Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED ALPHA-HELICAL PROTEIN - 31
BACKGROUND OF THE INVENTION
Hormones and polypeptide growth factors control proliferation,
maintenance, survival and differentiation of cells of multicellular organisms.
These
diffusable molecules allow cells to communicate with each other and act in
concert to
form cells and organs, and to repair and regenerate damaged tissue. Examples
of
hormones and ~.5x,:>wth 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 granulocyte-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.
The present invention addresses this need by providing novel polypeptides and
related
compositions and methods. These and other aspects of the invention will become
3 o evident upon reference to the following detailed description of the
invention.
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2
DETAILED DESCRIPTION OF THE INVENTION
The teachings of all the references cited herein are incorporated in their
entirety herein by reference.
Prior to setting forth the invention in detail, it may be helpful to the
understanding thereof to define the following terms:
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
l0 binding agent is available can be used as an affinity tag. Affinity tags
include a poly-
histidine tract, protein A, Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et
al.,
Methods Enzymol. 198:3 ( 1991 ), glutathione S transferase, Smith and Johnson,
Gene
67:31 (1988), Glu-Glu affinity tag, Grussenmeyer et al., Proc. Natl. Acad.
Sci. USA
82:7952-4 (1985), substance P, FIagTM peptide, Hopp et al., Biotechnology
6:1204-1210
(1988), streptavidin binding peptide, or other antigenic epitope or binding
domain. See,
in general, Ford et al., Protein Expression and Purification 2: 95-107 (1991).
DNAs
encoding affinity tags are available from commercial suppliers (e.g.,
Pharmacia
Biotech, Piscataway, NJ).
The term "allelic variant" is used herein to dery,ote any of two or more
2 0 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.
2 5 The terms "amino-terminal" and "carboxyl-terminal" are used herein to
denote positions within polypeptides. Where the context allows, these terms
are used
with reference to a particular sequence or portion of a polypeptide to denote
proximity
or relative position. For example, a certain sequence positioned carboxyl-
terminal to a
reference sequence within a polypeptide is located proximal to the carboxyl
terminus of
3 0 the reference sequence, but is not necessarily at the carboxyl terminus of
the complete
polypeptide.
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"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
migration, capillary sprout formation, and endothelial cell proliferation.
Tre term "complement/anti-complement pair" denotes non-identical
moieties that form a non-covalently associated, stable pair under appropriate
conditions.
For instance, biotin and avidin (or streptavidin) are prototypical members of
a
complement/anti-complement pair. Other exemplary complement/anti-complement
pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope)
pairs,
sense/antisense polynucleotide pairs, and the like. Where subsequent
dissociation of
the complement/anti-complement pair is desirable, the complement/anti-
complement
pair preferably has a binding affinity of <109 M-1.
The term "complements of a polynucleotide molecule" is a
polynucleotide molecule having a complementary base sequence and reverse
orientation as compared to a reference sequence. For example, the sequence 5'
ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
The term "contig" denotes a polynucleotide that has a contiguous stretch
of identical or complementary sequence to another polynucleotide. Contiguous
2 0 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'-ATGGAGCTT-3' are 5'-
AGCTTgagt-3' and 3'-tcgacTACC-5'.
The term "degenerate nucleotide sequence" denotes a sequence of
2 5 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
3 0 or circular, that comprises a segment encoding a polypeptide of interest
operably linked
to additional segments that provide for its transcription. Such additional
segments
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include promoter and terminator sequences, and may also include one or more
origins
of replication, one or more selectable markers, an enhancer, a polyadenylation
signal,
etc. Expression vectors are generally derived from plasmid or viral DNA, or
may
contain elements of both.
The term "isolated", when applied to a polynucleotide, denotes that the
polynucleotide has been removed from its natural genetic milieu and is thus
free of
other extraneous or unwanted coding sequences, and is in a form suitable for
use within
genetically engineered protein production systems. Such isolated molecules are
those
that are separated from their natural environment and include cDNA and genomic
1 o clones. Isolated DNA molecules of the present invention are free of other
genes with
which they are ordinarily associated, but may include naturally occurring 5'
and 3'
untranslated regions such as promoters and terminators. The identification of
associated regions will be evident to one of ordinary skill in the art (see
for example,
Dynan and Tijan, Nature 316:774-78 (1985).
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
2 0 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.
Tre term "operably linked", when referring to DNA segments, indicates
that the segments are arranged so that they function in concert for their
intended
purposes, e.g., transcription initiates in the promoter and proceeds through
the coding
segment to the terminator.
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.
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"Paralogs" are distinct but structurally related proteins made by an
organism. Paralogs are believed to arise through gene duplication. For
example, a-
globin, (3-globin, and myoglobin are paralogs of each other.
A "polynucleotide" is a single- or double-stranded polymer of
5 deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
Polynucleotides include RNA and DNA, and may be isolated from natural sources,
synthesized in vitro, or prepared from a combination of natural and synthetic
molecules.
Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides
("nt"), or kilobases ("kb"). Where the context allows, the latter two terms
may describe
polynucleotides that are single-stranded or double-stranded. When the term is
applied
to double-strandfv:l molecules it is used to denote overall length and will be
understood
to be equivalent to the term "base pairs". It will be recognized by those
skilled in the
art that the two strands of a double-stranded polynucleotide may differ
slightly in length
and that the ends thereof may be staggered as a result of enzymatic cleavage;
thus all
nucleotides within a double-stranded polynucleotide molecule may not be
paired.
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
2 0 denote a portion of a gene containing DNA sequences that provide for the
binding of
RNA polymerise and initiation of transcription. Promoter sequences are
commonly,
but not always, found in the 5' non-coding regions of genes.
A "protein" is a macromolecule comprising one or more polypeptide
chains. A protein may also comprise non-peptidic components, such as
carbohydrate
2 5 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.
3 0 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.
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Membrane-bound receptors are characterized by a mufti-domain structure
comprising
an extracellular ligand-binding domain and an intracellular effector domain
that is
typically involved in signal transduction. Binding of ligand to receptor
results in a
conformational change in the receptor that causes an interaction between the
effector
domain and other 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
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
receptor, erythropoietin receptor and IL-6 receptor).
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 dexvote alternative forms of
2 0 RNA transcribed from a gene. Splice variation arises naturally through use
of
alternative splicing sites within a transcribed RNA molecule, or less commonly
between separately transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode polypeptides having
altered amino acid sequence. The term splice variant is also used herein to
denote a
2 5 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 ~ 10%.
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The present invention provides novel cytokine polypeptides/proteins.
The novel cytokine, termed "alpha helical protein-31" hereinafter referred to
as
"Zalpha3l" was discovered and identified to be a cytokine by the presence of
polypeptide and polynucleotide features characteristic of four-helix-bundle
cytokines
(e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin and growth
hormone).
The sequence of the zalpha3l polypeptide was obtained from a single
clone believed to contain its corresponding polynucleotide sequence. Libraries
that
might be searched for such sequences include heart, brain, thyroid, liver,
spinal cord,
adrenal gland, testis, macrophages, lymphoid cells, activated immune cells,
and the
like.
The nucleotide sequence of a representative zalpha3l-encoding DNA is
described in SEQ ID NO:1, and its deduced 142 amino acid sequence is described
in
SEQ ID N0:2. In its entirety, the zalpha3l polypeptide (SEQ ID N0:2)
represents a
full-length polypeptide segment (residue 1 (Met) to residue 142 (Arg) of SEQ
ID
N0:2). The domains and structural features of zalpha3l are further described
below.
Analysis of the zalpha3l polypeptide encoded by the DNA sequence of
SEQ ID NO:1 revealed an open reading frame encoding 142 amino acids (SEQ ID
N0:2) comprising a predicted signal peptide of 19 amino acid residues (residue
1 (Met)
to residue 19 (Asp) of SEQ ID N0:2), and a mature polypeptide of 122 amino
acids
2 0 (residue 20 (Asp) to residue 142 (Arg) of SEQ ID N0:2).
In general, cytokines are predicted to have a four-alpha helix structure,
with helices A, C and D being most important in ligand-receptor interactions,
and are
more highly conserved among members of the family. Helices A-D in zalpha3l
define
a biologically active receptor-binding domain that comprises amino acids 37
(Ile) to
2 5 132 (Leu) of SEQ ID N0:2. The mature Zalpha31 polypeptide has an
unglycosylated
molecular weight of approximately 14,009 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:
1) "helix A" (corresponding to amino acids 37 (Ile) to 51 (Tyr) of SEQ
3 o ID N0:2);
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2) "helix B" (corresponding to amino acids 65 (Leu) to 79 (Glu) of SEQ
ID N0:2);
3) "helix C" (corresponding to amino acids 87 (Ile) to 101 (Leu) of SEQ
ID N0:2); and
4) "helix D" (corresponding to amino acids 118 (Leu) to 132 (Leu) of
SEQ ID N0:2).
Alternatively, Helix A can correspond to amino acids 26 (Ala) to 40
(Leu) of SEQ ID N0:2; and Helix B can correspond to amino acids 59 (Leu) to 73
(Thr) of SEQ ID N0:2. As such Helices A-D in zalpha3l can define an extended
biologically active receptor-binding domain that comprises amino acids 26
(Ala) to 132
(Leu) of SEQ ID N0:2.
Each helix contains an external region having amino acid residues which
are generally hydrophilic, and an internally located region which generally
contains
hydrophobic amino acid residues. The amino acid residues which 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 which are positioned on the interior of the helix may
be
changed to any hydrophobic amino acid residue.
In helix A, amino acid residues 37, 40, 41, 44, 47, 48 and 51 of SEQ ID
2 0 N0:2 are positioned towards the interior of helix A, while amino acid
residues 38, 39,
42, 43, 45, 46, 49 and 50 of SEQ ID N0:2 are positioned on the external
portion of
helix A.
In helix B, amino acid residues 65, 68, 69, 72, 75, 76, and 79 of SEQ ID
N0:2 are positioned towards the interior of helix A, while amino acid residues
66, 67,
70, 71, 73, 74, 77, and 78 of SEQ ID N0:2 are positioned on the external
portion of
helix B.
In helix C, amino acid residues 87, 90, 91, 94, 97, 98, and 101 of SEQ
ID N0:2 are positioned towards the interior of helix A, while amino acid
residues 88,
89, 92. 92, 95, 96, 99 and 100 of SEQ ID N0:2 are positioned on the external
portion of
3 0 helix C.
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In helix D, amino acid residues 118, 121, 122, 125, 128, 129, and 132 of
SEQ ID N0:2 are positioned towards the interior of helix A, while amino acid
residues
119, 120, 123, 1~.~T, 126, 127, 130, and 131 of SEQ ID N0:2 are positioned on
the
external portion of helix D.
Helices 1 through 4 are spaced apart from N-terminus to C-terminus in a
configuration represented by the following:
AspZO-{ 16}-H1-{ 13 }-H2-{7}-H3-{ 16}-H4-{9}- Arg,42,
where Aspzo is the starting residue of the mature polypeptide (as shown
in SEQ ID N0:2),
Arg,42 is the ending residue of the mature polypeptide(as shown in SEQ
ID N0:2),
H# denotes the specific helix disclosed above (e.g., H1 is Helix A, H2 is
Helix B etc.), and
{#} denotes the approximate number of amino acid residues between the
motifs, up to plus or minus 2 residues.
The corresponding polynucleotides encoding the zalpha3l polypeptide
regions, domains, motifs, residues and sequences described above are as shown
in SEQ
ID NO:1.
Four-helical bundle cytokines are also grouped by the length of their
2 0 component helices. "Long-helix" form cytokines generally consist of
between 24-30
residue helices, and include IL-6, ciliary neutrotrophic factor (CNTF),
leukemia
inhibitory factor (LIF) and human growth hormone (hGH). "Short-helix" form
cytokines generally consist of between 18-21 residue helices and include IL-2,
IL-4 and
GM-CSF. Zalpha3l is believed to be a new member of the short-helix form
cytokine
2 5 group. Studies using CNTF and IL-6 demonstrated that a CNTF helix can be
exchanged for the equivalent helix in IL-6, conferring CTNF-binding properties
to the
chimera. Thus, it appears that functional domains of four-helical cytokines
are
determined on the basis of structural homology, irrespective of sequence
identity, and
can maintain functional integrity in a chimera (Kallen et al., J. Biol. Chem.
274:11859-
3 0 11867, 1999). Therefore, the helical domains of zalpha31 will be useful
for preparing
chimeric fusion molecules, particularly with other short-helix form cytokines
to
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determine and modulate receptor binding specificity. Of particular interest
are fusion
proteins engineered with helix A and/or helix D, and fusion proteins that
combine
helical and loop domains from other short-form cytokines such as IL-2, IL-4,
IL-I S and
GM-CSF. The amino acid residues comprising helices A, B, C, and D, and loops
A/B,
5 B/C and C/D for zalpha3l, IL-2, IL-4, IL-15 and GM-CSF are shown in Table 1.
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Table 1
Helix A/B Helix B/C Helix C/D Helix
A Loop B Loop C Loop D
zalpha3l37-51 52-64 65-79 80-86 87-101 102- 118- SEQ
residues 117 132 ID
N0:2
IL-2 36-46 47-52 53-75 76-86 87-99 100- 103- SEQ
residues 102 121 ID
NO:
14
IL-4 29-43 44-64 65-83 84-94 95-118 119- 134- SEQ
residues 133 151 ID
NO:
15
IL-15 45-68 69-83 84-101 102- 107- 120- 134- SEQ
residues 106 119 133 160 ID
NO:
16
GM- 30-44 45-71 72-81 82-90 91-102 103- 120- SEQ
CSF 119 131 ID
residues NO:
17
POLYNUCLEOTIDES:
The present invention also provides polynucleotide molecules, including
DNA and RNA molecules, that encode the zalpha3l polypeptides disclosed herein.
Those skilled in the art will readily recognize that, in view of the
degeneracy of the
genetic code, considerable sequence variation is possible among these
polynucleotide
molecules. SEQ ID N0:3 is a degenerate DNA sequence that encompasses all DNAs
l0 that encode the zalpha3l polypeptide of SEQ ID N0:2. Those skilled in the
art will
recognize that the degenerate sequence of SEQ ID N0:3 also provides all RNA
sequences encoding SEQ ID N0:2 by substituting U for T. Thus, zalpha3l
polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 426
of
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SEQ ID N0:3 and their RNA equivalents are contemplated by the present
invention.
Table 2 sets forth the one-letter codes used within SEQ ID N0:3 to denote
degenerate
nucleotide positions. "Resolutions" are the nucleotides denoted by a code
letter.
"Complement" indicates the code for the complementary nucleotide(s). For
example,
the code Y denotes either C or T, and its complement R denotes A or G, A being
complementary to T, and G being complementary to C.
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TABLE 2
Nucleotide Resolution Complement Resolution
A A T T
C C G G
G G C C
T T A A
R A~G Y CST
Y CST R A~G
''~~I ABC K GET
K GET M ABC
S CMG S CMG
W ACT W ACT
H A~C~T D A~G~T
B C~G~T V A~C~G
V A~C~G B C~G~T
D A~G~T H A~C~T
N A~C~G~T N A~C~G~T
The degenerate codons used in SEQ ID N0:3, encompassing all possible
codons for a given amino acid, are set forth in Table 3.
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TABLE 3
One
Amino Letter Codons Degenerate
Acid Code Codon
Cys C TGC TGT TGY
Ser S AGC AGT TCA TCC TCG TCT WSN
Thr T ACA ACC ACG ACT ACN
Pro P CCA CCC CCG CCT CCN
Ala A GCA GCC GCG GCT GCN
Gly G GGA GGC GGG GGT GGN
Asn N AAC AAT AAY
Asp D GAC GAT GAY
Glu E GAA GAG GAR
Gln Q CAA CAG CAR
His H CAC CAT CAY
Arg R AGA AGG CGA CGC CGG CGT MGN
Lys K AAA AAG AAR
Met M ATG ATG
Ile I ATA ATC ATT ATH
Leu L CTA CTC CTG CTT TTA TTG YTN
Val V GTA GTC GTG GTT GTN
Phe F TTC TTT TTY
Tyr Y TAC TAT TAY
Trp W TGG TGG
Ter . TAA TAG TGA TRR
Asn~AspB RAY
Glu~GlnZ SAR
Any X NNN
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One of ordinary skill in the art will appreciate that some ambiguity is
introduced in determining a degenerate codon, representative of all possible
codons
encoding each amino acid. For example, the degenerate codon for serine (WSN)
can, in
some circumstances, encode arginine (AGR), and the degenerate codon for
arginine
5 (MGN) can, in some circumstances, encode serine (AGY). A similar
relationship exists
between codons encoding phenylalanine and leucine. Thus, some polynucleotides
encompassed by the degenerate sequence may encode variant amino acid
sequences,
but one of ordinary skill in the art can easily identify such variant
sequences by
reference to the amino acid sequence of SEQ ID N0:2. Variant sequences can be
10 readily tested for functionality as described herein.
One of ordinary skill in the art will also appreciate that different species
can exhibit "preferential codon usage." 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
al., Gene 13:355-364 (1981); Grosjean and Fiers, Gene 18:199-209 (1982); Holm,
Nuc.
15 Acids Res. 14:3075-3087 (1986); Ikemura, J. Mol. Biol. 158:573-597 (1982).
As used
herein, the term "preferential codon usage" or "preferential codons" is a term
of art
referring to protein translation codons that are most frequently used in cells
of a certain
species, thus favoring one or a few representatives of the possible codons
encoding
each amino acid (See Table 3). For example, the amino acid Threonine (Thr) may
be
2 o 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
2 5 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
3 0 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
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16
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
matched probe. Numerous equations for calculating T", are known in the art,
and are
specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences
of
varying length (see, for example, Sambrook et al., Molecular Cloning: A
Laboratory
Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al.,
(eds.),
Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987);
Berger and
Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc.
