Note: Descriptions are shown in the official language in which they were submitted.
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ZSIG67: A MEMBER OF THE HUMAN SECRETIN-GLUCAGON-VIP
s HORMONE FAMILY
TECHNICAL FIELD
The present invention relates generally to a new polypeptide hormone
1 o having diagnostic and therapeutic uses. In particular, the present
invention relates to a
novel hormone, designated "Zsig67," and to nucleic acid molecules encoding
Zsig67.
BACKGROUND OF THE INVENTION
Cellular differentiation of multicellular organisms is controlled by
15 hormones and polypeptide growth factors. These diffusable molecules allow
cells to
communicate with each other and act in concert to form tissues and organs, and
to
repair and regenerate damaged tissue. Examples of hormones and growth factors
include the steroid hormones, parathyroid hormone, follicle stimulating
hormone, the
interferons, the interleukins, platelet derived growth factor, epidermal
growth factor,
2o and granulocyte-macrophage colony stimulating factor, among others.
Hormones and growth factors influence cellular metabolism by binding
to receptor proteins. Certain receptors are integral membrane proteins that
bind with
the hormone or growth factor outside the cell, and that are linked to
signaling pathways
within the cell, such as second messenger systems. Other classes of receptors
are
25 soluble intracellular molecules.
Of special interest, are pancreatic hormones that are members of the
secretin-glucagon-vasoactive intestinal peptide (VIP) family, which are used
for
therapy and diagnosis (Geoghegan and Pappas, Ann. Surg. 225:145 (1997); Ulrich
et
al., Gastroenterol. 114:382 (1998); Walsh, "Hormones of Therapeutic Interest,"
in
3o Biopharmaceuticals: Biochemistry and Biotechnology, pages 256-292 (John
Wiley &
Sons 1998)). Secretin, for example, is used as an adjunct to the radiologic
assessment
of inflammatory diseases of the pancreas, to diagnose Zollinger-Ellison
syndrome, and
to treat intrahepatic cholestasis. Glucagon is used in the emergency treatment
of
insulin-induced hypoglycemia in diabetic patients, and to reduce pylrospasm
and
35 duodenal contractility during upper gastrointestinal endoscopy.
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2
Although new uses of known members of this family may be
discovered, a need exists for the provision of new polypeptidic hormones for
biopharmaceuticals.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a novel polypeptide, designated
"Zsig67." The present invention also provides variant Zsig67 polypeptides and
Zsig67
fusion proteins, as well as nucleic acid molecules encoding such polypeptides
and
proteins, and methods for using these nucleic acid molecules and amino acid
sequences.
to
DESCRIPTION OF THE INVENTION
1. Overview
The present invention provides nucleic acid molecules that encode a new
human polypeptide that is a member of the secretin-glucagon-VIP hormone
family. An
illustrative nucleic acid molecule containing a sequence that encodes the
"Zsig67"
polypeptide has the nucleotide sequence of SEQ ID NO:l. The encoded
polypeptide
has the following amino acid sequence: MRTPNTSFLV LASQPLLVLI SLSALILASY
SSPLLTRVSL ETVRTKEDGR HNDFNKIKDK DASRAGRERG YRNFLFHFHL
SLFPHNSPNS ISKGFKFHVS YRKK (SEQ ID N0:2). Thus, the ZSig67 gene
2o described herein encodes a polypeptide of 104 amino acids.
The Zsig67 protein has a number of structural features, including a
signal sequence located at amino acid residues 1 to 28 of SEQ ID N0:2. The
protein
also contains the following N-terminal cleavage site that is a motif of member
of the
secretin-glucagon-VIP hormone family: RH[SAN]D, wherein acceptable amino acids
for a given position are indicated within square brackets (amino acid residues
50 to 53
of SEQ ID N0:2). Cleavage at this site would produce a polypeptide having
Asn52 at
the N-terminus. There is also a potential di-basic peptide cleavage site at
Lys104.
Cleavage at this site would remove amino acid residues 102 to 104, and Tyr101
would
be converted to an amide. Accordingly, Zsig67 can occur with an amidated C-
terminus. Comparison with other members of the secretin-glucagon-VIP peptide
hormone family indicates that a polypeptide consisting of amino acid residues
52 to 85
of SEQ ID N0:2 can effectively bind to the cognate Zsig67 receptor.
