213>Homo Sapiens
<400> 28
Gln Arg Arg Leu Leu Gly Arg His Glu Gln Val Val Glu Arg Leu Leu
1 5 10 15
Glu Thr Gln Asp Gly Ala Glu Lys Gln Leu Arg Glu Ile Leu Thr Met
20 25 30
Glu Lys Glu Val Ala Gln Ser Leu Leu Asn Ala Lys Glu Gln Val His
35 40 45
Gln Gly Gly Ual Glu Leu Gln Gln Leu Glu Ala Gly Leu Gln Glu Ala
50 55 60
Gly Glu Glu Asp Thr Arg Leu Lys Ala Ser Leu Leu Gln Leu Thr Arg
65 70 75 80
Glu Leu Glu Glu Leu Lys Glu Ile Glu Ala Asp Leu Glu Arg Gln Glu
85 90 95
Lys Glu Val Asp Glu Asp Thr Thr Val Thr Ile Pro Ser Ala Val Tyr
100 105 110
Val Ala Gln Leu Tyr His Gln Val Ser Lys Ile Glu Trp Asp Tyr
115 120 125
<210>29
<211>32
<212>PRT
<213>Homo sapiens
<400> 29
Leu Gln Gln Leu Glu Ala Gly Leu Gln Glu Ala Gly Glu Glu Asp Thr
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
1 5 10 15
Arg Leu Lys Ala Ser Leu Leu Gln Leu Thr Arg Glu Leu Glu Glu Leu
25 30
<210>30
<211>74
<212>PRT
<213>Homo sapiens
<400> 30
LeuGlnGlnLeu GluAla GlyLeuGln GluAlaGly GluGluAsp Thr
1 5 10 15
ArgLeuLysAla SerLeu LeuGlnLeu ThrArgGlu LeuGluGlu Leu
20 25 30
LysGluIleGlu AlaAsp LeuGluArg GlnGluLys GluValAsp Glu
35 40 45
AspThrThrVal ThrIle ProSerAla ValTyrVal AlaGlnLeu Tyr
50 55 60
HisGlnValSer LysIle GluTrpAsp Tyr
65 70
<210>31
<211>57
<212>PRT
<213>Homo Sapiens
<400> 31
LeuLysAla SerLeuLeu GlnLeuThr ArgGlu Leu Glu Leu
Glu Lys
1 5 10 15
GluIleGlu AlaAspLeu GluArgGln GluLys Glu Asp Glu
Ual Asp
20 25 30
ThrThrVal ThrIlePro SerAlaVal TyrUal Ala Leu Tyr
Gln His
35 40 45
GlnValSer LysIleGlu TrpAspTyr
50 55
<210>32
<211>53
<212>PRT
<213>Homo Sapiens
<400> 32
Thr Gln Asp Gly Ala Glu Lys Gln Leu Arg Glu Ile Leu Thr Met Glu
1 5 10 15
CA 02374520 2001-11-23
WO 00/71717 PCT/US00/14563
16
Lys Glu Val Ala Gln Ser Leu Leu Asn Ala Lys Glu Gln Val His Gln
20 25 30
Gly Gly Ual Glu Leu Gln Gln Leu Glu Ala Gly Leu Gln Glu Ala Gly
35 40 45
Glu Glu Asp Thr Arg
<210>33
<211>42
<212>PRT
<213>Homo sapiens
<400> 33
GluAla Glu Glu Thr LeuLys Ala Ser Leu Leu Gln
Gly Asp Arg Leu
1 5 10 15
ThrArg Leu Glu Leu GluIle Glu Ala Asp Leu Glu
Glu Glu Lys Arg
20 25 30
GlnGlu Glu Val Glu ThrThr
Lys Asp Asp
35 40
<210>34
<211>47
<212>PRT
<213>Homo sapiens
<400> 34
GluAla LeuGlu Arg Gln LysGlu Val Asp AspThr
Asp Glu Glu Thr
1 5 10 15
ValThr ProSer Ala Val ValAla Gln Leu HisGln
Ile Tyr Tyr Val
20 25 30
SerLys GluTrp Asp Tyr CysGlu Pro Gly ValLys
Ile Glu Met
35 40 45