1987);
and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis
software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0
(Premier
Biosoft International; Palo Alto, CA), as well as sites on the Internet, are
available tools
for analyzing a given sequence and calculating T", based on user defined
criteria. Such
programs can also analyze a given sequence under defined conditions and
identify
suitable probe sequences. Typically, hybridization of longer polynucleotide
sequences,
>50 base pairs, is performed at temperatures of about 20-25°C below the
calculated Tm.
For smaller probes, <50 base pairs, hybridization is typically carried out at
the Tm or 5-
10°C below. This allows for the maximum rate of hybridization for DNA-
DNA and
DNA-RNA hybrids. Higher degrees of stringency at lower temperatures can be
2 0 achieved with the addition of formamide which reduces the Tm of the hybrid
about 1 °C
for each 1 % formamide in the buffer solution. Suitable stringent
hybridization
conditions are equivalent to about a 5 h to overnight incubation at about
42°C in a
solution comprising: about 40-50% formamide, up to about 6X SSC, about SX
Denhardt's solution, zero up to about 10% dextran sulfate, and about 10-20
~g/ml
2 5 denatured commercially-available carrier DNA. Generally, such stringent
conditions
include temperatures of 20-70°C and a hybridization buffer containing
up to 6x SSC
and 0-50% formamide; hybridization is then followed by washing filters in up
to about
2X SSC. For example, a suitable wash stringency is equivalent to O.1X SSC to
2X
SSC, 0.1% SDS, at 55°C to 65°C. Different degrees of stringency
can be used during
3 0 hybridization and washing to achieve maximum specific binding to the
target sequence.
Typically, the washes following hybridization are performed at increasing
degrees of
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17
stringency to remove non-hybridized polynucleotide probes from hybridized
complexes. Stringent hybridization and wash conditions depend on the length of
the
probe, reflected in the Tm, hybridization and wash solutions used, and are
routinely
determined empirically by one of skill in the art.
As previously noted, the isolated polynucleotides of the present
invention include DNA and RNA. Methods for preparing DNA and RNA are well
known in the art. In general, RNA is isolated from a tissue or cell that
produces large
amounts of Zalpha3l RNA. Such tissues and cells are identified by Northern
blotting,
Thomas, Proc. Nutl. Acad. Sci. USA 77:5201 ( 1980) and are discussed below.
Total
RNA can be prepared using guanidine HCl extraction followed by isolation by
centrifugation in v. CsCI gradient, Chirgwin et al., Biochemistry 18:52-94
(1979). Poly
(A)+ RNA is prepared from total RNA using the method of Aviv and Leder, Proc.
Natl.
Acad. Sci. USA 69:1408-1412 (1972). Complementary DNA (cDNA) is prepared from
poly(A)+ RNA using known methods. In the alternative, genomic DNA can be
isolated. Polynucleotides encoding Zalpha3l polypeptides are then identified
and
isolated by, for example, hybridization or PCR.
A full-length clone encoding Zalpha3l 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
2 0 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
thereof, for probing or priming a library. Expression libraries can be probed
with
antibodies to Zalpha31, receptor fragments, or other specific binding
partners.
2 5 The polynucleotides of the present invention can also be synthesized
using DNA synthesis machines. If chemically synthesized double stranded DNA is
required for an application such as the synthesis of a DNA or a DNA fragment,
then
each complementary strand is made separately, for example via the
phosphoramidite
method known in the art. The production of short polynucleotides (60 to 80 bp)
is
3 0 technically straightforward and can be accomplished by synthesizing the
complementary strands and then annealing them. However, for producing longer
CA 02374412 2001-11-28
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18
polynucleotides (longer than about 300 bp), special strategies are usually
employed.
For example, synthetic DNAs (double-stranded) are assembled in modular form
from
single-stranded fragments that are from 20 to 100 nucleotides in length. One
method
for building a synthetic DNA involves producing a set of overlapping,
complementary
oligonucleotides. Each internal section of the DNA has complementary 3' and 5'
terminal extensions designed to base pair precisely with an adjacent section.
After the
DNA is assembled, the process is completed by ligating the nicks along the
backbones
of the two strands. In addition to the protein coding sequence, synthetic DNAs
can be
designed with terminal sequences that facilitate insertion into a restriction
endonuclease
site of a cloning vector. Alternative ways to prepare a full-length DNA are
also known
in the art. See Glick and Pasternak, Molecular Biotechnology, Principles &
Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura
et
al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad.
Sci.
USA 87:633-7, 1990.
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 Zalpha3l pol~-peptides from
other
mammalian species, including murine, porcine, ovine, bovine, canine, feline,
equine,
2 0 and other primate polypeptides. Orthologs of human Zalpha31 can be cloned
using
information and compositions provided by the present invention in combination
with
conventional cloning techniques. For example, a cDNA can be cloned using mRNA
obtained from a tissue or cell type that expresses Zalpha3l as disclosed
herein.
Suitable sources of mRNA can be identified by probing Northern blots with
probes
2 5 designed from the sequences disclosed herein. A library is then prepared
from mRNA
of a positive tissue or cell line. A Zalpha3l-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.
3 0 4,683,202), using primers designed from the representative human Zalpha31
sequence
disclosed herein. Within an additional method, the cDNA library can be used to
CA 02374412 2001-11-28
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19
transform or transfect host cells, and expression of the cDNA of interest can
be detected
with an antibody to Zalpha3l 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 Zalpha3l 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,
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 Zalpha3l
polypeptide
are included within the scope of the present invention, as are polypeptides
encoded by
such cDNAs and mRNAs. Allelic variants and splice variants of these sequences
can
be cloned by probing cDNA or genomic libraries from different individuals or
tissues
according to standard procedures known in the art.
The present invention also provides isolated Zalpha3l polypeptides that
are substantially similar to the polypeptides of SEQ ID N0:2 and their
orthologs. The
2 0 term "substantially similar" is used herein to denote polypeptides having
70%,
preferably 80%, more preferably at least 85%, sequence identity to the
sequences
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 4 (amino acids are indicated by the
standard one
3 0 letter codes).
CA 02374412 2001-11-28
WO 00/73458 PCT/US00/14795
The percent identity is then calculated as:
Total number of identical matches
x 100
[length of the longer sequence plus the
5 number of gaps introduced into the longer
sequence in order to align the two sequences]
CA 02374412 2001-11-28
WO 00/73458 2~ PCT/US00/14795
rl N M
I
N Lf1 N N O
I I
M N N
I I
L~ r-i ,-I ~ M N
I I i
Ci a l0 ~ N N '-i M v-I
I I I
L(l O N f-I rl rl v-i rl
I ~ I I I
.'~. II1 rl M rl O rl M N N
I I I I I I I
a d' N N O M N ri N r-I v-1
I I I i I I
d~ N M v-I O M N rd M ri M
I I I I I I
x OD M M rl N rl N ~ N N N M
I I I I 1 I I I I I
lD N d~ ~ N M M N O N N M M
I I I I I I I I I I i
W Lll N O M M ri N M r-I O rl M N N
I I I I I I ~ I I I
Or Lf1 N N O M N rl O M rl O rl N r-I N
I ~ ~ I I I I I I
U 41 M ~ M M rl r-i M r1 N M rl rl N N rl
I t I I I I I I I I I I I I I
W lfl M O N rl rl M d~ rl M M rl O ri ~ M M
I I I I I I I I I I I I
z l17 rl M O O O r-i M M O N M N '-I O ~ N M
I ~ ~ I I I I I I
(Y., !11 O N M rl O N O M N N rl M N rl r1 M N M
I I i I I I I I I I i I I
~ rl N N O rl rl O N rl rl rl rl N ~ r~ O M N O
I I I I I I I I I I I I I i
~C rx z a v d w c7 x I--I a x ~ w w cra E-I 3 ~
H
In o m o
rl ~-I N
CA 02374412 2001-11-28
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22
Sequence identity of polynucleotide molecules is determined by similar methods
using
a ratio as disclosed above.
Those skilled in the art appreciate that there are many established
algorithms available to align two amino acid sequences. The "FASTA" similarity
search algorithm of Pearson and Lipman is a suitable protein alignment method
for
examining the level of identity shared by an amino acid sequence disclosed
herein and
the amino acid sequence of a putative variant zalpha3l. The FASTA algorithm is
described by Pearson and Lipman, Proc. Nat'1 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 trirr~~ned initial
regions are
2 o 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.
Preferred
parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap
extension
penalty=1, and substitution matrix=BLOSUM62, with other parameters set as
default.
These parameters can be introduced into a FASTA program by modifying the
scoring
matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol.
183:63 ( 1990).
3 0 FASTA can also be used to determine the sequence identity of nucleic
acid molecules using a ratio as disclosed above. For nucleotide sequence
comparisons,
CA 02374412 2001-11-28
WO 00/73458 PCT/US00/14795
23
the ktup value can range between one to six, preferably from three to six,
most
preferably three, with other parameters set as default.
The BLOSUM62 table (Table 4) is an amino acid substitution matrix
derived from about 2,000 local multiple alignments of protein sequence
segments,
representing highly conserved regions of more than 500 groups of related
proteins
(Henikoff and Henikoff, Proc. Nat'1 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. Althou~:h it is possible to design amino acid substitutions based
solely upon
1 o chemical properties (as discussed below), the language "conservative amino
acid
substitution" preferably refers to a substitution represented by a BLOSUM62
value of
greater than -1. For example, an amino acid substitution is conservative if
the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According
to this
system, preferred conservative amino acid substitutions are characterized by a
BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred
conservative
amino acid substitutions are characterized by a BLOSUM62 value of at least 2
(e.g., 2
or 3).
The present invention includes nucleic acid molecules that encode a
polypeptide having one or more conservative amino acid changes, compared with
the
2 0 amino acid sequence of SEQ ID N0:3. The BLOSUM62 table is an amino acid
substitution matrix derived from about 2,000 local multiple alignments of
protein
sequence segments, representing highly conserved regions of more than 500
groups of
related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915
(1992)).
Accordingly, the BLOSUM62 substitution frequencies can be used to define
2 5 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
3 0 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
CA 02374412 2001-11-28
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24
value of at least 2 (e.g., 2 or 3). Accordingly the present invention includes
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 Zalpha3l polypeptides or substantially homologous Zalpha3l
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 5) and other substitutions
that do not
significantly affect the folding or activity of the polypeptide; small
deletions, typically
of one to about 30 amino acids; and small amino- or carboxyl-terminal
extensions, such
as an amino-terminal methionine residue, a small linker peptide of up to about
20-25
residues, or an affinity tag. The present invention thus includes polypeptides
of from
about 30 to about 175 amino acid residues that comprise a sequence that is at
least 90%,
preferably at least 95%, and more preferably 99% or more identical to the
corresponding region of SEQ ID N0:2. Polypeptides comprising affinity tags can
further comprise a proteolytic cleavage site between the Zalpha3l polypeptide
and the
affinity tag. Preferred such sites include thrombin cleavage sites and factor
Xa
cleavage sites.
Table 5
Conservative amino acid substitutions
Basic: arginine
lysine
histidine
Acidic: glutamic acid
aspartic acid
Polar: glutamine
asparagine
Hydrophobic: leucine
isoleucine
valine
CA 02374412 2001-11-28
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Table 5 cont.
Aromatic: phenylalanine
tryptophan
tyrosine
5 Small: glycine
alanine
serine
threonine
methionine
The present invention further provides a variety of other polypeptide
fusions and rel.4t~ ~d multimeric proteins comprising one or more polypeptide
fusions.
For example, a Zalpha3l polypeptide can be prepared as a fusion to a
dimerizing
protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Preferred
dimerizing proteins in this regard include immunoglobulin constant region
domains.
Immunoglobulin-Zalpha3l polypeptide fusions can be expressed in genetically
engineered cells to produce a variety of multimeric Zalpha3l analogs.
Auxiliary
domains can be fused to Zalpha3l polypeptides to target them to specific
cells, tissues,
or macromolecules (e.g., collagen). For example, a Zalpha3l polypeptide or
protein
2 0 could be targeted to a predetermined cell type by fusing a Zalpha31
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
Zalpha3l 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
2 5 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
3 0 limitation, traps-3-methylproline, 2,4-methanoproline, cis-4-
hydroxyproline, traps-4-
hydroxyproline, N methylglycine, allo-threonine, methylthreonine,
hydroxyethylcysteme, hydroxyethylhomocysteine, nitroglutamine, homoglutamine,
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26
pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-
methylproline,
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 suppresser tRNAs.
Methods for synthesizing amino acids and aminoacylating tRNA are
known in the art. Transcription and translation of plasmids containing
nonsense
mutations is carried out in a cell-free system comprising an E. coli S30
extract and
commercially available enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722
( 1991 ); Ellman et al., Methods Enzymol. 202:301 ( 1991; Chung et al.,
Science 259:806-
809 (1993); and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-1019 (1993).
In a
second method, translation is carried out in Xenopus oocytes by microinjection
of
mutated mRNA and chemically aminoacylated suppresser 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
the presence of the desired non-naturally occurring amino acids) (e.g., 2-
azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, o~ 4-
Iluorophenylalanine).
2 0 The non-naturally occurring amino acid is incorporated into the protein in
place of its
natural counterpart. See, Koide et al., Biochem. 33:7470-7476 ( 1994).
Naturally
occurring amino acid residues can be converted to non-naturally occurring
species by in
vitro chemical modification. Chemical modification can be combined with site
directed mutagenesis to further expand the range of substitutions, Wynn and
Richards,
Protein Sci. 2:395-403 (1993).
A limited number of non-conservative amino acids, amino acids that are
not encoded by the genetic code, non-naturally occurring amino acids, and
unnatural
amino acids may be substituted for Zalpha31 amino acid residues.
Essential amino acids in the polypeptides of the present invention can be
3 0 identified according to procedures known in the art, such as site-directed
mutagenesis
or alanine-scanning mutagenesis, Cunningham and Wells, Science 244: 1081-1085
CA 02374412 2001-11-28
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27
(1989); Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502 (1991). In the
latter
technique, single alanine mutations are introduced at every residue in the
molecule, and
the resultant mutacit 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
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.
1 o Mol. Biol. 224:899-904 (1992); Wlodaver et al., FEBS Lett. 309:59-64
(1992).
Determination of amino acid residues that are within regions or domains
that are critical to maintaining structural integrity can be determined.
Within these
regions one can determine specific residues that will be more or less tolerant
of change
and maintain the overall tertiary structure of the molecule. Methods for
analyzing
sequence structure include, but are not limited to, alignment of multiple
sequences with
high amino acid or nucleotide identity and computer analysis using available
software
(e.g., the Insight II~ viewer and homology modeling tools; MSI, San Diego,
CA),
secondary structure propensities, binary patterns, complementary packing and
buried
polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and
Cordes et
2 o al., Current Opin. Struct. Biol. 6:3-10, 1996). In general, when designing
modifications
to molecules or identifying specific fragments determination of structure will
be
accompanied by evaluating activity of modified molecules.
Amino acid sequence changes are made in zalpha3l polypeptides so as
to minimize disruption of higher order structure essential to biological
activity. For
2 5 example, when the zalpha31 polypeptide comprises one or more helices,
changes in
amino acid residues will be made so as not to disrupt the helix geometry and
other
components of the molecule where changes in conformation abate some critical
function, for example, binding of the molecule to its binding partners. The
effects of
amino acid sequence changes can be predicted by, for example, computer
modeling as
3 o disclosed above or determined by analysis of crystal structure (see, e.g.,
Lapthorn et al.,
Nat. Struct. Biol. 2:266-268, 1995). Other techniques that are well known in
the art
CA 02374412 2001-11-28
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28
compare folding of a variant protein to a standard molecule (e.g., the native
protein).
For example, comparison of the cysteine pattern in a variant and standard
molecules
can be made. Mass spectrometry and chemical modification using reduction and
alkylation provide methods for determining cysteine residues which are
associated with
disulfide bonds or are free of such associations (Bean et al., Anal. Biochem.
201:216-
226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal.
Chem.
66:3727-3732, 1994). It is generally believed that if a modified molecule does
not have
the same disulfide bonding pattern as the standard molecule folding would be
affected.
Another well known and accepted method for measuring folding is circular
dichrosism
l0 (CD). Measuring and comparing the CD spectra generated by a modified
molecule and
standard molecule is routine (Johnson, Proteins 7:205-214, 1990).
Crystallography is
another well known method for analyzing folding and structure. Nuclear
magnetic
resonance (NMR), digestive peptide mapping and epitope mapping are also known
methods for analyzing folding and structural similarities between proteins and
polypeptides (Schaanan et al., Science 257:961-964, 1992).
A Hopp/Woods hydrophilicity profile of the zalpha3l protein sequence
as shown in SEQ ID N0:2 can be generated (Hopp et al., Proc. Natl. Acad.
Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et
al.,
Protein Engineering 11:153-169, 1998). The profile is based on a sliding six-
residue
2 0 window. Buried G, S, and T residues and exposed H, Y, and W residues were
ignored.
For example, in zalpha31, hydrophilic regions include: ( 1 ) amino acid
residues 14
(Asp) to 19 (Asp) of SEQ ID NO: 2, (2) amino acid residues 26 (Ala) to 31
(Glu) of
SEQ ID NO: 2, (3) amino acid 27 (Glu) to 32 (Pro) of SEQ ID NO: 2, (4) amino
acid
residues 136 (Tyr) to 141 (Lys) of SEQ ID NO: 2, and (5) amino acid residues
137
2 5 (Lys) to 142 (Arg) of SEQ ID NO: 2.
Those skilled in the art will recognize that hydrophilicity or
hydrophobicity will be taken into account when designing modifications in the
amino
acid sequence of a zalpha3l polypeptide, so as not to disrupt the overall
structural and
biological profile. Of particular interest for replacement are hydrophobic
residues
3 0 selected from the group consisting of Val, Leu and Ile or the group
consisting of Met,
Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant of substitution
could include
CA 02374412 2001-11-28
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29
such residues as shown in SEQ ID N0:2. However, Cysteine residues could be
relatively intolerant of substitution.