Additional Zsig67 peptides can be generated by cleavage of a variety of
monobasic sites or by a furin-like cleavage. Cleavage products include the
following
amino acid sequences of SEQ ID N0:2: amino acid residues 29 to 45, amino acid
residues 29 to 48, amino acid residues 29 to 48-amidated, amino acid residues
29 to 50,
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3
amino acid residues 29 to 55, amino acid residues 29 to 63, amino acid
residues 29 to
68, amino acid residues 51 to 63, amino acid residues 51 to 65-amidiated,
amino acid
residues 51 to 67, amino acid residues 51 to 92, amino acid residues 51 to
104, amino
acid residues 68 to 92, amino acid residues 68 to 101, amino acid residues 68
to 104,
amino acid residues 70 to 101, amino acid residues 73 to 92, amino acid
residues 73 to
101, and amino acid residues 73 to 104.
The Zsig67 gene resides in chromosome 8q24. As discussed below, this
region is associated with numerous diseases and disorders.
Studies were performed in which Zsig67 nucleic acid molecules were
1o amplified from various human tissues using the polymerase chain reaction.
The results
indicate that Zsig67 mRNA is present at high levels in the following tissues:
pituitary,
prostate, adrenal gland, uterus, liver, thyroid, and lymphoid node. In
addition, Zsig67
mRNA appeared to be present, although at a lower level, in testis, skeletal
muscle, and
spleen, while kidney, placenta, pancreas, and spinal cord seemed to contain
even lower
levels of Zsig67 mRNA. Accordingly, nucleic acid molecules that encode Zsig67
can
be used to differentiate between various tissues. In addition, the observation
that
Zsig67 is produced in numerous tissues, including pituitary, liver, prostate,
and thyroid,
indicates that the protein may be useful as a releasing hormone to regulate
metabolic
homeostasis, or contractility, in a variety of tissues.
As described herein, the present invention provides isolated polypeptides
having an amino acid sequence that is at least 70%, at least 80%, or at least
90%
identical to the amino acid sequence of SEQ ID N0:2, wherein such isolated
polypeptides can specifically bind with an antibody that specifically binds
with a
polypeptide consisting of the amino acid sequence of SEQ ID N0:2. An
illustrative
polypeptide is a polypeptide that comprises the amino acid sequence of SEQ ID
N0:2.
Additional exemplary polypeptides include the following: (a) a
polypeptide comprising the amino acid sequence of amino acid residues 29 to 45
of
SEQ ID N0:2, (b) a polypeptide comprising the amino acid sequence of amino
acid
residues S 1 to 63 of SEQ ID N0:2, (c) a polypeptide comprising the amino acid
sequence of amino acid residues 52 to 101 of SEQ ID N0:2, (d) a polypeptide
comprising the amino acid sequence of amino acid residues 68 to 92 of SEQ ID
N0:2,
(e) a polypeptide comprising the amino acid sequence of amino acid residues 70
to 101
of SEQ ID N0:2, and (f) a polypeptide comprising the amino acid sequence of
amino
acid residues 73 to 92 of SEQ ID N0:2.
Illustrative polypeptides also include: (a) a polypeptide comprising the
amino acid sequence of amino acid residues 29 to 48 of SEQ ID N0:2, (b) a
polypeptide comprising the amino acid sequence of amino acid residues 29 to 48
of
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SEQ ID N0:2, wherein the last amino acid residue of the recited sequence is
amidated,
(c) a polypeptide comprising the amino acid sequence of amino acid residues 29
to 50
of SEQ ID N0:2, (d) a polypeptide comprising the amino acid sequence of amino
acid
residues 29 to 55 of SEQ ID N0:2, (e) a polypeptide comprising the amino acid
sequence of amino acid residues 29 to 63 of SEQ ID N0:2, (f) a polypeptide
comprising the amino acid sequence of amino acid residues 29 to 68 of SEQ ID
N0:2,
(g) a polypeptide comprising the amino acid sequence of amino acid residues 29
to 101
of SEQ ID N0:2, wherein the last amino acid residue of the recited sequence is
amidated, and (h) a polypeptide comprising the amino acid sequence of amino
acid
1o residues 29 to 104 of SEQ ID N0:2.
Further exemplary polypeptides include: (a) a polypeptide comprising
the amino acid sequence of amino acid residues S 1 to 65 of SEQ ID N0:2,
wherein the
last amino acid residue of the recited sequence is amidated, (b) a polypeptide
comprising the amino acid sequence of amino acid residues 51 to 67 of SEQ ID
N0:2,
(c) a polypeptide comprising the amino acid sequence of amino acid residues 51
to 92
of SEQ ID N0:2, (d) a polypeptide comprising the amino acid sequence of amino
acid
residues 51 to 104 of SEQ ID N0:2, (e) a polypeptide comprising the amino acid
sequence of amino acid residues 52 to 101 of SEQ ID N0:2, wherein the last
amino
acid residue of the recited sequence is amidated, (fJ a polypeptide comprising
the amino
2o acid sequence of amino acid residues 52 to 104 of SEQ ID N0:2, (g) a
polypeptide
comprising the amino acid sequence of amino acid residues 68 to 101 of SEQ ID
N0:2,
(h) a polypeptide comprising the amino acid sequence of amino acid residues 68
to 104
of SEQ ID N0:2, (i) a polypeptide comprising the amino acid sequence of amino
acid
residues 73 to 101 of SEQ ID N0:2, and (j) a polypeptide comprising the amino
acid
sequence of amino acid residues 73 to 104 of SEQ ID N0:2.