The identities of essential amino acids can also be inferred from analysis
of sequence similarity between other cytokine family members with zalpha3l.
Using
methods such as "FASTA" analysis described previously, regions of high
similarity are
identified within a family of proteins and used to analyze amino acid sequence
for
conserved regions. An alternative approach to identifying a variant zalpha3l
polynucleotide on the basis of structure is to determine whether a nucleic
acid molecule
encoding a potential variant zalpha3l polynucleotide can hybridize to a
nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1, as discussed above.
(>t~ier methods of identifying essential amino acids in the polypeptides
of the present invention are procedures known in the art, such as site-
directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081 (1989), Bass et al., Proc. Natl Acad. Sci. USA 88:4498 (1991), Coombs
and
Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and
Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the
latter
technique, single alanine mutations are introduced at every residue in the
molecule, and
the resultant mutant molecules are tested for biological activity as disclosed
below to
identify amino acid residues that are critical to the activity of the
molecule. See also,
Hilton et al., J. Biol. Chem. 271:4699 (1996).
The present invention also includes functional fragments of zalpha3l
polypeptides and nucleic acid molecules encoding such functional fragments. A
"functional" zalpha3l or fragment thereof defined herein is characterized by
its
proliferative or differentiating activity, by its ability to induce or inhibit
specialized cell
2 5 functions, or by its ability to bind specifically to an anti- zalpha31
antibody or zalpha31
receptor (either soluble or immobilized). As previously described herein,
zalpha3l is
characterized by a four-helical-bundle structure comprising helix A (amino
acid
residues 37-51), helix B (amino acid residues 65-79), helix C (amino acid
residues 87-
101) and helix D (amino acid residues 118-132), as shown in SEQ ID NO: 2.
Thus, the
3 0 present invention further provides fusion proteins encompassing: (a)
polypeptide
molecules comprising one or more of the helices described above; and (b)
functional
CA 02374412 2001-11-28
WO 00/73458 PCT/US00/14795
fragments comprising one or more of these helices. The other polypeptide
portion of
the fusion protein may be contributed by another four-helical-bundle cytokine,
such as
IL-15, IL-2, IL-4 and GM-CSF, or by a non-native and/or an unrelated secretory
signal
peptide that facilitates secretion of the fusion protein.
5 Routine deletion analyses of nucleic acid molecules can be performed to
obtain functional fragments of a nucleic acid molecule that encodes a zalpha3l
polypeptide. As an illustration, DNA molecules having the nucleotide sequence
of
SEQ ID NO:1 or fragments thereof, can be digested with Ba131 nuclease to
obtain a
series of nested deletions. These DNA fragments are then inserted into
expression
10 vectors in proper reading frame, and the expressed polypeptides are
isolated and tested
for zalpha3l activity, or for the ability to bind anti-zalpha3l antibodies or
zalpha3l
receptor. One alternative to exonuclease digestion is to use oligonucleotide-
directed
mutagenesis to introduce deletions or stop codons to specify production of a
desired
zalpha3l fragment. Alternatively, particular fragments of a zalpha3l
polynucleotide
15 can be synthesized using the polymerase chain reaction.
Standard methods for identifying functional domains are well-known to
those of skill in the art. For example, studies on the truncation at either or
both termini
of interferons have been summarized by Horisberger and Di Marco, Pharmac.
Ther.
66:507 (1995). Moreover, standard techniques for functiona~ analysis of
proteins are
2 0 described by, for example, Treuter et al., Molec. Gen. Genet. 240:113 (
1993); Content
et al., "Expression and preliminary deletion analysis of the 42 kDa 2-5A
synthetase
induced by human interferon," in Biological Interferon Systems, Proceedings of
ISIR-T'NO Meeting on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff
1987);
Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation 1
Boynton
2 5 et al., (eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J.
Biol. Chem.
270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al.,
Biochem. Pharmacol. 50:1295 (1995); and Meisel et al., Plant Molec. Biol. 30:1
( 1996).
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 02374412 2001-11-28
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31
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 Zalpha3l 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. Preferred assays in this regard include cell
proliferation
assays and biosensor-based ligand-binding assays, which are described below.
Mutagenized DNA molecules that encode active polypeptides can be recovered
from
2 5 the host cells and rapidly sequenced using modern equipment. These methods
allow
the rapid determination of the importance of individual amino acid residues in
a
polypeptide of interest, and can be applied to polypeptides of unknown
structure.
In addition, the proteins of the present invention (or polypeptide
fragments thereof) can be joined to other bioactive molecules, particularly
other
3 0 cytokines, to provide multi-functional molecules. For example, one or more
helices
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32
from zalpha3l can be joined to other cytokines to enhance their biological
properties or
efficiency of production.
The present invention thus provides a series of novel, hybrid molecules
in which a segment comprising one or more of the helices of zalpha3l is fused
to
another polypeptide. Fusion is preferably done by splicing at the DNA level to
allow
expression of chimeric molecules in recombinant production systems. The
resultant
molecules are then assayed for such properties as improved solubility,
improved
stability, prolonged clearance half life, improved expression and secretion
levels, and
pharmacodynamics. Such hybrid molecules may further comprise additional amino
acid residues (e.g. a polypeptide linker) between the component proteins or
polypeptides.
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 Zalpha3l protein.
For any
Zalpha3l polypeptide, including variants and fusion proteins, one of ordinary
skill in
the art can readily generate a fully degenerate polynucleotide sequence
encoding that
variant using the information set forth in Tables 2 and 3 above.
2 0 PROTEIN PRODUCTION
The Zalpha3l 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
2 5 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
3 0 Laboratory Press, Cold Spring Harbor, NY, 1989), and Ausubel et al., eds.,
Current
Protocols in Molecular Biology (John Wiley and Sons, Inc., NY, 1987).
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33
In general. a DNA sequence encoding a Zalpha3l polypeptide is
operably linked to other genetic elements required for its expression,
generally
including a transcription promoter and terminator, within an expression
vector. The
vector will also commonly contain one or more selectable markers and one or
more
origins of replication, although those skilled in the art will recognize that
within certain
systems selectable markers may be provided on separate vectors, and
replication of the
exogenous DNA may be provided by integration into the host cell genome.
Selection
of promoters, terminators, selectable markers, vectors and other elements is a
matter of
routine design within the level of ordinary skill in the art. Many such
elements are
described in the literature and are available through commercial suppliers.
T~~ direct a Zalpha3l polypeptide into the secretory pathway of a host
cell, a secretory signal sequence (also known as a leader sequence, prepro
sequence or
pre sequence) is provided in the expression vector. The secretory signal
sequence may
be that of Zalpha3l, 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
Zalpha3l
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. A
2 5 signal fusion polypeptide can be made wherein a secretory signal sequence
that encodes
a signal peptide from amino acids 1 (Met) to 19 (Asp) of SEQ ID N0:2 is
operably
linked to another DNA segment encoding a polypeptide using methods known in
the
art and disclosed herein. The secretory signal sequence contained in the
fusion
polypeptides of the present invention is preferably fused amino-terminally to
an
3 0 additional peptide to direct the additional peptide into the secretory
pathway. Such
constructs have numerous applications known in the art. For example, these
novel
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34
secretory signal sequence fusion constructs can direct the secretion of an
active
component of a normally non-secreted protein. Such fusions can 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
and Pearson, Somatic Cell Genetics 7:603 (1981); Graham and Van der Eb,
Virology
52:456 (1973), electroporation, Neumann et al., EMBO J. 1:841-845 (1982), DEAE-
dextran mediated transfection (Ausubel et al., ibid., and liposome-mediated
transfection, Hawley-Nelson et al., Focus 15:73 (1993); Ciccarone et al.,
Focus 15:80
(1993), and viral vectors, Miller and Rosman, BioTechnique.s 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-I (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK
(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL
1573; Graham et c:l., J. Gen. Virol. 36:59 (1977) and Chinese ha.rnster ovary
(e.g. CHO-
KI; ATCC No. CCL 61) cell lines. Additional suitable cell l~rAes are known in
the art
2 0 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.
2 5 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
3 o 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
CA 02374412 2001-11-28
WO 00/73458 PCT/US00/14795
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
5 marker is dihydrofolate reductase, which confers resistance to methotrexate.
Other
drug resistance genes (e.g. hygromycin resistance, multi-drug resistance,
puromycin
acetyltransferase) can also be used. Alternative markers that introduce an
altered
phenotype, such as green fluorescent protein, or cell surface proteins such as
CD4,
CDB, Class I MHU', placental alkaline phosphatase may be used to sort
transfected cells
10 from untransfected cells by such means as FACS sorting or magnetic bead
separation
technology.
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.
15 (Ban alore 11:47-58, 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
WIPO publication WO 94/06463. Insect cells can be infected with recombinant
baculovirus, commonly derived from Autographa californica nuclear polyhedrosis
virus (AcNPV). See, King, L.A. and Possee, R.D., The Baculovirus Expression
2 o System: A Laboratory Guide. London, Chapman & Hall; O'Reilly, D.R. et al.,
Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford
University
Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols.
Methods
in Molecular Biology, Totowa, NJ, Humana Press, 1995. The second method of
making
recombinant baculovirus utilizes a transposon-based system described by Luckow
25 (Luckow, V.A, et al., J Virol 67:4566-79, 1993). This system is sold in the
Bac-to-
BacTM kit (Life Technologies, Rockville, MD). This system utilizes a transfer
vector,
pFastBaclT"" (Life Technologies) containing a Tn7 transposon to move the DNA
encoding the zalpha31 polypeptide into a baculovirus genome maintained in E.
coli as a
large plasmid called a "bacmid." The pFastBaclT"' transfer vector utilizes the
AcNPV
3 0 polyhedrin promoter to drive the expression of the gene of interest, in
this case
zalpha3l. However, pFastBaclT"' can be modified to a considerable degree. The
CA 02374412 2001-11-28
WO 00/73458 PCT/US00114795
36
polyhedrin promoter can be removed and substituted with the baculovirus basic
protein
promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier
in the
baculovirus infection, and has been shown to be advantageous for expressing
secreted
proteins. See, Hill-Perkins, M.S. and Possee, R.D., J. Gen. Virol. 71:971-6,
1990;
Bonning, B.C. et al., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk, G.D.,
and
Rapoport, B., J. Biol. Chem. 270:1543-9, 1995. In such transfer vector
constructs, a
short or long version of the basic protein promoter can be used. Moreover,
transfer
vectors can be constructed which replace the native zalpha31 secretory signal
sequences
with secretory signal sequences derived from insect proteins. For example, a
secretory
1 o 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 zalpha3l 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 zalpha3l polypeptide, for example, a Glu-
Glu
epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985).
Using a
technique known in the art, a transfer vector containing zalpha3l 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
2 0 frugiperda cells, e.g. Sf~ cells. Recombinant virus that expresses
zalpha31 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 armyworm, Spodoptera frugiperda. See, in general, Glick
and
Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant
DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High
FiveOT"' cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No.
5,300,435). Commercially available serum-free media are used to grow and
maintain
the cells. Suitable media are Sf~00 IIT"" (Life Technologies) or ESF 921T"'
(Expression
3 0 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
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37
inoculation density of approximately 2-5 x 105 cells to a density of 1-2 x 106
cells at
which time a recombinant viral stock is added at a multiplicity of infection
(MOI) of
0.1 to 10, more typically near 3. Procedures used are generally described in
available
laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et
al., ibid.;
Richardson, C. D., ibid. . Subsequent purification of the zalpha3l 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 ch ;closed 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
4,661,454. Transformation systems for other yeasts, including Hansenula
polymorpha,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,
Ustilago
maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida
maltosa are known in the art. See, for example, Gleeson et al., J. Gen.
Microbiol.
132:3459 (1986) and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be
utilized according to the methods of McKnight et al., U.S. Patent No.
4,935,349.
Methods for transforming Acremonium chrysogenum are disclosed by Sumino et
al.,
3 0 U.S. Patent No. 5,162,228. Methods for transforming Neurospora are
disclosed by
Lambowitz, U.S. Patent No. 4,486,533.
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38
The use of Pichia methanolica as host for the production of recombinant
proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO
98/02536, and WO 98/02565. DNA molecules for use in transforming P.
methanolica
will commonly be prepared as double-stranded, circular plasmids, which are
preferably
linearized prior to transformation. For polypeptide production in P.
methanolica, it is
preferred that the promoter and terminator in the plasmid be that of a P.
methanolica
gene, such as a P. methanolica alcohol utilization gene (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 methanolica is a P. methanolica ADE2 gene, which encodes
phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21 ), which allows
ade2 host cells to grow in the absence of adenine. For large-scale, industrial
processes
where it is desirable to minimize the use of methanol, it is preferred to use
host cells in
which both methanol utilization genes (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. ;zet~anolica cells.
It is
preferred to transform P. methanolica cells by electroporation using an
exponentially
decaying, pulsed electric field having a field strength of from 2.5 to 4.5
kV/cm,
preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40
milliseconds, most
preferably about 20 milliseconds.
Prokaryotic host cells, including strains of the bacteria Escherichia coli,
2 5 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
Zalpha3l 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
3 0 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
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39
urea. The denatured polypeptide can then be refolded and dimerized by diluting
the
denaturant, such as by dialysis against a solution of urea and a combination
of reduced
and oxidized glutathione, followed by dialysis against a buffered saline
solution. In the
latter case, the polypeptide can be recovered from the periplasmic space in a
soluble
and functional form by disrupting the cells (by, for example, sonication or
osmotic
shock) to release the contents of the periplasmic space and recovering the
protein,
thereby obviating the need for denaturation and refolding.
Transformed or transfected host cells are cultured according to
conventional procedures in a culture medium containing nutrients and other
l0 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
2 0 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), I% BactoTM yeast extract
(Difco
Laboratories), 0.004% adenine and 0.006% L-leucine).
2 5 Another embodiment of the present invention provides for a peptide or
polypeptide comprising an epitope-bearing portion of a Zalpha3l polypeptide of
the
invention. The epitope of the this polypeptide portion is an immunogenic or
antigenic
epitope of a polypeptide of the invention. A region of a protein to which an
antibody
can bind is defined as an "antigenic epitope". See for instance, Geysen, H.M.
et al.,
3 0 Proc. Natl. Acad Sci. 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
CA 02374412 2001-11-28
WO 00/73458 PCT/US00/14795
molecule to which an antibody can bind), it is well known in the art that
relatively short
synthetic peptides that mimic part of a protein sequence are routinely capable
of
eliciting an antiserum that reacts with the partially mimicked protein. See
Sutcliffe, J.G.
et al. Science 219:660-666 (1983). Peptides capable of eliciting protein-
reactive sera
5 are frequently represented in the primary sequence of a protein, can be
characterized by
a set of simple chemical rules, and are confined neither to immunodominant
regions of
intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl
terminals.
Peptides that are extremely hydrophobic and those of six or fewer residues
generally are
ineffective at inducing antibodies that bind to the mimicked protein; longer
soluble
10 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
polypeptides of the present invention contain a sequence of at least nine,
preferably
15 between 15 to about 30 amino acids contained within the amino acid sequence
of a
polypeptide of the invention. However, peptides or polypeptides comprising a
larger
portion of an amino acid sequence of the invention, containing from 30 to 50
amino
acids, or any length up to and including the entire amino acid sequence of a
polypeptide
of the invention, also are useful for inducing antibodies that react with the
protein.
2 0 Preferably, the amino acid sequence of the epitope-bearing peptide is
selected to
provide substantial solubility in aqueous solvents (i.e., the sequence
includes relatively
hydrophilic residues and hydrophobic residues are preferably avoided); and
sequences
containing proline residues are particularly preferred. All of the
polypeptides shown in
the sequence listing contain antigenic epitopes to be used according to the
present
2 5 invention, however, specifically designed antigenic epitopes include the
peptides
predicted, for example, from a Jameson-Wolf plot, comprising: (1) amino acid
residues
11 (Thr) to 20 (Asp) of SEQ ID N0:2; (2) amino acid residues 60 (Ser) to 64
(Lys) of
SEQ ID N0:2; (3) amino acid residues 88 (Ser) to 96 (Gln) of SEQ ID N0:2; (4)
amino acid residues 127 (Ala) to 135 (Lys) of SEQ ID N0:2; and (5) amino acid
3 0 residues 127 (Ala) to 139 (Leu) of SEQ ID N0:2. . The present invention
also
provides polypeptide fragments or peptides comprising an epitope-bearing
portion of a
CA 02374412 2001-11-28
WO 00/73458 PCT/US00/14795
41
Zalphal polypeptide described herein. Such fragments or peptides may comprise
an
"immunogenic epitope," which is a part of a protein that elicits an antibody
response
when the entire protein is used as an immunogen. Immunogenic epitope-bearing
peptides can be identified using standard methods (see, for example, Geysen et
al.,
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.
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 prc:eF~r-~ed 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 Zalpha3l polypeptides (or chimeric Zalpha3l
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
2 0 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,
DEAF, QAE
and Q derivatives are preferred. Exemplary chromatographic media include those
media
derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF
2 5 (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-
Sepharose
(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71
(Toso
Haas) and the like. Suitable solid supports include glass beads, silica-based
resins,
cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene
beads, cross-
linked polyacrylamide resins and the like that are insoluble under the
conditions in
3 0 which they are to be used. These supports may be modified with reactive
groups that
allow attachment of proteins by amino groups, carboxyl groups, sulflrydryl
groups,
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42
hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries
include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide
activation, sulfliydryl activation, hydrazide activation, and carboxyl and
amino
derivatives for carbodiimide coupling chemistries. These and other solid media
are
well known and widely used in the art, and are available from commercial
suppliers.
Methods for binding receptor polypeptides to support media are well known in
the art.
Selection of a particular method is a matter of routine design and is
determined in part
by the properties of the chosen support. See, for example, Amity
Chromatography:
Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988).
The polypeptides of the present invention can be isolated by exploitation
of their structural and biological properties. For example, immobilized metal
ion
adsorption (IMAC) chromatography can be used to purify histidine-rich
proteins,
including those comprising polyhistidine tags. Briefly, a gel is first charged
with
divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3:1
(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 Enrymol., Vol. l:'~1, "Guide to Protein
2 0 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
2 5 hybrid Zalpha31 proteins, are constructed using regions or domains of the
inventive
Zalpha3l, Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin.
Biology,
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
3 0 cellular localization of a polypeptide, and can be applied to polypeptides
of unknown
structure.
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43
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
biological function may be swapped between Zalpha3l of the present invention
with
the functionally equivalent domains) from another family member, e.g., IL-2,
IL-4,
GM-CSF, or other four-helix bundle cytokine family member. Such domains
include,
but are not limited to, the secretory signal sequence, helices A through D,
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.
Using the methods discussed herein, one of ordinary skill in the art can
identify and/or prepare a variety of polypeptides that have substantially
similar
sequence identity to residues 1-142 or 20-142 of SEQ ID NO: 2, or functional
fragments and fusions thereof, wherein such polypeptides or fragments or
fusions retain
the properties of the wild-type protein such as the ability to stimulate
proliferation,
2 0 differentiation, induce specialized cell function or bind a cell or
zalpha31 receptor or
anti-zalpha3l antibodies.
Standard molecular biological and cloning techniques can be used to
swap the equivalent domains between the zalpha3l polypeptide and those
polypeptides
to which they are fused. Generally, a DNA segment that encodes a domain of
interest,
2 5 e.g., a zalpha31 helix A through D, or motif described herein, is operably
linked in
frame to at least one other DNA segment encoding an additional polypeptide and
inserted into an appropriate expression vector, as described herein. Generally
DNA
constructs are made such that the several DNA segments that encode the
corresponding
regions of a polypeptide are operably linked in frame to make a single
construct that
3 0 encodes the entire fusion protein, or a functional portion thereof. For
example, a DNA
construct would encode from N-terminus to C-terminus a fusion protein
comprising a
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44
signal polypeptide followed by a mature polypeptide; or a DNA construct would
encode from N-terminus to C-terminus a fusion protein comprising a signal
polypeptide
followed by Helix A, followed by Helix B, followed by Helix C, followed by
Helix D,
or as interchanged with equivalent regions from another protein. Such fusion
proteins
can be expressed, isolated, and assayed for activity as described herein.
Zalpha3l polypeptides or fragments thereof may also be prepared
through chemical synthesis. Zalpha3l 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.
Polypeptides of the present invention can also be synthesized by
exclusive solid phase synthesis, partial solid phase methods, fragment
condensation or
classical solution synthesis. Methods for synthesizing polypeptides are well
known in
the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser
et al.,
Anal. Biochem. 34:595, 1970. After the entire synthesis of the desired peptide
on a
solid support, the peptide-resin is with a reagent which cleaves the
polypeptide from the
resin and removes most of the side-chain protecting groups. Such methods are
well
established in the art. The activity of molecules of the present invention can
be
measured using a variety of assays that measure for example, signal
transduetion, Ig
binding or cAMP modulation. Such assays are well known in the art. For a
general
2 0 reference, see Nihei, Y., et al., supra.; and Rindisbacher, L., et al.,
supra..
ASSAYS
The activity of molecules of the present invention can be measured using
a variety of assays. Of particular interest are changes in steroidogenesis,
2 5 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 example in increasing
sperm production, treating thyroid, adrenal, lymphoid, inflammatory,
pancreatic, blood
or bone disorders, can be measured in vitro using cultured cells or in vivo by
3 0 administering molecules of the present invention to the appropriate animal
model. For
instance, host cells expressing a secreted form of zalpha3l polypeptide can be
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embedded in an alginate environment and injected (implanted) into recipient
animals.
Alginate-poly-L-lysine microencapsulation, permselective membrane
encapsulation and
diffusion chambers are a means to entrap transfected mammalian cells or
primary
mammalian cells. These types of non-immunogenic "encapsulations" permit the
5 diffusion of proteins and other macromolecules secreted or released by the
captured
cells to the recipient animal. Most importantly, the capsules mask and shield
the
foreign, embedded cells from the recipient animal's immune response. Such
encapsulations can extend the life of the injected cells from a few hours or
days (naked
cells) to several weeks (embedded cells). Alginate threads provide a simple
and quick
10 means for generating embedded cells.
Tl;:~ materials needed to generate the alginate threads are known in the
art. 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 5 x 105 to
about 5 x
15 107 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. The thread is then
rinsed
with deionized water before coating the thread by incubating in a 0.01 %
solution of
2 0 poly-L-lysine. Finally, the thread is rinsed with Lactated Ringer's
Solution and drawn
from solution into a syringe barrel (without needle). A large bore needle is
then
attached to the syringe, and the thread is intraperitoneally injected into a
recipient in a
minimal volume of the Lactated Ringer's Solution.
An alternative in vivo approach for assaying proteins of the present
2 5 invention involves viral delivery systems. Exemplary viruses for this
purpose include
adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated
virus
(AAV). Adenovirus, a double-stranded DNA virus, is currently the best studied
gene
transfer vector for delivery of heterologous nucleic acid (for review, see
T.C. Becker et
al., Meth. Cell Biol. 43:161-89, 1994; and J.T. Douglas and D.T. Curiel,
Science &
3 o Medicine 4:44-53, 1997). The adenovirus system offers several advantages:
(i)
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46
adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to
high
titer; (iii) infect a broad range of mammalian cell types; and (iv) can be
used with many
different promoters including ubiquitous, tissue specific, and regulatable
promoters.
Also, because adenoviruses are stable in the bloodstream, they can be
administered by
intravenous injection.
Using adenovirus vectors where portions of the adenovirus genome are
deleted, inserts are incorporated into the viral DNA by direct ligation or by
homologous
recombination with a co-transfected plasmid. In an exemplary system, the
essential E1
gene has been deleted from the viral vector, and the virus will not replicate
unless the
El 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 E1 gene deletion, the virus cannot
replicate in the
host cells. However, the host's tissue (e.g., liver) will express and process
(and, if a
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.
Moreover, adenoviral vectors containing various deletions of viral genes
can be used in an attempt to reduce or eliminate immune responses to the
vector. Such
adenoviruses are E 1 deleted, and in addition contain deletions c; 1=;2A or E4
(Lusky, M.
2 0 et al., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene
Therapy 9:671-
679, 1998). In addition, deletion of E2b is reported to reduce immune
responses
(Amalfitano, A. et al., J. Virol. 72:926-933, 1998). Moreover, by deleting the
entire
adenovirus genome, very large inserts of heterologous DNA can be accommodated.
Generation of so called "gutless" adenoviruses where all viral genes are
deleted are
2 5 particularly advantageous for insertion of large inserts of heterologous
DNA. For
review, see Yeh, P. and Perricaudet, M., FASEB J. 11:615-623, 1997.
The adenovirus system can also be used for protein production in vitro.
By culturing ader, virus-infected non-293 cells under conditions where the
cells are not
rapidly dividing, the cells can produce proteins for extended periods of time.
For
3 o 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
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47
under serum-free conditions, which allows infected cells to survive for
several weeks
without significant cell division. Alternatively, adenovirus vector infected
293 cells
can be grown as adherent cells or in suspension culture at relatively high
cell density to
produce significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-
55,
1994). With either protocol, an expressed, secreted heterologous protein can
be
repeatedly isolated from the cell culture supernatant, lysate, or membrane
fractions
depending on the disposition of the expressed protein in the cell. Within the
infected
293 cell production protocol, non-secreted proteins may also be effectively
obtained.
The activity of molecules of the present invention can be measured using
1 o a variety of assays that measure proliferation of and/or binding to cells.
Of particular
interest are changes in zalpha3l-dependent cells. Suitable cell lines to be
engineered to
be zalpha3l-dependent include the IL-3-dependent BaF3 cell line (Palacios and
Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:
4133-4135,
1986), FDC-P 1 (Hapel et al., Blood 64: 786-790, 1984), and M07e (Kiss et al.,
Leukemia 7: 235-240, 1993). However, other growth factor-dependent cell lines,
such
as FDC-P1 (Hapel et al., Blood 64: 786-790, 1984), and M07e (Kiss et al.,
Leukemia
7: 235-240, 1993) are suitable for this purpose. Growth factor-dependent cell
lines can
be established according to published methods (e.g. Greenberger et al.,
Leukemia Res.
8: 363-375, 1984; Dexter et al., in Baum et al. Eds., Experimental Hematology
Today,
2 0 8th Ann. Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980).
Proteins of the present invention are useful for stimulating proliferation,
activation, differentiation and/or induction or inhibition of specialized cell
function of
cells of the involved homeostasis of the hematopoiesis and immune function. In
particular, zalpha3l polypeptides are useful for stimulating proliferation,
activation,
2 5 differentiation, induction or inhibition of specialized cell functions of
cells of the
hematopoietic lineages, including, but not limited to, T cells, B cells, NK
cells,
dendritic cells, monocytes, and macrophages, as well as epithelial cells.
Proliferation
and/or differentiation of hematopoietic cells can be measured in vitro using
cultured
cells or in vivo by administering molecules of the present invention to the
appropriate
3 0 animal model. Assays measuring cell proliferation or differentiation are
well known in
the art. For example, assays measuring proliferation include such assays as
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48
chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New
Drugs
8:347-354, 1990, incorporated herein by reference), incorporation of
radiolabelled
nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989, incorporated
herein by
reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of
proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985,
incorporated herein by reference), and use of tetrazolium salts (Mosmann, J.
Immunol.
Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall
et al.,
Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833,
1988; all
incorporated herein by reference). Assays measuring differentiation include,
for
example, measuring cell-surface markers associated with stage-specific
expression of a
tissue, enzymatic activity, functional activity or morphological changes
(Watt. FASEB,
5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim.
Cell Biol.
Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
As a ligand, the activity of zalpha3l polypeptide can be measured by a
silicon-based biosensor microphysiometer which measures the extracellular
acidification rate or proton excretion associated with receptor binding and
subsequent
physiologic cellular responses. An exemplary device is the CytosensorT""
Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety
of
2 0 cellular responses, such as cell proliferation, ion transport, energy
production,
inflammatory response, regulatory and receptor activation, and the like, can
be
measured by this method. See, for example, McConnell, H.M. et al., Science
257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997;
Arimilli,
S. et al., J. Immunol. Meth. 212:49-59, 1998; Van Liefde, I. et al., Eur. J.
Pharmacol.
2 5 346:87-95, 1998. The microphysiometer can be used for assaying adherent or
non-
adherent eukaryotic or prokaryotic cells. By measuring extracellular
acidification
changes in cell media over time, the microphysiometer directly measures
cellular
responses to various stimuli, including zalpha3l polypeptide, its agonists, or
antagonists. Preferably, the microphysiometer is used to measure responses of
a
3 o zalpha3l-responsive eukaryotic cell, compared to a control eukaryotic cell
that does not
respond to zalpha3l polypeptide. ZALPHA31-responsive eukaryotic cells comprise
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49
cells into which a receptor for zalpha3l has been transfected creating a cell
that is
responsive to zalpha31; or cells naturally responsive to zalpha31 such as
cells derived
from small intestine, PBLs, or bone marrow tissue. Differences, measured by a
change,
for example, an increase or diminution in extracellular acidification, in the
response of
cells exposed to zalpha3l polypeptide, relative to a control not exposed to
zalpha3l, are
a direct measurement of zalpha3l-modulated cellular responses. Moreover, such
zalpha3l-modulated responses can be assayed under a variety of stimuli. Using
the
microphysiometer, there is provided a method of identifying agonists of
zalpha3l
polypeptide, comprising providing cells responsive to a zalpha3l polypeptide,
culturing
a first portion of the cells in the absence of a test compound, culturing a
second portion
of the cells in thc: presence of a test compound, and detecting a change, for
example, an
increase or diminution, in a cellular response of the second portion of the
cells as
compared to the first portion of the cells. The change in cellular response is
shown as a
measurable change extracellular acidification rate. Moreover, culturing a
third portion
of the cells in the presence of zalpha3l polypeptide and the absence of a test
compound
can be used as a positive control for the zalpha3l-responsive cells, and as a
control to
compare the agonist activity of a test compound with that of the zalpha3l
polypeptide.
Moreover, using the microphysiometer, there is provided a method of
identifying
antagonists of zalpha3l polypeptide, comprising providing cells responsive to
a
2 0 zalpha3 I polypeptide, culturing a first portion of the cells in the
presence of zalpha31
and the absence of a test compound, culturing a second portion of the cells in
the
presence of zalpha3l and the presence of a test compound, and detecting a
change, for
example, an increase or a diminution in a cellular response of the second
portion of the
cells as compared to the first portion of the cells. The change in cellular
response is
2 5 shown as a measurable change extracellular acidification rate. Antagonists
and
agonists, for zalpha3l polypeptide, can be rapidly identified using this
method.
Moreover, zalpha3l can be used to identify cells, tissues, or cell lines
which respond tc a zalpha3l-stimulated pathway. The microphysiometer,
described
above, can be used to rapidly identify ligand-responsive cells, such as cells
responsive
3 0 to zalpha31 of the present invention. Cells can be cultured in the
presence or absence
of zalpha3l polypeptide. Those cells which elicit a measurable change in
extracellular
CA 02374412 2001-11-28
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acidification in the presence of zalpha3l are responsive to zalpha3l. Such
cell lines,
can be used to identify antagonists and agonists of zalpha3l polypeptide as
described
above.
5 Antagonists are also useful as research reagents for characterizing sites
of ligand-receptor interaction. Also as a treatment for prostate cancer.
Inhibitors of
Zalpha3l activity (Zalpha3l antagonists) include anti-Zalpha3l antibodies and
soluble
Zalpha3l receptors, as well as other peptidic and non-peptidic agents
(including
ribozymes).
10 Zalpha3l 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 Zalpha3l. In addition to those assays
disclosed
herein, samples can be tested for inhibition of Zalpha3l activity within a
variety of
assays designed to measure receptor binding or the stimulation/inhibition of
Zalpha3l-
15 dependent cellular responses. For example, Zalpha3l-responsive cell lines
can be
transfected with a reporter gene construct that is responsive to a Zalpha3l-
stimulated
cellular pathway. Reporter gene constructs of this type are known in the art,
and will
generally comprise a Zalpha3l-DNA response element operably linked to a gene
encoding a protein which can be assayed, such as luciferase. .~al'~A response
elements
2 0 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.
Acac~ 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).
25 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
activity of Zalpha3l on the target cells as evidenced by a decrease in
Zalpha3l
stimulation of reporter gene expression. Assays of this type will detect
compounds that
directly block Zalpha3l binding to cell-surface receptors, as well as
compounds that
3 0 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
Zalpha3l
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51
binding to receptor using Zalpha3l tagged with a detectable label (e.g., 'ZSI,
biotin,
horseradish peroxidase, FITC, and the like). Within assays of this type, the
ability of a
test sample to inhibit the binding of labeled Zalpha3l 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 Zalpha3l 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 Zalpha3l 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,
2 0 epoxide activation, sulfhydryl activation, and hydrazide activation. The
resulting
medium will generally be configured in the form of a column, and fluids
containing
ligand are passed through the column one or more times to allow ligand to bind
to the
receptor polypeptide. The ligand is then eluted using changes in salt
concentration,
chaotropic agents (guanidine HCl), or pH to disrupt ligand-receptor binding.
2 5 An assay system that uses a ligand-binding receptor (or an antibody, one
member of a complement/ anti-complement pair) or a binding fragment thereof,
and a
commercially available biosensor instrument (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
3 0 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,
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52
antibody, member or fragment is covalently attached, using amine or
sulflrydryl
chemistry, to dextran fibers that are attached to gold film within the flow
cell. A test
sample is passed through the cell. If a ligand, epitope, or opposite member of
the
complement/anti-complement pair is present in the sample, it will bind to the
immobilized receptor, antibody or member, respectively, causing a change in
the
refractive index of the medium, which is detected as a change in surface
plasmon
resonance of the gold film. This system allows the determination of on- and
off rates,
from which binding affinity can be calculated, and assessment of stoichiometry
of
binding.
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. NY Acad. Sci. 51: 660
(1949) and
calorimetric assays, Cunningham et al., Science 253:545 ( 1991 ); Cunningham
et al.,
Science 245:821 ( 1991 ).
Zalpha3l polypeptides can also be used to prepare antibodies that bind
to zalpha3l epitopes, peptides or polypeptides. The zalpha3l polypeptide or a
fragment
thereof serves as an antigen (immunogen) to inoculate an animal and elicit an
immune
response. One of skill in the art would recognize that antigenic, epitope-
bearing
2 o polypeptides contain a sequence of at least 6, preferably at least 9, and
more preferably
at least 15 to about 30 contiguous amino acid residues of a zalpha3l
polypeptide (e.g.,
SEQ ID N0:2). Polypeptides comprising a larger portion of a zalpha3l
polypeptide,
i.e., from 30 to 10 residues up to the entire length of the amino acid
sequence are
included. Antigens or immunogenic epitopes can also include attached tags,
adjuvants
2 5 and carriers, as described herein. Suitable antigens include the mature
zalpha31
polypeptide encoded by SEQ ID N0:2 from amino acid number 1 (Met) to 142 (Arg)
of
SEQ ID N0:2, or a contiguous 9 to 122 amino acid fragment thereof. Other
suitable
antigens include alpha helical domains, extracellular domains, motifs,
regions, epitopes,
etc., as disclosed herein. Preferred peptides to use as antigens are
hydrophilic peptides
3 0 such as those predicted by one of skill in the art from a hydrophobicity
plot. Zalpha3l
hydrophilic peptides include peptides comprising amino acid sequences selected
from
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53
the group consisting of: those predicted 6 amino acid antigenic epitopes
determined
from a Hopp/Woods hydrophilicity profile based on a sliding six-residue
window, with
buried G, S, and T residues and exposed H, Y, and W residues ignored. For
example, in
zalpha3l, suitable hydrophilic regions include: (1) amino acid residues 14
(Asp) to 19
(Asp) of SEQ ID NO: 2, (2) amino acid residues 26 (Ala) to 31 (Glu) of SEQ ID
NO: 2,
(3) amino acid 27 (Glu) to 32 (Pro) of SEQ ID NO: 2, (4) amino acid residues
136
(Tyr) to 141 (Lys) of SEQ ID NO: 2, and (5) amino acid residues 137 (Lys) to
142
(Arg) of SEQ ID NO: 2. Suitable hydrophilic peptides also include those
antigenic
epitopes predicted from a Jameson-Wolf plot, comprising: (1) amino acid
residues 11
l0 (Thr) to 20 (Asp) of SEQ ID N0:2; (2) amino acid residues 60 (Ser) to 64
(Lys) of SEQ
ID N0:2; (3) ar-i:no acid residues 88 (Ser) to 96 (Gln) of SEQ ID N0:2; (4)
amino acid
residues 127 (AIa) to 135 (Lys) of SEQ ID N0:2; and (5) amino acid residues
127 (Ala)
to 139 (Leu) of SEQ ID N0:2. Antibodies from an immune response generated by
inoculation of an animal with these antigens can be isolated and purified as
described
herein. Methods for preparing and isolating polyclonal and monoclonal
antibodies are
well known in the art. See, for example, Current Protocols in Immunology,
Cooligan,
et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995;
Sambrook
et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor,
NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:
Techniques
2 o and Applications, CRC Press, Inc., Boca Raton, FL, 1982.