The present invention also provides isolated polypeptides comprising an
amino acid sequence that is at least 70%, at least 80%, or at least 90%
identical to the
amino acid sequence of amino acid residues 52 to 85 of SEQ ID N0:2, wherein
such
isolated polypeptides can specifically bind with an antibody that specifically
binds with
a polypeptide consisting of amino acid residues 52 to 85 of SEQ ID N0:2.
The present invention also includes isolated polypeptides comprising at
least 15, or at least 30, contiguous amino acid residues of an amino acid
sequence of
SEQ ID N0:2 selected from the group consisting of: (a) amino acid residues
amino acid
residues 29 to 104, (b) amino acid residues 52 to 104, (c) amino acid residues
52 to 85,
(d) amino acid residues 68 to 104, and (e) amino acid residues 73 to 104.
Illustrative
polypeptides include polypeptides that either comprise, or consist of, amino
acid
residues (a) to (e).
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The present invention further provides antibodies and antibody
fragments that specifically bind with such polypeptides. Exemplary antibodies
include
polyclonal antibodies, marine monoclonal antibodies, humanized antibodies
derived
from marine monoclonal antibodies, and human monoclonal antibodies.
Illustrative
5 antibody fragments include F(ab')z, F(ab)z, Fab', Fab, Fv, scFv, and minimal
recognition
units. The present invention further includes compositions comprising a
carrier and a
peptide, polypeptide, or antibody described herein.
The present invention also provides isolated nucleic acid molecules that
encode a Zsig67 polypeptide, wherein the nucleic acid molecule is selected
from the
1 o group consisting of (a) a nucleic acid molecule having the nucleotide
sequence of SEQ
ID N0:3, (b) a nucleic acid molecule encoding the amino acid sequence of SEQ
ID
N0:2, and (c) a nucleic acid molecule that remains hybridized following
stringent wash
conditions to a nucleic acid molecule having the nucleotide sequence of
nucleotides
111-422 of SEQ ID NO:1, or the complement of nucleotides 111-422 of SEQ ID
NO:1.
Illustrative nucleic acid molecules include those in which any difference
between the amino acid sequence encoded by the nucleic acid molecule and the
corresponding amino acid sequence of SEQ ID N0:2 is due to a conservative
amino
acid substitution. The present invention further contemplates isolated nucleic
acid
molecules that comprise a nucleotide sequence of nucleotides 195-422 of SEQ ID
2o NO: l .
The present invention also includes vectors and expression vectors
comprising such nucleic acid molecules. Such expression vectors may comprise a
transcription promoter, and a transcription terminator, wherein the promoter
is operably
linked with the nucleic acid molecule, and wherein the nucleic acid molecule
is
operably linked with the transcription terminator. The present invention
further
includes recombinant host cells comprising these vectors and expression
vectors.
Illustrative host cells include bacterial, yeast, fungal, insect, mammalian,
and plant
cells. Recombinant host cells comprising such expression vectors can be used
to
produce Zsig67 polypeptides by culturing such recombinant host cells that
comprise the
3o expression vector and that produce the Zsig67 protein, and, optionally,
isolating the
Zsig67 protein from the cultured recombinant host cells.
The present invention also contemplates methods for detecting the
presence of Zsig67 RNA in a biological sample, comprising the steps of (a)
contacting a
Zsig67 nucleic acid probe under hybridizing conditions with either (i) test
RNA
molecules isolated from the biological sample, or (ii) nucleic acid molecules
synthesized from the isolated RNA molecules, wherein the probe has a
nucleotide
sequence comprising a portion of the nucleotide sequence of nucleotides 111-
422 of
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6
SEQ ID NO:I, or its complement, and (b) detecting the formation of hybrids of
the
nucleic acid probe and either the test RNA molecules or the synthesized
nucleic acid
molecules, wherein the presence of the hybrids indicates the presence of
Zsig67 RNA in
the biological sample. As an illustration, the biological sample can be a
human
biological sample, such as a biopsy or autopsy specimen.