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
zalpha3l
polypeptide or a fragment thereof. The immunogenicity of a zalpha3l
polypeptide may
2 5 be increased through the use of an adjuvant, such as alum (aluminum
hydroxide) or
Freund's complete or incomplete adjuvant. Polypeptides useful for immunization
also
include fusion polypeptides, such as fusions of zalpha3l or a portion thereof
with an
immunoglobulin polypeptide or with maltose binding protein. The polypeptide
immunogen may be a full-length molecule or a portion thereof. If the
polypeptide
3 0 portion is "hapten-like", such portion may be advantageously joined or
linked to a
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54
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. Moreover,
human antibodies can be produced in transgenic, non-human animals that have
been
engineered to contain human immunoglobulin genes as disclosed in WIPO
Publication
WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these
animals be inactivated or eliminated, such as by homologous recombination.
2 0 Antibodies are considered to be specifically binding if: 1 ) they exhibit
a
threshold level of binding activity, and 2) they do not significantly cross-
react with
related polypeptide molecules. A threshold level of binding is determined if
anti-
zalpha3l antibodies herein bind to a zalpha3l polypeptide, peptide or epitope
with an
affinity at least 10-fold greater than the binding affinity to control (non-
zalpha3l)
2 5 polypeptide. It is preferred that the antibodies exhibit a binding
affinity (Ka) of 106 M
1 or 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
(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).
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Whether anti-zalpha3l antibodies do not significantly cross-react with
related polypeptide molecules is shown, for example, by the antibody detecting
zalpha3l polypeptide but not known related polypeptides using a standard
Western blot
analysis (Ausubel et al., ibid.). Examples of known related polypeptides are
those
5 disclosed in the prior art, such as known orthologs, and paralogs, and
similar known
members of a protein family (e.g., other four-helix bundle cytokines),
Screening can
also be done using non-human zalpha3l, and zalpha3l mutant polypeptides.
Moreover,
antibodies can be "screened against" known related polypeptides, to isolate a
population that specifically binds to the zalpha3l polypeptides. For example,
10 antibodies raised to zalpha3l are adsorbed to related polypeptides adhered
to insoluble
matrix; antibodies specific to zalpha3l will flow through the matrix under the
proper
buffer conditions. Screening allows isolation of polyclonal and monoclonal
antibodies
non-crossreactive to known closely related polypeptides (Antibodies: A
Laboratory
Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988;
Current
15 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.
2 0 Immunol. 2: 67-101, 1984. Specifically binding anti-zalpha3l antibodies
can be
detected by a number of methods in the art, and disclosed below.
A variety of assays known to those skilled in the art can be utilized to
detect antibodies which bind to zalpha3l proteins or polypeptides. Exemplary
assays
are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane
(Eds.),
2 5 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 zalpha3l protein or
polypeptide.
3 0 Alternative techniques for generating or selecting antibodies useful
herein include in vitro exposure of lymphocytes to zalpha3l protein or
peptide, and
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selection of antibody display libraries in phage or similar vectors (for
instance, through
use of immobilized or labeled zalpha3l protein or peptide). Genes encoding
polypeptides having potential zalpha3l polypeptide binding domains can be
obtained
by screening random peptide libraries displayed on phage (phage display) or on
bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can
be
obtained in a number of ways, such as through random mutagenesis and random
polynucleotide synthesis. These random peptide display libraries can be used
to screen
for peptides which interact with a known target which can be a protein or
polypeptide,
such as a ligand or receptor, a biological or synthetic macromolecule, or
organic or
inorganic substances. Techniques for creating and screening such random
peptide
display libraries are known in the art (Ladner et al., US Patent NO.
5,223,409; Ladner
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)
and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display
libraries can be screened using the zalpha3l sequences disclosed herein to
identify
proteins which bind to zalpha3l. These "binding polypeptides" which interact
with
zalpha3l polypeptides can be used for tagging cells; for isolatira~; homolog
polypeptides
2 0 by affinity purification; they can be directly or indirectly conjugated to
drugs, toxins,
radionuclides and the like. These binding polypeptides can also be used in
analytical
methods such as for screening expression libraries and neutralizing activity,
e.g., for
blocking interaction between ligand and receptor, or viral binding to a
receptor. The
binding polypeptides can also be used for diagnostic assays for determining
circulating
2 5 levels of zalpha31 polypeptides; for detecting or quantitating soluble
zalpha31
polypeptides as marker of underlying pathology or disease. These binding
polypeptides
can also act as zalpha3l "antagonists" to block zalpha3l binding and signal
transduction in vitro and in vivo. These anti-zalpha3l binding polypeptides
would be
useful for inhibiting zalpha31 activity or protein-binding.
3 0 Antibodies to zalpha31 may be used for tagging cells that express
zalpha3l; for isolating zalpha3l by affinity purification; for diagnostic
assays for
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57
determining circulating levels of zalpha3l polypeptides; for detecting or
quantitating
soluble zalpha3l as marker of underlying pathology or disease; in analytical
methods
employing FACS; for screening expression libraries; for generating anti-
idiotypic
antibodies; and as neutralizing antibodies or as antagonists to block zalpha3l
activity in
vitro and in vivo. Suitable direct tags or labels include radionuclides,
enzymes,
substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent
markers,
magnetic particles and the like; indirect tags or labels may feature use of
biotin-avidin
or other complement/anti-complement pairs as intermediates. Antibodies herein
may
also be directly or indirectly conjugated to drugs, toxins, radionuclides and
the like, and
l0 these conjugates used for in vivo diagnostic or therapeutic applications.
Moreover,
antibodies to z~.ii ha31 or fragments thereof may be used in vitro to detect
denatured
zalpha3l 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
2 0 corresponding anti-complementary molecule (receptor or antigen,
respectively, for
instance). More specifically, Zalpha3l polypeptides or anti-Zalpha3l
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.
2 5 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
3 0 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
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58
to the polypeptide or antibody, or indirectly attached through means of a
chelating
moiety, for instance). Polypeptides or antibodies may also be conjugated to
cytotoxic
drugs, such as adriamycin. For indirect attachment of a detectable or
cytotoxic
molecule, the detectable or cytotoxic molecule can be conjugated with a member
of a
complementary/ anticomplementary pair, where the other member is bound to the
polypeptide or antibody portion. For these purposes, biotin/streptavidin is an
exemplary complementary/ anticomplementary pair.
In another embodiment, polypeptide-toxin fusion proteins or antibody-
toxin fusion proteins can be used for targeted cell or tissue inhibition or
ablation (for
instance, to treat cancer cells or tissues). Alternatively, if the polypeptide
has multiple
functional domains (i.e., an activation domain or a 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, Zalpha3l-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 Zalpha3l polypeptide or anti-
Zalpha3l antibody targets the hyperproliferative blood or bone marrow cell.
See,
generally, Hornick et al., Blood 89:4437 (1997). They described fusion
proteins enable
2 5 targeting of a cytokine to a desired site of action, thereby providing an
elevated local
concentration of cytokine. Suitable Zalpha3l polypeptides or anti-Zalpha3l
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-
3 0 CSF), for instance.
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In yet another embodiment, if the Zalpha3l polypeptide or anti-
Zalpha3l 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
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
predicted with targeting of a bioactive conjugate containing a radionuclide,
as described
herein.
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.
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, thyroid, heart
and
adrenal function. For example, proteins and peptides of the present invention
can be
immobilized on a column and membrane preparations run over the column,
2 0 Immobilized Affinity 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. '_ 82, "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)
2 5 and specific cell-surface proteins can be identified.
The molecules of the present invention will be useful for testing
disorders of the reproductive system, thyroid, adrenal, heart and
immunological
systems.
Zalpha3l represents a novel polypeptide with a putative signal peptide
3 0 leader sequence and alpha helical structure. Therefore this gene may
encode a secreted
polypeptide with secondary structure indicating it is a member of the four
helix bundle
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cytokine family. Alternatively, this polypeptide may have other activities
associated
with other biological functions including: enzymatic activity, association
with the cell
membrane, or function as a carrier protein.
Most four-helix bundle cytokines as well as other proteins produced by
5 activated T lymphocytes play an important biological role in cell
differentiation,
activation, recruitment and homeostatsis of cells throughout the body.
Therapeutic
utility includes treatment of diseases which require immune regulation
including
autoimmune diseases such as rheumatoid arthritis, multiple sclerosis,
myasthenia
gravis, systemic lupus erythomatosis and diabetes. Zalpha3l may be important
in the
1 o regulation of inflammation and therefore would be useful in treating
rheumatoid
arthritis, asthma and sepsis. There may be a role of zalpha3l in mediating
tumorgenesis and therefore would be useful in the treatment of cancer.
Zalpha3l may
be a potential therapeutic in suppressing the immune system which would be
important
for reducing graft rejection. Alternatively zalpha3l may activate the immune
system
15 which would be important in boosting immunity to infectious diseases or in
improving
vaccines.
Thyroid malfunction and some of the currently associated therapies can
elicit detrimental effects on bone in vivo. Thus, given the thyroidal and
pituitary
localization of the present invention, assays that measure '~~oaie formation
and/or
2 0 resorption are important assays to assess zalpha31 activity. One example
is an assay
system that permits rapid identification of substances having selective
calcitonin
receptor activity on cells expressing the calcitonin receptor. The calcitonin
receptor is a
member of the G-protein receptor family and transducer signal via activation
of
adenylate cyclase, leading to elevation of cellular cAMP levels (Lin et al.,
Science
2 5 254:1022-24, 1991 ). This assay system exploits the receptor's ability to
elevate cAMP
levels as a way to detect other molecules that are able to stimulate the
calcitonin
receptor and initiate signal transduction. Other assays that measure bone
formation or
resorption include calvarial assays, QCT, and assays that measure osteoblast
size and
number. Such assays are known in the art and discussed below.
3 0 Receptor activation can be detected by: ( 1 ) measurement of adenylate
cyclase activity (Salomon et al., Anal. Biochem. 58:541-48, 1974; Alvarez and
Daniels,
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61
Anal. Biochem. 187:98-103, 1990); (2) measurement of change in intracellular
cAMP
levels using conventional radioimmunoassay methods (Steiner et al., J. Biol.
Chem.
247:1106-13, 1972; Harper and Brooker, J. Cyc. Nucl. Res. 1:207-18, 1975); or
(3)
through use of a CAMP scintillation proximity assay (SPA) method (Amersham
Corp.,
Arlington Heights, IL). While these methods provide sensitivity and accuracy,
they
involve considerable sample processing prior to assay, are time consuming,
involve the
use of radioisotopes, and would be cumbersome for large scale screening
assays.
An alternative assay system involves selection of polypeptides that are
able to induce expression of a cyclic AMP response element (CRE)-luciferase
reporter
gene, as a consequence of elevated cAMP levels, in cells expressing a
calcitonin
receptor, but nor. in cells lacking calcitonin receptor expression, as
described in U.S.
patent No. 5,622,839, U.S. Patent No. 5,674,689, and U.S. patent No.
5,674,981.
Well established animal models are available to test in vivo efficacy of
zalpha3l polypeptides, agonists or antagonists, that interact with the
calcitonin
receptor. Moreover, these models may be used to test effects of zalpha3l on
bone other
than through the calcitonin receptor. For example, the hypocalcemic rat or
mouse
model can be used to determine the effect on serum calcium, and the
ovariectomized rat
or mouse can be used as a model system for osteoporosis. Bone changes seen in
these
models and in humans during the early stages of estrogen deficiency are
qualitatively
2 0 similar. Calcitonin has been shown to be an effective agent for the
prevention of bone
loss in ovariectomized women and rats (Mazzuoli et al., Calcif. Tissue Int.
47:209-14,
1990; Wronski et al., Endocrinology 129:2246-50, 1991). High dose estrogen has
been
shown to inhibit bone resorption and to stimulate bone formation in an
ovariectomized
mouse model (Bain et al., J. Bone Miner. Res. 8:435-42, 1993).
2 5 Biologically active zalpha31 polypeptides, agonists or antagonists, of the
present invention that interact with the calcitonin receptor, or exert other
effects on
bone, are therefore contemplated to be advantageous for use in therapeutic
applications
for which calcitonin is useful. Such applications, for example, are in the
treatment of
osteoporosis, Paget's disease, hyperparathyroidism, osteomalacia, idiopathic
3 0 hypercalcemia of infancy and other conditions. Additional applications are
to inhibit
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62
gastric secretion in the treatment of acute pancreatitis and gastrointestinal
disorders, and
uses as analgesics, in particular for bone pain.
In vivo assays for measuring changes in bone formation rates include
performing bone histology (see, Recker, R., eds. Bone Histomorphometry:
Techniques
and Interpretation. Boca Raton: CRC Press, Inc., 1983) and quantitative
computed
tomography (QCT; Ferretti,J. Bone 17:353S-3645, 1995; Orphanoludakis et al.,
Investig. Radiol. 14:122-130, 1979; and Durand et al., Medical Physics 19:569-
573,
1992). An exemplary ex vivo assay for measuring changes in bone formation is a
calavarial assay (Gowen et al., J. Immunol. 136:2478-2482, 1986) or resorption
calvarial assay (Linkhart, T.A., and Mohan, S., Endocrinology 125:1484-1491,
1989).
In addition, polypeptides of the present invention can be assayed and
used for their ability to modify inflammation. Methods to determine
proinflammatory
and antiinflammatory qualities of zalpha3l are known in the art and discussed
herein.
For example, suppression of cAMP production is an indication of anti-
inflammatory
effects (Nihei, Y., et al., Arch. Dermatol. Res., 287:546-552, 1995).
Suppression of
cAMP and inhibition of ICAM and HLA-Dr induced by IFN-y in keratinocytes can
be
used to assess inhibition of inflammation. Alternatively, enhancement of CAMP
production and induction of ICAM and HLA-Dr in this system can be an
measurement
of proinflammatory effects of a protein. Zalpha3l, likewise can exhibit
similar
2 0 inflammatory effects, as shown in vivo (Example 8) and may exert these
effects in
tissues in which it is expressed, or in other tissues. For example, zalpha3l
is expressed
in the colon, and can be useful in promoting wound healing in this tissue, or
exhibit
anti-bacterial or anti-viral effects. Moreover, zalpha3l or its antagonists
can be useful
in treatment of inflammatory bowel disease, diverticulitis, inflammation
during and
after intestinal surgery, and the like. In addition, zalpha3l, expressed in
thyroid, can
have wound-healing or antimicrobial or antiviral actions in tissues outside of
thyroid,
such, as heart, brain, liver, kidney, and the like. Moreover, direct
measurement of
zalpha3l polypeptide and zalpha3l antibodies can be useful in diagnosing
inflammatory diseases such as melanoma, inflammatory bowel disease,
diverticulitis,
3 o asthma, pelvic inflammatory disease, (PID), psoriasis, arthritis,
reperfusion ischemia,
and other inflammatory diseases. Moreover zalpha3l, antagonists can be useful
in
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63
treatment of myocarditis, atherosclerosis, pelvic inflammatory disease, (PID),
psoriasis,
arthritis, eczema, scleroderma, and other inflammatory diseases.
As such, zalpha3l polypeptide, agonists or its antagonists, have potential
uses in inflammatory diseases such as asthma and arthritis. For example, if
zalpha3l is
proinflammatory, antagonists would be valuable in asthma therapy or other anti
inflammatory therapies where migration of lymphocytes is damaging. In
addition,
zalpha3l can serve other important roles in lung function, for instance,
bronchodilation,
tissue elasticity, recruitment of lymphocytes in lung infection and damage.
Assays to
assess the activity of zalpha3l in lung cells are discussed in Laberge, S. et
al., Am. J.
l0 Respir. Cell Mol. Biol. 17:193-202, 1997; Rumsaeng, V. et al., J. Immunol.,
159:2904-
2910, 1997; and Schluesener, H.J. et al., J. Neurosci. Res. 44:606-611, 1996.
Methods
to determine proinflammatory and antiinflammatory qualities of zalpha3l its
agonists
or its antagonist are known in the art. Moreover, other molecular biological,
immunological, and biochemical techniques known in the art and disclosed
herein can
be used to determine zalpha31 activity and to isolate agonists and
antagonists.
While the Northern blot (Example 2) for zalpha3l shows relatively
ubiquitous distribution of the gene, the electronic northern is very
informative. The
large size of the accessible EST database is such that the incidence of a
gene, such as
zalpha3l, in the data is suggestive of the expression levels found in the
respective
2 0 library (i.e. rare, regulated genes are underrepresented in most libraries
while highly
abundant or inducible genes have high copy number).