The present invention further provides methods for detecting the
presence of Zsig67 polypeptide in a biological sample, comprising the steps
of: (a)
contacting the biological sample with an antibody or an antibody fragment that
specifically binds with a polypeptide consisting of the amino acid sequence of
SEQ ID
1 o N0:2, wherein the contacting is performed under conditions that allow the
binding of
the antibody or antibody fragment to the biological sample, and (b) detecting
any of the
bound antibody or bound antibody fragment. Such an antibody or antibody
fragment
may further comprise a detectable label selected from the group consisting of
radioisotope, fluorescent label, chemiluminescent label, enzyme label,
bioluminescent
label, and colloidal gold. An exemplary biological sample is a human
biological
sample, such as a biopsy or autopsy specimen.
The present invention also provides kits for performing these detection
methods. For example, a kit for detection of Zsig67 gene expression may
comprise a
container that comprises a nucleic acid molecule, wherein the nucleic acid
molecule is
2o selected from the group consisting of (a) a nucleic acid molecule
comprising the
nucleotide sequence of nucleotides 111-422 of SEQ ID NO:1, (b) a nucleic acid
molecule comprising the complement of nucleotides 111-422 of the nucleotide
sequence of SEQ ID NO: l , (c) a nucleic acid molecule that is a fragment of
(a)
consisting of at least eight nucleotides, and (d) a nucleic acid molecule that
is a
fragment of (b) consisting of at least eight nucleotides. Such a kit may also
comprise a
second container that comprises one or more reagents capable of indicating the
presence of the nucleic acid molecule. On the other hand, a kit for detection
of Zsig67
protein may comprise a container that comprises an antibody, or an antibody
fragment,
that specifically binds with a polypeptide consisting of the amino acid
sequence of SEQ
3o ID N0:2.
The present invention also contemplates anti-idiotype antibodies, or anti-
idiotype antibody fragments, that specifically bind an antibody or antibody
fragment
that specifically binds a polypeptide consisting of the amino acid sequence of
SEQ ID
N0:2.
In addition, the present invention provides fusion proteins that comprise
a Zsig67 polypeptide moiety, and an immunoglobulin moiety, such as an
immunoglobulin heavy chain constant region. Illustrative Zsig67 polypeptide
moieties
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include: (a) a polypeptide consisting of the amino acid sequence of amino acid
residues
1 to 101 of SEQ ID N0:2, (b) a polypeptide consisting of the amino acid
sequence of
amino acid residues 1 to 104 of SEQ ID N0:2, (c) a polypeptide consisting of
the
amino acid sequence of amino acid residues 29 to 104 of SEQ ID N0:2, (d) a
polypeptide consisting of the amino acid sequence of amino acid residues 29 to
101 of
SEQ ID N0:2, (e) a polypeptide consisting of the amino acid sequence of amino
acid
residues 52 to 104 of SEQ ID N0:2, (fJ a polypeptide consisting of the amino
acid
sequence of amino acid residues 52 to 101 of SEQ ID N0:2, and (g) a
polypeptide
consisting of the amino acid sequence of amino acid residues 52 to 85 of SEQ
ID N0:2.
1 o Additional suitable polypeptides are described herein.
These and other aspects of the invention will become evident upon
reference to the following detailed description.
1 s 2. Definitions
In the description that follows, a number of terms are used extensively.
The following definitions are provided to facilitate understanding of the
invention.
As used herein, "nucleic acid" or "nucleic acid molecule" refers to
2o polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid
(RNA),
oligonucleotides, fragments generated by the polymerase chain reaction (PCR),
and
fragments generated by any of ligation, scission, endonuclease action, and
exonuclease
action. Nucleic acid molecules can be composed of monomers that are naturally-
occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring
25 nucleotides (e.g., a-enantiomeric forms of naturally-occurring
nucleotides), or a
combination of both. Modified riuclentic~ec ran haVP altPrat~nnc m ennar
mniAtio~
and/or in pyrimidine or purine base moieties. Sugar modifications include, for
example, replacement of one or more hydroxyl groups with halogens, alkyl
groups,
amines, and azido groups, or sugars can be functionalized as ethers or esters.
3o Moreover, the entire sugar moiety can be replaced with sterically and
electronically
similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples
of
modifications in a base moiety include alkylated purines and pyrimidines,
acylated
purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic
acid
monomers can be linked by phosphodiester bonds or analogs of such linkages.
Analogs
35 of phosphodiester linkages include phosphorothioate, phosphorodithioate,
phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,
phosphoranilidate,
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phosphoramidate, and the like. The term "nucleic acid molecule" also includes
so-
called "peptide nucleic acids," which comprise naturally-occurring or modified
nucleic
acid bases attached to a polyamide backbone. Nucleic acids can be either
single
stranded or double stranded.
The term "complement of a nucleic acid molecule" refers to a nucleic
acid molecule having a complementary nucleotide sequence and reverse
orientation as
compared to a reference nucleotide sequence. For example, the sequence 5'
ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
The term "contig" denotes a nucleic acid molecule that has a contiguous
to stretch of identical or complementary sequence to another nucleic acid
molecule.
Contiguous sequences are said to "overlap" a given stretch of a nucleic acid
molecule
either in their entirety or along a partial stretch of the nucleic acid
molecule.
The term "degenerate nucleotide sequence" denotes a sequence of
nucleotides that includes one or more degenerate codons as compared to a
reference
nucleic acid molecule that encodes a polypeptide. Degenerate codons contain
different
triplets of nucleotides, but encode the same amino acid residue (i. e., GAU
and GAC
triplets each encode Asp).
The term "structural gene" refers to a nucleic acid molecule that is
transcribed into messenger RNA (mRNA), which is then translated into a
sequence of
2o amino acids characteristic of a specific polypeptide.
An "isolated nucleic acid molecule" is a nucleic acid molecule that is not
integrated in the genomic DNA of an organism. For example, a DNA molecule that
encodes a growth factor that has been separated from the genomic DNA of a cell
is an
isolated DNA molecule. Another example of an isolated nucleic acid molecule is
a
chemically-synthesized nucleic acid molecule that is not integrated in the
genome of an
organism. A nucleic acid molecule that has been isolated from a particular
species is
smaller than the complete DNA molecule of a chromosome from that species.
A "nucleic acid molecule construct" is a nucleic acid molecule, either
single- or double-stranded, that has been modified through human intervention
to
3o contain segments of nucleic acid combined and juxtaposed in an arrangement
not
existing in nature.
"Linear DNA" denotes non-circular DNA molecules having free 5' and
3' ends. Linear DNA can be prepared from closed circular DNA molecules, such
as
plasmids, by enzymatic digestion or physical disruption.
"Complementary DNA (cDNA)" is a single-stranded DNA molecule that
is formed from an mRIVA template by the enzyme reverse transcriptase.
Typically, a
primer complementary to portions of mRNA is employed for the initiation of
reverse
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transcription. Those skilled in the art also use the term "cDNA" to refer to a
double-
stranded DNA molecule consisting of such a single-stranded DNA molecule and
its
complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA
molecule synthesized from an RNA template.
A "promoter" is a nucleotide sequence that directs the transcription of a
structural gene. Typically, a promoter is located in the 5' non-coding region
of a gene,
proximal to the transcriptional start site of a structural gene. Sequence
elements within
promoters that function in the initiation of transcription are often
characterized by
consensus nucleotide sequences. These promoter elements include RNA polymerase
1o binding sites, TATA sequences, CART sequences, differentiation-specific
elements
(DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclic AMP response
elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer
Biol.
1:47 (1990)), glucocorticoid response elements (GREs), and binding sites for
other
transcription factors, such as CRE/ATF (O'Reilly et al., J. Biol. Chem.
267:19938
(1992)), AP2 (Ye et al., J. Biol. Chem. 269:25728 (1994)), SP1, CAMP response
element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamer
factors
(see, in general, Watson et al., eds., Molecular Biology of the Gene, 4th ed.
(The
Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau,
Biochem. J. 303:1 (1994)). If a promoter is an inducible promoter, then the
rate of
2o transcription increases in response to an inducing agent. In contrast, the
rate of
transcription is not regulated by an inducing agent if the promoter is a
constitutive
promoter. Repressible promoters are also known.
A "core promoter" contains essential nucleotide sequences for promoter
function, including the TATA box and start of transcription. By this
definition, a core
2s promoter may or may not have detectable activity in the absence of specific
sequences
that may enhance the activity or confer tissue specific activity.
A "regulatory element" is a nucleotide sequence that modulates the
activity of a core promoter. For example, a regulatory element may contain a
nucleotide sequence that binds with cellular factors enabling transcription
exclusively
30 or preferentially in particular cells, tissues, or organelles. These types
of regulatory
elements are normally associated with genes that are expressed in a "cell-
specific,"
"tissue-specific," or "organelle-specific" manner.
An "enhancer" is a type of regulatory element that can increase the
e~ciency of transcription, regardless of the distance or orientation of the
enhancer
35 relative to the start site of transcription.
"Heterologous DNA" refers to a DNA molecule, or a population of
DNA molecules, that does not exist naturally within a given host cell. DNA
molecules
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heterologous to a particular host cell may contain DNA derived from the host
cell
species (i. e., endogenous DNA) so long as that host DNA is combined with non-
host
DNA (i.e., exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA segment
5 comprising a transcription promoter is considered to be a heterologous DNA
molecule.