The data for zalpha3l suggests a highly inducible gene that is prevalent
in B-cells (tonsils) and those cells of the monocyte/macrophage/dendritic
lineage,
(consistent with a ubiquitous distribution in the "normal", non-induced state)
and
2 5 particularly following treatment with a proinflammatory stimulus such as
PMA, TNF,
or LPS. This conclusion is based on its presence at higher than expected
frequency in
the following tissues: germinal center B cell (tonsil) library, multiple
sclerosis lesions;
stimulated THP-1 cells (pro-monocyte line); and peripheral blood dendritic
cells
(stimulated). The gene was also found in a T-cell line (stimulated) and
peripheral blood
3 0 mononuclear cells (stimulated), fetal liver CD34+ progenitor cells and
cartilage from an
osteoarthritis patient showing a tendency to be associated with inflammatory
events.
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64
Moreover, there was a significant number of ESTs found in libraries of
neural origin, both tissues (normal and diseased) and differentiated cell
lines (neurons).
The disease association shows up with: multiple sclerosis, Huntingtons,
gliosis,
oligoastrocytoma, brain tumor (e.g., mets hypernephroma), and spinal cord
w/lymphoma, of which zalpha3l may be associated with activated immune
responses
associated with those diseases. Other proinflammatory cytokines are produced
by brain
tumors, found in MS lesions, and exhibit other neuropathies (Fontana, A. et
al., J.
Immunol. 132:1837-1844, 1984; Suarez, GA et al., Neurology 46:559-561, 1996)
Moreover, abnormalities in single cytokines can lead to neurological disease,
such as by
inducing immune cell infiltration into neurological tissues (Hanisch, UK et
al., Synapse
24:104-114, 1996; Sugita, Y et al., J. Neuropathol. Exp. Neurol. 58:480-488,
1999); or
both neurological and immune disease (Zhu, J. et al., J. Neurol. Sci. 125:132-
137, 1994;
Zhu, J. et al., J. Neurosci. Res. 54:373-381, 1998).
Moreover, an antagonist of zalpha3l would be predicted to be an anti-
inflammatory agent. Agonists and antagonists may be useful for a broad range
on
neural diseases such as; MS or Huntington's Disease, while a cytokine-
radionuclide (or
similar) may be useful for neural tumors. Moreover, zalpha3l may act
indirectly or in
conjunction with other cytokines in exerting it's effects. For example, an
interferon,
IL-1 or IL-2 could exacerbate a disease in which there is an ir.;~iammation
component.
2 0 As such, zalpha31 antagonists that dissociate or block the combined
effects of various
cytokines, are also useful.
Moreover, there appears to be a "neurological disease cluster" present at
the chromosomal localization site for zalpha3l as discussed below. This
suggests that
zalpha3l polypeptides, polynucleotides or antibodies can be used as
diagnostics for
2 5 neurologic diseases or to determine genetic susceptibility to such
diseases.
The molecules of the present invention can be useful for proliferation of
cardiac tissue cells, such as cardiac myocytes or myoblasts; skeletal myocytes
or
myoblasts and smooth muscle cells; chrondrocytes; endothelial cells;
adipocytes and
osteoblasts in vitro. For example, molecules of the present invention are
useful as
3 0 components of def ned cell culture media, and can be used alone or in
combination with
other cytokines and hormones to replace serum that is commonly used in cell
culture.
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Molecules of the present invention are particularly useful in specifically
promoting the
growth and/or development of myocytes in culture, and may also prove useful in
the
study of cardiac myocyte hyperplasia and regeneration.
The polypeptides, nucleic acids and/or antibodies of the present
5 invention can be used in treatment of disorders associated with myocardial
infarction,
congestive heart failure, hypertrophic cardiomyopathy and dilated
cardiomyopathy.
Molecules of the present invention may also be useful for limiting infarct
size following
a heart attack, aiding in recovery after heart transplantation, promoting
angiogenesis
and wound healing following angioplasty or endarterectomy, to develop coronary
10 collateral circulation, for revascularization in the eye, for complications
related to poor
circulation such o diabetic foot ulcers, for stroke, following coronary
reperfusion using
pharmacologic methods, and other indications where angiogenesis is of benefit.
Molecules of the present invention may be useful for improving cardiac
function, either
by inducing cardiac myocyte neogenesis and/or hyperplasia, by inducing
coronary
15 collateral development, or by inducing remodeling of necrotic myocardial
area. Other
therapeutic uses for the present invention include induction of skeletal
muscle
neogenesis and/or hyperplasia, kidney regeneration and/or for treatment of
systemic
and pulmonary hypertension.
Zalpha3l induced coronary collateral development is measured in
2 0 rabbits, dogs or pigs using models of chronic coronary occlusion (Landau
et al., Amer.
Heart J. 29:924-931, 1995; Sellke et al., Surgery 120(2):182-188, 1996; and
Lazarous et
al., 1996, ibid. Zalpha3l efficacy for treating stroke is tested in vivo in
rats, utilizing
bilateral carotid artery occlusion and measuring histological changes, as well
as maze
performance (Gage et al., Neurobiol. Aging 9:645-655, 1988). Zalpha3l efficacy
in
2 5 hypertension is tested in vivo utilizing spontaneously hypertensive rats
(SHR) for
systemic hypertension (Marche et al., Clin. Exp. Pharmacol. Physiol. Suppl.
1:S114-
116, 1995).
Moreover, based on high expression in thyriod, zalpha3l polypeptide
may exhibit antiviral activity by inhibiting viral replication by specific
signaling via it's
3 0 receptors) on a host cell (e.g. T-cell). Zalpha31 can exhibit immune cell
proliferative
activity (See, example 8), can be assayed for this activity as disclosed
herein, and may
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66
stimulate the imw>une system to fight viral infections. Moreover, zalpha3l may
bind
CD4 or another lymphocyte receptor and exhibit antiviral effects, for example,
against
human immunodeficiency virus (HIV) or human T-cell lymphotropic virus (HTLV).
Alternatively, zalpha3l polypeptide may compete for a viral receptor or co-
receptor to
block viral infection. Zalpha3l may be given parentally to prevent viral
infection or to
reduce ongoing viral replication and re-infection (Gayowski, T. et al.,
Transplantation
64:422-426, 1997). Thus, zalpha3l may be used as an antiviral therapeutic, for
example, for viral leukemias (HTLV), AIDS (HIV), or gastrointestinal viral
infections
caused by, for example, rotavirus, calicivirus (e.g., Norwalk Agent) and
certain strains
l0 of pathogenic adenovirus.
Both zalpha3l modulated direct and indirect inflammation can be
assayed by methods in the art. Foe example see, Hamada, T. et al. J. Exp. Med.
188:539-548, 1998; and Liu, L. et al., J. Immunol. 161:3064-3070, 1998. For
example,
proinflammatory effects of zalpha3l polypeptide can be directly tested in
assays using a
TranswellT"" (Costar), wherein endothelial cells are plated on a semi-
permeable
membrane and zalpha3l polypeptide is present in the lower chamber of the
transwell
and Cr5' or fluorescently-labeled neutrophils (PMNs), lymphocytes, HL60 cells,
K562
cells, or the like are added on to the upper chamber of the transwell.
Migration of the
PMNs and the like to the lower chamber of the transwell in the presence of
zalpha3l
2 0 polypeptide, but not its absence (Negative control), would demonstrate
zalpha31
polypeptide as a direct chemoattractant of the PMNs. Moreover, IL-8 could be
employed in this assay as a positive control. To test zalpha3l as indirect
stimulator of
inflammatory response, a similar method can be employed. For example, an
experiment can be set up as per above where in addition to the presence of
zalpha3l on
2 5 the lower chamber of the transwell, fibroblast or adipocytes are plated
there. In this
way, effects of zalpha3l polypeptide in inducing these cells to secrete
factors that
enhance migration of PMNs, i.e., inflammation, can be measured. The bFGF can
be
used as a positive control for indirect assay. Anti-inflammatory effects of
zalpha3l
polypeptide can also be measured when added on the upper chamber in the
presence of
3 0 PMN's using a similar transwell assay known in the art.
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67
The activity of molecules of the present invention may be measured
using a variety of assays that, for example, measure neogenesis or hyperplasia
(i.e.,
proliferation) of cardiac cells based on the potential effects of
extrathyroidal activity of
zalpha3l. Additional activities likely associated with the polypeptides of the
present
invention include proliferation of endothelial cells, cardiomyocytes,
fibroblasts, skeletal
myocytes directly or indirectly through other growth factors; action as a
chemotaxic
factor for endothelial cells, fibroblasts and/or phagocytic cells; osteogenic
factor; and
factor for expanding mesenchymal stem cell and precursor populations.
Proliferation can be measured using cultured cardiac cells or in vivo by
administering molecules of the present invention to the appropriate animal
model.
Generally, proliferative effects are seen as an increase in cell number, and
may include
inhibition of apoptosis as well as stimulation of mitogenesis. Cultured cells
for use in
these assays include cardiac fibroblasts, cardiac myocytes, skeletal myocytes,
and
human umbilical vein endothelial cells from primary cultures. Suitable
established cell
lines include: NIH 3T3 fibroblasts (ATCC No. CRL-1658), CHH-1 chum heart cells
(ATCC No. CRL-1680), H9c2 rat heart myoblasts (ATCC No. CRL-1446), Shionogi
mammary carcinoma cells (Tanaka et al., Proc. Natl. Acad. Sci. 89:8928-8932,
1992),
and LNCap.FGC adenocarcinoma cells (ATCC No. CRL-1740.) Assays measuring cell
proliferation are well known in the art. For example, assays measuring
proliferation
2 0 include such assays as chemosensitivity to neutral red dye (Cavanaugh et
al.,
Investigational New Drugs 8:347-354, 1990), incorporation of radiolabeled
nucleotides
(Cook et al., Analytical Biochem. 179:1-7, 1989), incorporation of 5-bromo-2'-
deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J.
Immunol.
Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol.
Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall
et al.,
Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833,
1988).
Differentiation is a progressive and dynamic process, beginning with
pluripotent stem cells and ending with terminally differentiated cells.
Pluripotent stem
cells that can regenerate without commitment to a lineage express a set of
3 0 differentiation markers that are lost when commitment to a cell lineage is
made.
Progenitor cells express a set of differentiation markers that may or may not
continue to
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68
be expressed as the cells progress down the cell lineage pathway toward
maturation.
Differentiation markers that are expressed exclusively by mature cells are
usually
functional properties such as cell products, enzymes to produce cell products,
and
receptors. The stage of a cell population's differentiation is monitored by
identification
of markers present in the cell population. Myocytes, osteoblasts, adipocytes,
chrondrocytes, fibroblasts and reticular cells are believed to originate from
a common
mesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988). Markers
for
mesenchymal stem cells have not been well identified (Owen et al., J. of Cell
Sci.
87:731-738, 1987), so identification is usually made at the progenitor and
mature cell
1 o stages. The existence of early stage cardiac myocyte progenitor cells
(often referred to
as cardiac myocyte stem cells) has been speculated, but not demonstrated, in
adult
cardiac tissue. The novel polypeptides of the present invention may be useful
for
studies to isolate mesenchymal stem cells and cardiac myocyte progenitor
cells, both in
vivo and ex vivo.
There is evidence to suggest that factors that stimulate specific cell types
down a pathway towards terminal differentiation or dedifferentiation affect
the entire
cell population originating from a common precursor or stem cell. Thus, the
present
invention includes stimulating or inhibiting the proliferation of myocytes,
smooth
muscle cells, osteoblasts, adipocytes, chrondrocytes and endoti~ulial cells.
Molecules of
2 0 the present invention may, while stimulating proliferation or
differentiation of cardiac
myocytes, inhibit proliferation or differentiation of adipocytes, by virtue of
the affect on
their common precursor/stem cells. Thus molecules of the present invention may
have
use in inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.
Assays measuring differentiation include, for example, measuring cell-
2 5 surface markers associated with stage-specific expression of a tissue,
enzymatic
activity, functional activity or morphological changes (Watt, FASEB. 5:281-
284, 1991;
Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol.
Bioprocesses, 161-171, 1989; all incorporated herein by reference).
In vivo assays for evaluating cardiac neogenesis or hyperplasia include
3 o treating neonatal and mature rats with the molecules of the present
invention. The
animals' cardiac function is measured as heart rate, blood pressure, and
cardiac output
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69
to determine left ventricular function. Post-mortem methods for assessing
cardiac
decline or improvement include: increased or decreased cardiac weight,
nuclei/cytoplasmic volume, and staining of cardiac histology sections to
determine
proliferating cell nuclear antigen (PCNA) vs. cytoplasmic actin levels (Quaini
et al.,
Circulation Res. 75:1050-1063, 1994 and Reiss et al., Proc. Natl. Acad. Sci.
93:8630-
8635, 1996.)
Proteins of the present invention are useful for stimulating proliferation,
activation, differentiation and/or induction or inhibition of specialized cell
function of
cells of the involved homeostasis of the hematopoiesis and immune function. In
particular, zalpha3l polypeptides are useful for stimulating proliferation,
activation,
differentiation, i.r5:-luction or inhibition of specialized cell functions of
cells of the
hematopoietic iineages, including, but not limited to, T cells, B cells, NK
cells,
dendritic cells, monocytes, and macrophages, as well as epithelial cells.
Proliferation
and/or differentiation of hematopoietic cells can be measured in vitro using
cultured
cells or in vivo b5 administering molecules of the claimed invention to the
appropriate
animal model. Assays measuring cell proliferation or differentiation are well
known in
the art. For example, assays measuring proliferation include such assays as
chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New
Drugs
8:347-354, 1990, incorporated herein by reference), incorporation of
radiolabelled
nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989, incorporated
herein by
reference), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of
proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985,
incorporated herein by reference), and use of tetrazolium salts (Mosmann, J.
Immunol.
Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall
et al.,
Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833,
1988; all
incorporated herein by reference). Assays measuring differentiation include,
for
example, measuring cell-surface markers associated with stage-specific
expression of a
tissue, enzymatic activity, functional activity or morphological changes
(Watt, FASEB,
5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim.
Cell Biol.
3 0 Technol. Bioprocesses, 161-171, 1989; all incorporated herein by
reference).
Alternatively, zalpha3l polypeptide itself can serve as an additional cell-
surface or
CA 02374412 2001-11-28
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secreted marker associated with stage-specific expression of a tissue. As
such, direct
measurement of zalpha3l polypeptide, or its loss of expression in a tissue as
it
differentiates, can serve as a marker for differentiation of tissues.
Similarly, direct measurement of zalpha3l polypeptide, or its loss of
5 expression in a tissue can be determined in a tissue or cells as they
undergo tumor
progression. Increases in invasiveness and motility of cells, or the gain or
loss of
expression of zalpha3l in a pre-cancerous or cancerous condition, in
comparison to
normal tissue, can serve as a diagnostic for transformation, invasion and
metastasis in
tumor progression. As such, knowledge of a tumor's stage of progression or
metastasis
10 will aid the physician in choosing the most proper therapy, or
aggressiveness of
treatment, for a given individual cancer patient. Methods of measuring gain
and loss of
expression (of either mRNA or protein) are well known in the art and described
herein
and can be applied to zalpha3l expression. For example, appearance or
disappearance
of polypeptides that regulate cell motility can be used to aid diagnosis and
prognosis of
15 prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev.
17:449-458,
1999). As an effector of cell motility, zalpha3l gain or loss of expression
may serve as
a diagnostic for lymphoid, B-cell, endothelial, hematopoietic and other
cancers.
Moreover, the activity and effect of zalpha3l on tumor progression and
2 o metastasis can be measured in vivo. Several syngeneic mouse models have
been
developed to study the influence of polypeptides, compounds or other
treatments on
tumor progression. In these models, tumor cells passaged in culture are
implanted into
mice of the same strain as the tumor donor. The cells will develop into tumors
having
similar characteristics in the recipient mice, and metastasis will also occur
in some of
2 5 the models. Appropriate tumor models for our studies include the Lewis
lung
carcinoma (ATCC No. CRL-1642) and B 16 melanoma (ATCC No. CRL-6323),
amongst others. These are both commonly used tumor lines, syngeneic to the
C57BL6
mouse, that are readily cultured and manipulated in vitro. Tumors resulting
from
implantation of either of these cell lines are capable of metastasis to the
lung in
3 0 C57BL6 mice. The Lewis lung carcinoma model has recently been used in mice
to
identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-
328,1994).
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71
C57BL6/J mice are treated with an experimental agent either through daily
injection of
recombinant protein, agonist or antagonist or a one time injection of
recombinant
adenovirus. Three days following this treatment, 105 to 106 cells are
implanted under
the dorsal skin. Alternatively, the cells themselves may be infected with
recombinant
adenovirus, such as one expressing zalpha3l, before implantation so that the
protein is
synthesized at the tumor site or intracellularly, rather than systemically.
The mice
normally develop visible tumors within 5 days. The tumors are allowed to grow
for a
period of up to 3 weeks, during which time they may reach a size of 1500 -
1800 mm3
in the control treated group. Tumor size and body weight are carefully
monitored
throughout the experiment. At the time of sacrifice, the tumor is removed and
weighed
along with the lungs and the liver. The lung weight has been shown to
correlate well
with metastatic tumor burden. As an additional measure, lung surface
metastases are
counted. The resected tumor, lungs and liver are prepared for
histopathological
examination, immunohistochemistry, and in situ hybridization, using methods
known
in the art and described herein. The influence of the expressed polypeptide in
question,
e.g., zalpha3l, on the ability of the tumor to recruit vasculature and undergo
metastasis
can thus be assessed. In addition, aside from using adenovirus, the implanted
cells can
be transiently transfected with zalpha3l . Use of stable zalpha3l
transfectants as well as
use of induceable promoters to activate zalpha3l expression in vivo are known
in the art
2 0 and can be used in this system to assess zalpha31 induction of metastasis.