Conversely, a heterologous DNA molecule can comprise an endogenous gene
operably
linked with an exogenous promoter. As another illustration, a DNA molecule
comprising a gene derived from a wild-type cell is considered to be
heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks the wild-type
gene.
1o 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."
A "protein" is a macromolecule comprising one or more polypeptide
chains. A protein may also comprise non-peptidic components, such as
carbohydrate
groups. Carbohydrates and other non-peptidic substituents may be added to a
protein
by the cell in which the protein is produced, and will vary with the type of
cell.
Proteins are defined herein in terms of their amino acid backbone structures;
substituents such as carbohydrate groups are generally not specified, but may
be present
nonetheless.
2o A peptide or polypeptide encoded by a non-host DNA molecule is a
"heterologous" peptide or polypeptide.
An "integrated genetic element" is a segment of DNA that has been
incorporated into a chromosome of a host cell after that element is introduced
into the
cell through human manipulation. Within the present invention, integrated
genetic
elements are most commonly derived from linearized plasmids that are
introduced into
the cells by electroporation or other techniques. Integrated genetic elements
are passed
from the original host cell to its progeny.
A "cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid,
or bacteriophage, that has the capability of replicating autonomously in a
host cell.
3o Cloning vectors typically contain one or a small number of restriction
endonuclease
recognition sites that allow insertion of a nucleic acid molecule in a
determinable fashion
without loss of an essential biological function of the vector, as well as
nucleotide
sequences encoding a marker gene that is suitable for use in the
identification and
selection of cells transformed with the cloning vector. Marker genes typically
include
genes that provide tetracycline resistance or ampicillin resistance.
An "expression vector" is a nucleic acid molecule encoding a gene that is
expressed in a host cell. Typically, an expression vector comprises a
transcription
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promoter, a gene, and a transcription terminator. Gene expression is usually
placed under
the control of a promoter, and such a gene is said to be "operably linked to"
the promoter.
Similarly, a regulatory element and a core promoter are operably linked if the
regulatory
element modulates the activity of the core promoter.
A "recombinant host" is a cell that contains a heterologous nucleic acid
molecule, such as a cloning vector or expression vector. In the present
context, an
example of a recombinant host is a cell that produces Zsig67 from an
expression vector.
In contrast, Zsig67 can be produced by a cell that is a "natural source" of
Zsig67, and
that lacks an expression vector.
to "Integrative transformants" are recombinant host cells, in which
heterologous DNA has become integrated into the genomic DNA of the cells.
A "fusion protein" is a hybrid protein expressed by a nucleic acid
molecule comprising nucleotide sequences of at least two genes. For example, a
fusion
protein can comprise at least part of a Zsig67 polypeptide fused with a
polypeptide that
binds an affinity matrix. Such a fusion protein provides a means to isolate
large
quantities of Zsig67 using affinity chromatography.
The term "receptor" denotes a cell-associated protein that binds to a
bioactive molecule termed a "ligand." This interaction mediates the effect of
the ligand
on the cell. Receptors can be membrane bound, cytosolic or nuclear; monomeric
(e.g.,
thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric
(e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF
receptor, erythropoietin receptor and IL-6 receptor). Membrane-bound receptors
are
characterized by a mufti-domain structure comprising an extracellular ligand-
binding
domain and an intracellular effector domain that is typically involved in
signal
transduction. In certain membrane-bound receptors, ~ the extracellular ligand-
binding
domain and the intracellular effector domain are located in separate
polypeptides that
comprise the complete functional receptor.
In general, the binding of ligand to receptor results in a conformational
change in the receptor that causes an interaction between the effector domain
and other
3o molecules) in the cell, which in turn leads to an alteration in the
metabolism of the cell.
Metabolic events that are often linked to receptor-ligand interactions include
gene
transcription, phosphorylation, dephosphorylation, increases in cyclic AMP
production,
mobilization of cellular calcium, mobilization of membrane lipids, cell
adhesion,
hydrolysis of inositol lipids and hydrolysis of phospholipids.
The term "secretory signal sequence" denotes a nucleotide sequence that
encodes a peptide (a "secretory peptide") that, as a component of a larger
polypeptide,
directs the larger polypeptide through a secretory pathway of a cell in which
it is
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12
synthesized. The larger polypeptide is commonly cleaved to remove the
secretory
peptide during transit through the secretory pathway.
An "isolated polypeptide" is a polypeptide that is essentially free from
contaminating cellular components, such as carbohydrate, lipid, or other
proteinaceous
impurities associated with the polypeptide in nature. Typically, a preparation
of isolated
polypeptide contains the polypeptide in a highly purified form, i. e., at
least about 80%
pure, at least about 90% pure, at least about 95% pure, greater than 95% pure,
or greater
than 99% pure. One way to show that a particular protein preparation contains
an
isolated polypeptide is by the appearance of a single band following sodium
dodecyl
l0 sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation
and
Coomassie Brilliant Blue staining of the gel. However, the term "isolated"
does not
exclude the presence of the same polypeptide in alternative physical forms,
such as
dimers or alternatively glycosylated or derivatized forms.
The terms "amino-terminal" and "carboxyl-terminal" are used herein to
denote positions within polypeptides. Where the context allows, these terms
are used
with reference to a particular sequence or portion of a polypeptide to denote
proximity
or relative position. For example, a certain sequence positioned carboxyl-
terminal to a
reference sequence within a polypeptide is located proximal to the carboxyl
terminus of
the reference sequence, but is not necessarily at the carboxyl terminus of the
complete
2o polypeptide.
The term "expression" refers to the biosynthesis of a gene product. For
example, in the case of a structural gene, expression involves transcription
of the
structural gene into mRNA and the translation of mRNA into one or more
polypeptides.
The term "splice variant" is used herein to denote alternative forms of
RNA transcribed from a gene. Splice variation arises naturally through use of
alternative splicing sites within a transcribed RNA molecule, or less commonly
between separately transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode polypeptides having
altered amino acid sequence. The term splice variant is also used herein to
denote a
3o polypeptide encoded by a splice variant of an mRNA transcribed from a gene.
As used herein, the term "immunomodulator" includes cytokines, stem
cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic
factors, and
synthetic analogs of these molecules. Examples of immunomodulators include
tumor
necrosis factor, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4,
IL-S, IL-6, IL-
7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15), colony
stimulating factors
(e.g., granulocyte-colony stimulating factor and granulocyte macrophage-colony
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13
stimulating factor), interferons (e.g., interferons-a, -(3, -'y, -~, -s, and -
i), the stem cell
growth factor designated "S 1 factor," erythropoietin, and thrombopoietin.
The term "complement/anti-complement pair" denotes non-identical
moieties that form a non-covalently associated, stable pair under appropriate
conditions.
For instance, biotin and avidin (or streptavidin) are prototypical members of
a
complement/anti-complement pair. Other exemplary complement/anti-complement
pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope)
pairs,
sense/antisense polynucleotide pairs, and the like. Where subsequent
dissociation of
the complement/anti-complement pair is desirable, the complement/anti-
complement
1 o pair preferably has a binding affinity of less than 1 O9 M-' .
An "anti-idiotype antibody" is an antibody that binds with the variable
region domain of an immunoglobulin. In the present context, an anti-idiotype
antibody
binds with the variable region of an anti-Zsig67 antibody, and thus, an anti-
idiotype
antibody mimics an epitope of Zsig67.
An "antibody fragment" is a portion of an antibody such as F(ab')Z, F(ab)2,
Fab', Fab, and the like. Regardless of structure, an antibody fragment binds
with the same
antigen that is recognized by the intact antibody. For example, an anti-Zsig67
monoclonal antibody fragment binds with an epitope of Zsig67.
The term "antibody fragment" also includes a synthetic or a genetically
2o engineered polypeptide that binds to a specific antigen, such as
polypeptides consisting of
the light chain variable region, "Fv" fragments consisting of the variable
regions of the
heavy and light chains, recombinant single chain polypeptide molecules in
which light
and heavy variable regions are connected by a peptide linker ("scFv
proteins"), and
minimal recognition units consisting of the amino acid residues that mimic the
hypervariable region.
A "chimeric antibody" is a recombinant protein that contains the variable
domains and complementary determining regions derived from a rodent antibody,
while
the remainder of the antibody molecule is derived from a human antibody.
"Humanized antibodies" are recombinant proteins in which marine
3o complementarity determining regions of a monoclonal antibody have been
transferred
from heavy and light variable chains of the marine immunoglobulin into a human
variable domain.
As used herein, a "therapeutic agent" is a molecule or atom which is
conjugated to an antibody moiety to produce a conjugate which is useful for
therapy.
Examples of therapeutic agents include drugs, toxins, immunomodulators,
chelators,
boron compounds, photoactive agents or dyes, and radioisotopes.
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A "detectable label" is a molecule or atom which can be conjugated to
an antibody moiety to produce a molecule useful for diagnosis. Examples of
detectable
labels include chelators, photoactive agents, radioisotopes, fluorescent
agents,
paramagnetic ions, or other marker moieties.