Moreover,
purified zalpha3l or zalpha3l conditioned media can be directly injected in to
this
mouse model, and hence be used in this system. For general reference see,
O'Reilly
MS, et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models of
Liver
Metastasis. Invasion Metastasis 14:349-361, 1995.
The activity of zalpha3l and its derivatives (conjugates) on growth and
dissemination of tumor cells derived from human hematologic malignancies can
also be
measured in vivo in a mouse Xenograft model Several mouse models have been
developed in which human tumor cells are implanted into immunodeficient mice,
collectively referred to as xenograft models. See Caftan, AR and Douglas, E
Leuk.
3 0 Res. 18:513-22, 1994; and Flavell, DJ, Hematological Oncology 14:67-82,
1996. The
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72
characteristics of the disease model vary with the type and quantity of cells
delivered to
the mouse. Typically, the tumor cells will proliferate rapidly and can be
found
circulating in the blood and populating numerous organ systems. Therapeutic
strategies
appropriate for testing in such a model include antibody induced toxicity,
ligand-toxin
conjugates or cell-based therapies. The latter method, commonly referred to
adoptive
immunotherapy, involves treatment of the animal with components of the human
immune system (i.e. lymphocytes, NK cells) and may include ex vivo incubation
of
cells with zalpha3l or other immunomodulatory agents.
Polynucleotides encoding Zalpha3l polypeptides are useful within gene
therapy applications where it is desired to increase or inhibit Zalpha3l
activity. If a
mammal has a mutated or absent Zalpha3l gene, the Zalpha3l gene can be
introduced
into the cells of the mammal. In one embodiment, a gene encoding a Zalpha3l
polypeptide is introduced in vivo in a viral vector. Such vectors include an
attenuated
or defective DNA virus, such as, but not limited to, herpes simplex virus
(HSV),
papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus
(AAV),
and the like. Defective viruses, which entirely or almost entirely lack viral
genes, are
preferred. A defective virus is not infective after introduction into a cell.
Use of
defective viral vectors allows for administration to cells in a specific,
localized area,
2 0 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 (HSV 1 )
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
(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 Zalpha3l 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.
Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120 (1988); Temin et
al., U.S.
3 0 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).
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73
Alternatively, the vector can be introduced by lipofection in vivo using
liposomes.
Synthetic cationic lipids can be used to prepare liposomes for in vivo
transfection of a
gene encoding a marker, Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413
(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
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. Tare,: ted peptides (e.g., hormones or neurotransmitters), proteins
such as
antibodies, or non-peptide molecules can be coupled to liposomes chemically.
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
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.
2 0 Antisense methodology can be used to inhibit Zalpha31 gene
transcription, such as to inhibit cell proliferation in vivo. Polynucleotides
that are
complementary to a segment of a Zalpha3l-encoding polynucleotide (e.g., a
polynucleotide as set froth in SEQ ID NO:1) are designed to bind to Zalpha3l-
encoding
mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides
are
2 5 used to inhibit expression of Zalpha31 polypeptide-encoding genes in cell
culture or in
a subject.
The present invention also provides reagents which will find use in
diagnostic applications. For example, the zalpha3l gene, a probe comprising
zalpha3l
3 0 DNA or RNA or a subsequence thereof can be used to determine if the
zalpha31 gene is
present on human chromosome 10 or if a mutation has occurred. Zalpha3l is
located at
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74
the 1Oq23-q24 region of chromosome 10 (See, Example 3). Detectable chromosomal
aberrations at the zalpha3l 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 108:255-65, 1995).
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 chromosomal region; and 3) cross-
referencing
model organisms, such as mouse, which may aid in determining what function a
particular gene might have.
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
2 0 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
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
2 5 with a gene sequence of interest for the mapping data contained within
these short
genomic landmark STS sequences.
The zalpha3l gene is located at the 1Oq23-q24 region of chromosome
10. Several disease related genes in a cluster in this region that are
associated with
neuropathies, brain cancer, and other neural effects. For example, phosphatase
and
3 0 tensin homolog (PTEN; loss linked to brain tumors; l Oq23.3), Bannayan-
Riley-
Ruvalcaba syndrome (1Oq23), glioma-inactivated leucine rich gene (LGI1;
1Oq24);
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macrocephaly, ( 1 Oq23.3); partial epilepsy ( 1 Oq23.3-q24.1 ); infant-onset
spinocerebellar
ataxia (IOSCA; 1Oq24); urofacial syndrome (Ochoa syndrome) (1Oq23-q24); and
autosomal dominant spastic paraplegia 9 (1Oq23.3-q24.1) all map to this region
of
chromosome 10. In addition, several of these diseases are linked to large
chromosomal
5 rearrangements, such as chromosome loss or loss of heterogeneity in the
1Oq23-q24
region chromosome 10. Moreover, Loss of 1 copy of chromosome 10 is the most
common genetic event in high grade glioma, wherein rearrangement and loss of
at least
some parts of the second copy of the chromosome, particularly in the l Oq23-26
region,
has been demonstrated in approximately 80% of glioblastoma tumors (Bigner, S.
and
1 o Vogelstein, B. Brain Path. 1:12-18, 1990). Moreover, loss of heterogeneity
at l Oq23
occurs in about 70% of glioblastomas and 60% of advanced prostate cancers (Li,
J et al.
Science 275:1943-1946, 1997), as well as other cancers. Moreover, a
significant
percentage (e.g., 7%) of childhood T-cell acute leukemia is accompanied by
translocation within the 1Oq24 locus (Dube, ID et al., Blood 78:2996-3003,
1991). As
15 the zalpha3l gene is also located at the 1Oq23-q24 region zalpha3l,
polynucleotide
probes can be used to detect chromosome 1Oq23-q24 loss or translocation
associated
with human diseases, such as glioblastoma, macrocephaly, and T-cell leukemia,
or
other cancers, neurologic or immune diseases.
Further, zalpha3l polynucleotide probes can be used to detect
2 0 abnormalities or genotypes associated with chromosome 10 trisomy. For
example,
split-hand/foot malformation, type 3 (SHFM3) appears to be a result of a
trisomy at
1Oq24-q25 (Nunes, ME et al., Hum. Molec. Genet. 4:2165-2170, 1995). As the
zalpha3l gene is also located at the 1Oq23-q24 region zalpha3l, polynucleotide
probes
can be used to detect chromosome 1Oq23-q24 gain, or trisomy associated with
such
2 5 human diseases. Moreover, amongst other genetic loci, those for dilated
cardiomyopathy (1Oq21-q23), autoimmune diseases associated with the FAS ligand
which maps to 1Oq24.1, retinitis pigmentosa associated with retinal G-protein
coupled
receptor (1Oq23), Cytochrome P450-2C9 (CYP2C9) (1Oq24) all manifest themselves
in
human disease states as well as map to this region of the human genome. See
the
3 0 Online Mendellian Inheritance of Man (OMIM) gene map, and references
therein, for
this region of chromosome 10 on a publicly available WWW server
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76
(http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?chromosome=1 Oq23-q24).
All of these serve as possible candidate genes for an inheritable disease that
show
linkage to the same chromosomal region as the zalpha3l gene.
Similarly, defects or over expression in the zalpha3l locus itself may
result in a heritable human disease state. For example, zalha3l is located in
a
chromosomal region that is associated with neural and brain implications as
well as
several tumors. As discussed herein a significant number of ESTs for zalpha3l
are
found in libraries of neural origin with disease association with multiple
sclerosis,
Huntington's Disease, gliosis, oligoastrocytoma, brain tumors (e.g., mets
hypernephroma), and spinal cord w/lymphoma. Moreover, proinflammatory
cytokines
are produced by brain tumors, found in MS lesions, and exhibit other
neuropathies.
Molecules of the present invention, such as the polypeptides,
antagonists, agonists, polynucleotides and antibodies of the present invention
would aid
in the detection, diagnosis prevention, and treatment associated with a
zalpha3l genetic
defect.
A diagnostic could assist physicians in determining the type of disease
and appropriate associated therapy, or assistance in genetic counseling. As
such, the
inventive anti-zalpha3l antibodies, polynucleotides, and polypeptides can be
used for
the detection of zalpha31 polypeptide, mRNA or anti-zalpha3 ~ antibodies, thus
serving
2 0 as markers and be directly used for detecting or genetic diseases or
cancers, as
described herein, using methods known in the art and described herein.
Further,
zalpha3l polynucleotide probes can be used to detect abnormalities or
genotypes
associated with chromosome 1Oq23-q24 deletions and translocations associated
with
human diseases, other translocations involved with malignant progression of
tumors or
2 5 other 1 Oq23-q24 mutations, which are expected to be involved in
chromosome
rearrangements in malignancy; or in other cancers, or in spontaneous abortion.
Similarly, zalpha3l polynucleotide probes can be used to detect abnormalities
or
genotypes associated with chromosome 1Oq23-q24 trisomy and chromosome loss
associated with human diseases or spontaneous abortion. Thus, zalpha3l
3 0 polynucleotide probes can be used to detect abnormalities or genotypes
associated with
these defects.
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As discussed above, defects in the zalpha3l gene itself may result in a
heritable human disease state. Molecules of the present invention, such as the
polypeptides, antagonists, agonists, polynucleotides and antibodies of the
present
invention would aid in the detection, diagnosis prevention, and treatment
associated
with a zalpha3l genetic defect. In addition, zalpha3l polynucleotide probes
can be
used to detect allelic differences between diseased or non-diseased
individuals at the
zalpha3l chromosomal locus. As such, the zalpha3l sequences can be used as
diagnostics in forensic DNA profiling.
In general, the diagnostic methods used in genetic linkage analysis, to
detect a genetic abnormality or aberration in a patient, are known in the art.
Most
diagnostic meth~.;ds comprise the steps of (a) obtaining a genetic sample from
a
potentially diseased patient, diseased patient or potential non-diseased
carrier of a
recessive disease allele; (b) producing a first reaction product by incubating
the genetic
sample with a ZSMF 16 polynucleotide probe wherein the polynucleotide will
hybridize
to complementary polynucleotide sequence, such as in RFLP analysis or by
incubating
the genetic sample with sense and antisense primers in a PCR reaction under
appropriate PCR reaction conditions; (iii) Visualizing the first reaction
product by gel
electrophoresis and/or other known method such as visualizing the first
reaction
product with a ZSMF 16 polynucleotide probe wherein the polynucleotide will
2 0 hybridize to the complementary polynucleotide sequence of the first
reaction; and (iv)
comparing the visualized first reaction product to a second control reaction
product of a
genetic sample from wild type patient. A difference between the first reaction
product
and the control reaction product is indicative of a genetic abnormality in the
diseased or
potentially diseased patient, or the presence of a heterozygous recessive
carrier
2 5 phenotype for a non-diseased patient, or the presence of a genetic defect
in a tumor
from a diseased patient, or the presence of a genetic abnormality in a fetus
or pre-
implantation embryo. For example, a difference in restriction fragment
pattern, length
of PCR products, length of repetitive sequences at the zalpha3l genetic locus,
and the
like, are indicative of a genetic abnormality, genetic aberration, or allelic
difference in
3 0 comparison to the normal wild type control. Controls can be from
unaffected family
members, or unrelated individuals, depending on the test and availability of
samples.
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Genetic samples for use within the present invention include genomic DNA,
mRNA,
and cDNA isolated form any tissue or other biological sample from a patient,
such as
but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and
the like.
The polynucleotide probe or primer can be RNA or DNA, and will comprise a
portion
of SEQ ID NO:1, the complement of SEQ ID NO:1, or an RNA equivalent thereof.
Such methods of showing genetic linkage analysis to human disease phenotypes
are
well known in the art. For reference to PCR based methods in diagnostics see
see,
generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humans Press,
Inc.
1991 ), White (ed.), PCR Protocols: Current Methods and Applications (Humans
Press,
Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humans Press, Inc.
1996),
Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humans Press, Inc.
1998),
Lo (ed.), Clinical Applications of PCR (Humans Press, Inc. 1998), and Meltzer
(ed.),
PCR in Bioanalysis (Humans Press, Inc. 1998)).
Mutations associated with the zalpha3l locus can be detected using
nucleic acid molecules of the present invention by employing standard methods
for
direct mutation analysis, such as restriction fragment length polymorphism
analysis,
short tandem repeat analysis employing PCR techniques, amplification-
refractory
mutation system analysis, single-strand conformation polymorphism detection,
RNase
cleavage methods, denaturing gradient gel electrophoresis, fluorescence-
assisted
2 0 mismatch analysis, and other genetic analysis techniques known in the art
(see, for
example, Mathew (ed.), Protocols in Human Molecular Genetics (Humans Press,
Inc.
1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular
Diagnostics
(Human Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases
(Humans Press, Inc. 1996), Landegren (ed.), Laboratory Protocols for Mutation
2 5 Detection (Oxford University Press 1996), Birren et al. (eds.), Genome
Analysis, Vol. 2:
Detecting Genes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al.
(eds.),
Current Protocols in Human Genetics (John Wiley & Sons 1998), and Richards and
Ward, "Molecular Diagnostic Testing," in Principles of Molecular Medicine,
pages 83-
88 (Humans Press, Inc. 1998)). Direct analysis of an zalpha3l gene for a
mutation can
3 0 be performed using a subject's genomic DNA. Methods for amplifying genomic
DNA,
obtained for example from peripheral blood lymphocytes, are well-known to
those of
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79
skill in the art (see, for example, Dracopoli et al. (eds.), Current Protocols
in Human
Genetics, at pages 7.1.6 to 7.1.7 (John Wiley & Sons 1998)).
Mice engineered to express the zalpha3l gene, referred to as "transgenic
mice," and mice that exhibit a complete absence of zalpha3l gene function,
referred to
as "knockout mice," may also be generated (Snouwaert et al., Science 257:1083,
1992;
Lowell et al., Nature 366:740-42, 1993; Capecchi, M.R., Science 244: 1288-
1292,
1989; Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,
transgenic mice that over-express zalpha3l, either ubiquitously or under a
tissue-
1 o specific or tissue-restricted promoter can be used to ask whether over-
expression causes
a phenotype. For example, over-expression of a wild-type zalpha3l polypeptide,
polypeptide fragment or a mutant thereof may alter normal cellular processes,
resulting
in a phenotype that identifies a tissue in which zalpha3l expression is
functionally
relevant and may indicate a therapeutic target for the zalpha31, its agonists
or
antagonists. For example, a preferred transgenic mouse to engineer is one that
over-
expresses the zalpha3l mature polypeptide (approximately amino acid residue 20
(Asp)
to residue 142 (Arg) of SEQ ID N0:2). Moreover, such over-expression may
result in
a phenotype that shows similarity with human diseases. Similarly, knockout
zalpha3l
mice can be used to determine where zalpha3l is absolutely required in vivo.
The
2 0 phenotype of knockout mice is predictive of the in vivo effects of that a
zalpha31
antagonist, such as those described herein, may have. The human zalpha3l cDNA
can
be used to isolate murine zalpha3l mRNA, cDNA and genomic DNA, which are
subsequently used to generate knockout mice. These mice may be employed to
study
the zalpha3l gene and the protein encoded thereby in an in vivo system, and
can be
2 5 used as in vivo models for corresponding human diseases. Moreover,
transgenic mice
expression of zalpha3l antisense polynucleotides or ribozymes directed against
zalpha3l, described herein, can be used analogously to transgenic mice
described
above.
3 0 For pharmaceutical use, the proteins of the present invention are
formulated for parenteral, particularly intravenous or subcutaneous, delivery
according
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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 Zalpha3l protein in combination with a
pharmaceutically
acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or
the like.
5 Formulations may further include one or more excipients, preservatives,
solubilizers,
buffering agents, albumin to prevent protein loss on vial surfaces, etc.
Methods of
formulation are well known in the art and are disclosed, for example, in
Remington:
The Science and Practice of Pharmacy, Gennaro, ed.,(Mack Publishing Co.,
Easton,
PA, 19th ed., 1995). Therapeutic doses will generally be in the range of 0.1
to 100
10 ~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
15 may be used in chronic treatment, over several months or years.
The invention is further illustrated by the following non-limiting
examples.
2 0 Example 1
Identification of zalpha3l
Using an EST Sequence to Obtain Full-length zalpha3l
Scanning of translated DNA databases of predicted full-length
assemblies using a signal sequence and alpha helices as a query resulted in
25 identification of an assembly having a 5' EST sequence (EST362610; Image
Consortium).
Confirmation of the EST sequence was made by sequence analyses of
the cDNA from which the EST originated. This cDNA was contained in a plasmid,
and
was sequenced using the following primers to generate complete double stranded
3 o sequence of this clone: ZC694 (SEQ ID N0:4), ZC7625 (SEQ ID NO:S), ZC22487
(SEQ ID N0:6), ZC22488 (SEQ ID N0:7), ZC22249 (SEQ ID N0:8). The
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EST362610 sequence (SEQ ID NO:1) encoded a full-length protein designated
zalpha3l, as described herein and in SEQ ID N0:2.
Example 2
Tissue Distribution
Northerns were performed using Human Multiple Tissue Blots(MTN1,
MTN2 and MTN3) and Master Dot blot (Clontech, Palo Alto, CA). An cDNA probe
was prepared using PCR. Oligo nucleotides ZC22,230 (SEQ ID N0:9) and ZC22,249
(SEQ ID N0:8), designed off the EST INC515639H2 (Incyte Pharmaceuticals, Palo
Alto, CA), were used as primers. The template was human brain Marathon cDNA
(Clontech) mad.~~ in house using manufacturer's instructions. PCR thermocycler
conditions were as follows: one cycle at 94°C for 1.5 min.; 35 cycles
at 94°C for 10
sec., 62°C for 20 sec., 72°C for 30 sec.; one cycle at
72°C for 10 min.; followed by a
4°C hold. The approximately SOObp by probe was purified using a Gel
Extraction Kit
(Qiagen, Chatsworth, CA) according to manufacturer's instructions. The probe
was
radioactively labeled using a Rediprime II DNA labeling kit (Amersham,
Arlington
Heights, IL) according to the manufacturer's specifications. The probe was
purified
using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA).
EXPRESSHYB (Clontech) solution was used for prehybridization and as a
hybridizing
2 0 solution for the Northern blots. Hybridization took place overnight at
55°C, using 1.5 x
10-6 cpm/ml labeled probe. The blots were then washed in 2XSSC and 0.1% SDS at
room temperature, then with 2XSSC and 0.1% SDS at 65°C, followed by a
wash in
O.1X SSC and 0.1% SDS at 65°C. Two transcript sizes were observed on
the blots at
-~-1.35kb and ~2kb in all tissues with strongest expression in heart, thyroid,
spinal cord,
2 5 adrenal gland, brain, and testis.
Dot Blots were also performed using Human RNA Master Blots~'~"~'
(Clontech). The methods and conditions for the Dot Blots are the same as for
the
Multiple Tissue Blots disclosed above. The Dot blots showed signals in all
tissues with
strongest in brain, liver and heart.
3 0 Th.; EST electronic northern data for zalpha31 suggests that it is a
highly
inducible gene that is prevalent in B-cells (tonsils) and those cells of the
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monocyte/macrophage/dendritic lineage, (consistent with a ubiquitous
distribution in
the "normal", non-induced state) and particularly following treatment with a
proinflammatory stimulus such as PMA, TNF, or LPS. This conclusion is based on
its
presence at higher than expected frequency in the following tissues: germinal
center B
cell (tonsil) library, multiple sclerosis lesions; stimulated THP-1 cells (pro-
monocyte
line); and peripheral blood dendritic cells (stimulated). The gene was also
found in a T-
cell line (stimulated) and peripheral blood mononuclear cell s(stimulated),
fetal liver
CD34+ progenitor cells and cartilage from an osteoarthritis patient. Moreover,
there
was a significant number of ESTs found in libraries of neural origin, both
tissues
(normal and diseased) and differentiated cell lines (neurons).
Example 3
Chromosomal Assignment and Placement of Zalpha31
Zalpha3l was mapped to chromosome 10 using the commercially
available "GeneBridge 4 Radiation Hybrid (RH) Mapping Panel"(Research
Genetics,
Inc., Huntsville, AL). The GeneBridge 4 RH panel contains DNA from each of 93
radiation hybrid clones, plus two control DNAs (the HFL donor and the A23
recipient).
A publicly available WWW server (http://www-genome.wi.mit.edu/cgi-
bin/contig/rhmapper.pl) allows mapping relative to the Whitehead Institute/MIT
Center
2 0 for Genome Research's radiation hybrid map of the human genome (the
"WICGR"
radiation hybrid map) which was constructed with the GeneBridge 4 RH panel.
For the mapping of Zalpha3l with the GeneBridge 4 RH panel, 20 ~l
reactions were set up in a 96-well microtiter plate compatible for PCR
(Stratagene, La
Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene).
Each of
2 5 the 95 PCR reactions consisted of 2 ql 1 OX KlenTaq PCR reaction buffer
(CLONTECH
Laboratories, Inc., Palo Alto, CA), 1.6 ~1 dNTPs mix (2.5 mM each, PERKIN-
ELMER,
Foster City, CA), 1 pl sense primer, ZC 23,469 (SEQ ID NO:10), 1 ~1 antisense
primer,
ZC 23,470 (SEQ ID NO:11 ), 2 g l "RediLoad" (Research Genetics, Inc.,
Huntsville, AL),
0.4 ~l SOX Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25
ng of
3 0 DNA from an individual hybrid clone or control and distilled water for a
total volume of
~ 1. The reactions were overlaid with an equal amount of mineral oil and
sealed. The
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PCR cycler conditions were as follows: an initial 1 cycle 5 minute
denaturation at 94oC,
35 cycles of a 45 seconds denaturation at 94oC, 45 seconds annealing at 58oC
and 1
minute and 15 seconds extension at 72oC, followed by a final 1 cycle extension
of 7
minutes at 72oC. The reactions were separated by electrophoresis on a 2%
agarose gel
(EM Science, Gibbstown, NJ) and visualized by staining with ethidium bromide.
The results showed that Zalpha3l maps 0.90 cR 3000 distal from the
framework marker WI-8488 on the chromosome 10 WICGR radiation hybrid map. The
use of surrounding genes/markers positions Zalpha3l in the 1Oq23-q24
chromosomal
region.
Example 4
Generation of untag~ed zalpha3l Recombinant Adenovirus
A. Generation of expression vector construct for Adenovirus expression
The protein coding region of human zalpha3l was amplified by PCR
using primers that added FseI and AscI restriction sties at the 5' and 3'
termini
respectively. PCR primers ZC23457 (SEQ ID N0:12) and ZC23458 (SEQ ID N0:13)
were used with template pT7T3D plasmid containing the full-length human
zalpha3l
cDNA in a PCR reaction as follows: one cycle at 95°C for 5 minutes;
followed by 15
cycles at 95°C for 0.5 min., 58°C for 0.5 min., and 72°C
for 0.5 min.; followed by 72°C
2 0 for 7 min.; followed by a 4°C soak. The PCR reaction product was
loaded onto a 1.2
(low melt) SeaPlaque GTG (FMC, Rockland, ME) gel in TAE buffer. The zalpha3l
PCR product was excised from the gel, melted at 65°C, phenol extracted
twice and then
ethanol precipitated. The PCR product was then digested with FseI-AscI,
phenol/chloroform extracted, EtOH precipitated, and rehydrated in TE
(Tris/EDTA pH
8). The 429bp :aalpha3l fragment was then ligated into the FseI-AscI sites of
a
modified pAdTrack CMV (He, T-C. et al., PNAS 95:2509-2514, 1998). This
construct
also contains the GFP marker gene. The CMV promoter driving GFP expression was
replaced with the SV40 promoter and the SV40 polyadenylation signal was
replaced
with the human growth hormone polyadenylation signal. In addition, the native
3 o polylinker was replaced with FseI, EcoRV, and AscI sites. This modified
form of
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pAdTrack CMV was named pZyTrack. Ligation was performed using the Fast-LinkTM
DNA ligation and screening kit (Epicentre Technologies, Madison, WI). Clones
containing the zalpha3l cDNA were identified by standard mini prep procedures.
In
order to linearize the plasmid, approximately 5 p,g of the pZyTrack zalpha3l
plasmid
was digested with PmeI. Approximately 1 ~g of the linearized plasmid was
cotransformed with 200ng of supercoiled pAdEasy (He et al., su ra. into BJ5183
cells.
The co-transformation was done using a Bio-Rad Gene Pulser at 2.5kV, 200 ohms
and
25mFa. The entire co-transformation was plated on 4 LB plates containing 25
pg/ml
kanamycin. The smallest colonies were picked and expanded in LB/kanamycin and
recombinant adenovirus DNA identified by standard DNA miniprep procedures.
Digestion of the recombinant adenovirus DNA with FseI-AscI confirmed the
presence
of zalpha31. The recombinant adenovirus miniprep DNA was transformed into DH 1
OB
competent cells and DNA prepared using a Qiagen maxi prep kit as per kit
instructions.
B. Transfection of 293a Cells with Recombinant DNA
Approximately 5 ~g of recombinant adenoviral DNA was digested with
PacI enzyme (New England Biolabs) for 3 hours at 37°C in a reaction
volume of 100 p.l
containing 20-30t ~ of PacI. The digested DNA was extracted twice with an
equal
volume of phenol/chloroform and precipitated with ethanol. ~: he DNA pellet
was
resuspended in 5 pl distilled water. A T25 flask of QBI-293A cells (Quantum
2 0 Biotechnologies, Inc. Montreal, Qc. Canada), inoculated the day before and
grown to
60-70% confluence, were transfected with the PacI digested DNA. The PacI-
digested
DNA was diluted up to a total volume of 50 ~1 with sterile HBS (150mM NaCI,
20mM
HEPES). In a separate tube, 25 ~l DOTAP (Boehringer Mannheim, lmg/ml) was
diluted to a total volume of 100 pl with HBS. The DNA was added to the DOTAP,
2 5 mixed gently by pipeting up and down, and left at room temperature for 15
minutes.
The media was removed from the 293A cells and washed with 5 ml serum-free
MEMalpha (Gibco BRL) containing 1mM Sodium Pyruvate (GibcoBRL), 0.1 mM
MEM non-essential amino acids (GibcoBRL) and 25mM HEPES buffer (GibcoBRL).
5 ml of serum-free MEM was added to the 293A cells and held at 37°C.
The DNA/lipid
3 0 mixture was added drop-wise to the T25 flask of 293A cells, mixed gently
and
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incubated at 37°C for 4 hours. After 4 hours the media containing the
DNA/lipid
mixture was aspirated off and replaced with 5 ml complete MEM containing 5%
fetal
bovine serum. The transfected cells were monitored for Green Fluorescent
Protein
(GFP) expression and formation of foci, i.e., viral plaques.
5 Seven days after transfection of 293A cells with the recombinant
adenoviral DNA, the cells expressed the GFP protein and started to form foci.
These
foci are viral "plaques" and the crude viral lysate was collected by using a
cell scraper
to collect all of the 293A cells. The lysate was transferred to a SOmI conical
tube. To
release most of the virus particles from the cells, three freeze/thaw cycles
were done in
10 a dry ice/ethanol bath and a 37° waterbath.
C. Amplificat<~~~_: of Recombinant Adenovirus (rAdV)
The crude lysate was amplified (Primary (1°) amplification) to
obtain a
working "stock" of zalpha3l rAdV lysate. Ten lOcm plates of nearly confluent
(80-
90%) 293A cells were set up 20 hours previously, 200m1 of crude rAdV lysate
added to
15 each lOcm plate and monitored for 48 to 72 hours looking for CPE under the
white
light microscope and expression of GFP under the fluorescent microscope. When
all of
the 293A cells showed CPE (Cytopathic Effect) this 1 ° stock lysate was
collected and
freeze/thaw cycles performed as described under Crude rAdV Lysate.
Secondary (2°) Amplification of zalpha3l rAdV was obtained as
2 0 follows: Twenty l5cm tissue culture dishes of 293A cells were prepared so
that the
cells were 80-90% confluent. All but 20 mls of 5%MEM media was removed and
each
dish was inoculated with 300-SOOmI 1° amplified rAdv lysate. After 48
hours the
293A cells were lysed from virus production and this lysate was collected into
250m1
polypropylene centrifuge bottles and the rAdV purified.
2 5 D. Purification of recombinant Adenovirus
NP-40 detergent was added to a final concentration of 0.5% to the
bottles of crude lysate in order to lyse all cells. Bottles were placed on a
rotating
platform for 10 min. agitating as fast as possible without the bottles falling
over. The
debris was pelleted by centrifugation at 20,000 X G for 15 minutes. The
supernatant
3 0 was transferred to 250m1 polycarbonate centrifuge bottles and 0.5 volumes
of
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20%PEG8000/2.5M NaCI solution added. The bottles were shaken overnight on ice.
The bottles were : ~ntrifuged at 20,000 X G for 15 minutes and supernatant
discarded
into a bleach solution. The white precipitate in two vertical lines along the
wall of the
bottle on either side of the spin mark is the precipitated virus/PEG. Using a
sterile cell
scraper, the precipitate from 2 bottles was resuspended in 2.5 ml PBS. The
virus
solution was placed in 2 ml microcentrifuge tubes and centrifuged at 14,000 X
G in the
microfuge for 10 minutes to remove any additional cell debris. The supernatant
from
the 2m1 microcentrifuge tubes was transferred into a l5ml polypropylene
snapcap tube
and adjusted to a density of 1.34g/ml with cesium chloride (CsCI). The volume
of the
virus solution was estimated and 0.55 g/ml of CsCI added. The CsCI was
dissolved
and 1 ml of this solution weighed 1.34 g. The solution was transferred
polycarbonate
thick-walled centrifuge tubes 3.2m1 (Beckman No. 362305) and spin at 80,000
rpm
(348,000 X G) for 3-4 hours at 25°C in a Beckman Optima TLX
microultracentrifuge
with the TLA-100.4 rotor. The virus formed a white band. Using wide-bore
pipette
tips, the virus band was collected.
The virus from the gradient has a large amount of CsCI which must be
removed before it can be used on cells. Pharmacia PD-10 columns prepacked with
Sephadex G-25M (Pharmacia) were used to desalt the virus preparation. The
column
was equilibrated with 20 ml of PBS. The virus was loaded and allowed to run
into the
column. 5 ml of PBS was added to the column and fractions of 8-10 drops
collected.
The optical densities of 1:50 dilutions of each fraction was determined at
260nm on a
spectrophotometer. A clear absorbance peak was present between fractions 7-12.
These fractions were pooled and the optical density (OD) of a 1:25 dilution
determined.
A formula is used to convert OD into virus concentration: (OD at
260nm)(25)(1.1 x
2 5 1012) = virions/ml. The OD of a 1:25 dilution of the zalpha31 rAdV was
0.059,
giving a virus concentration of 3.0 X 1012 virions/ml.
To store the virus, glycerol was added to the purified virus to a final
concentration of 15%, mixed gently but effectively, and stored in aliquots at -
80°C.
E. Tissue Culture Infectious Dose at 50% CPE (TCID 50) Viral Titration Assay
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A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Qc.
Canada) was followed to measure recombinant virus infectivity. Briefly, two 96-
well
tissue culture plates were seeded with 1X104 293A cells per well in MEM
containing
2% fetal bovine serum for each recombinant virus to be assayed. After 24 hours
10-
fold dilutions of each virus from 1X10 2 to 1X10 14 were made in MEM
containing
2% fetal bovine serum. 100 ~1 of each dilution was placed in each of 20 wells.
After 5
days at 37°C, wells were read either positive or negative for
Cytopathic Effect (CPE)
and a value for "Plaque Forming Units/ml" (PFU) is calculated.
TCID50 formulation used was as per Quantum Biotechnologies, Inc.,
above. The titer (T) is determined from a plate where virus used is diluted
from 10 2 to
10 14, and read 5 days after the infection. At each dilution a ratio (R) of
positive wells
for CPE per the total number of wells is determined.
To Calculate titer of the undiluted virus sample: the factor, "F" = 1+d(S-
0.5); where "S" is the sum of the ratios (R); and "d" is LoglO of the dilution
series, for
example, "d" is equal to 1 for a ten-fold dilution series. The titer of the
undiluted
sample is T = 10(1+F) = TCID50/ml. To convert TCID50/ml to pfu/ml, 0.7 is
subtracted from the exponent in the calculation for titer (T).
The zalpha31 adenovirus had a titer of 1.3 X 1010 pfu/ml.
2 0 Example 5
Construct for generating human zalpha3l Transgenic Mice
A. Construct for expressing human zalpha3l from the MT-1 promoter
Approximately 10 °g Zytrack vector containing the sequence
confirmed
zalpha3l coding region (Example 4) was digested with FseI and AscI. The vector
was
2 5 then ethanol precipitated and the pellet was resuspended in TE. The
released 429 by
zalpha3l fragment was isolated by running the digested vector on a 1.2%
SeaPlaque gel
and exicising the fragment. DNA was purified using the QiaQuick (Qiagen) gel
extraction kit.
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as
The zalpha3l fragment was then ligated into our standard transgenic
vector pTGl2-8, which was previously digested with FseI and AscI. The pTGl2-8
plasmid, designed for expression of a gene of interest in transgenic mice,
contains an
expression cassette flanked by 10 kb of MT-1 5' DNA and 7 kb of MT-1 3' DNA.
The
expression cassette comprises the MT-1 promoter, the rat insulin II intron, a
polylinker
for the insertion of the desired clone, and the human growth hormone poly A
sequence.
About one microliter of the ligation reaction was electroporated into
DH10B ElectroMax~ competent cells (GIBCO BRL, Gaithersburg, MD) according to
manufacturer's direction, plated onto LB plates containing 100 ~g/ml
ampicillin, and
incubated overnight at 37°C. Colonies were picked and grown in LB media
containing
100 ug/ml ampicillin. Miniprep DNA was prepared from the picked clones and
screened for the zalpha3l insert by restriction digestion with EcoRI, and
subsequent
agarose gel electrophoresis. Maxipreps of the correct pTGl2-8 zalpha3l
construct
were performed.
A SaII fragment containing with 5' and 3' flanking sequences, the MT
promoter, the rat insulin II intron, zalpha31 cDNA and the human growth
hormone poly
A sequence was prepared using standard techniques described herein, and used
for
microinjection into fertilized marine oocytes. Microinjection and production
of
transgenic mice were produced as described in Hogan, B. et al. ,~%lanipulating
the Mouse
2 0 Embryo, 2"d ed., Cold Spring Harbor Laboratory Press, NY, 1994.
B. Construct for expressing human zalpha3l from the lymphoid-specific E~,LCK
promoter
The zalpha3l DNA fragment digested with FseI and AscI (Example SA)
is cloned into pKF051, a lymphoid-specific transgenic vector, previously
digested with
FseI and AscI as described above. The pKF051 transgenic vector is derived from
p1026X (Iritani, B.M., et al., EMBO J. 16:7019-31, 1997) and contains the T
cell-
specific lck proximal promoter, the B/T cell-specific immunoglobulin ~ heavy
chain
enhancer, a polylinker for the insertion of the desired clone, and a mutated
hGH gene
3 0 that encodes an inactive growth hormone protein (providing 3' introns and
a
polyadenylation signal). About one microliter of each ligation reaction is
CA 02374412 2001-11-28
WO 00/73458 PCT/US00/14795
89
electroporated, plated, clones picked and screened for the human zalpha3l
insert by
restriction digesti«n as described above. A correct clone of pKF051-zalpha3l
is
verified by sequencing, and a maxiprep of this clone is performed. A NotI
fragment,
containing the lck proximal promoter and immunoglobulin ~ enhancer (Eq,LCK),
zalpha3l cDNA, and the mutated hGH gene is prepared to be used for
microinjection
into fertilized murine oocytes.
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. Accnr:~lingly, the invention is not limited except as by the
appended claims.