The term "affinity tag" is used herein to denote a polypeptide segment
that can be attached to a second polypeptide to provide for purification or
detection of
the second polypeptide or provide sites for attachment of the second
polypeptide to a
substrate. In principal, any peptide or protein for which an antibody or other
specific
binding agent is available can be used as an affinity tag. Affinity tags
include a poly-
1o histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson
et al.,
Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith and Johnson,
Gene
67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad
Sci. USA
82:7952 (1985)), substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204
(1988)), streptavidin binding peptide, or other antigenic epitope or binding
domain.
See, in general, Ford et al., Protein Expression and Purification 2:95 (1991).
Nucleic
acid molecules encoding affinity tags are available from commercial suppliers
(e.g.,
Pharmacia Biotech, Piscataway, NJ).
A "naked antibody" is an entire antibody, as opposed to an antibody
fragment, which is not conjugated with a therapeutic agent. Naked antibodies
include
2o both polyclonal and monoclonal antibodies, as well as certain recombinant
antibodies,
such as chimeric and humanized antibodies.
As used herein, the term "antibody component" includes both an entire
antibody and an antibody fragment.
An "immunoconjugate" is a conjugate of an antibody component with a
therapeutic agent or a detectable label.
As used herein, the term "antibody fusion protein" refers to a
recombinant molecule that comprises an antibody component and a therapeutic
agent.
Examples of therapeutic agents suitable for such fusion proteins include
immunomodulators ("antibody-immunomodulator fusion protein") and toxins
("antibody-toxin fusion protein").
A "tumor associated antigen" is a protein normally not expressed, or
expressed at lower levels, by a normal counterpart cell. Examples of tumor
associated
antigens include alpha-fetoprotein, carcinoembryonic antigen, and Her-2/neu.
Many
other illustrations of tumor associated antigens are known to those of skill
in the art.
See, for example, Urban et al., Ann. Rev. Immunol. 10:617 (1992).
A "target polypeptide" or a "target peptide" is an amino acid sequence
that comprises at least one epitope, and that is expressed on a target cell,
such as a
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tumor cell, or a cell that carries an infectious agent antigen. T cells
recognize peptide
epitopes presented by a major histocompatibility complex molecule to a target
polypeptide or target peptide and typically lyse the target cell or recruit
other immune
cells to the site of the target cell, thereby killing the target cell.
5 An "antigenic peptide" is a peptide which will bind a maj or
histocompatibility complex molecule to form an MHC-peptide complex which is
recognized by a T cell, thereby inducing a cytotoxic lymphocyte response upon
presentation to the T cell. Thus, antigenic peptides are capable of binding to
an
appropriate major histocompatibility complex molecule and inducing a cytotoxic
T
to cells response, such as cell lysis or specific cytokine release against the
target cell
which binds or expresses the antigen. The antigenic peptide can be bound in
the
context of a class I or class II major histocompatibility complex molecule, on
an
antigen presenting cell or on a target cell.
In eukaryotes, RNA polymerise II catalyzes the transcription of a
15 structural gene to produce mRNA. A nucleic acid molecule can be designed to
contain
an RNA polymerise II template in which the RNA transcript has a sequence that
is
complementary to that of a specific mRNA. The RNA transcript is termed an
"anti
sense RNA" and a nucleic acid molecule that encodes the anti-sense RNA is
termed an
"anti-sense gene." Anti-sense RNA molecules are capable of binding to mRNA
2o molecules, resulting in an inhibition of mRNA translation.
An "anti-sense oligonucleotide specific for Zsig67" or an "Zsig67 anti-
sense oligonucleotide" is an oligonucleotide having a sequence (a) capable of
forming a
stable triplex with a portion of the Zsig67 gene, or (b) capable of forming a
stable
duplex with a portion of an mRNA transcript of the Zsig67 gene.
A "ribozyme" is a nucleic acid molecule that contains a catalytic center.
The term includes RNA enzymes, self splicing RNAs, self cleaving RNAs, and
nucleic
acid molecules that perform these catalytic functions. A nucleic acid molecule
that
encodes a ribozyme is termed a "ribozyme gene."
An "external guide sequence" is a nucleic acid molecule that directs the
3o endogenous ribozyme, RNase P, to a particular species of intracellular
mRNA, resulting
in the cleavage of the mRNA by RNase P. A nucleic acid molecule that encodes
an
external guide sequence is termed an "external guide sequence gene."
The term "variant Zsig67 gene" refers to nucleic acid molecules that
encode a polypeptide having an amino acid sequence that is a modification of
SEQ ID
N0:2. Such variants include naturally-occurring polymorphisms of Zsig67 genes,
as
well as synthetic genes that contain conservative amino acid substitutions of
the amino
acid sequence of SEQ ID N0:2. Additional variant forms of Zsig67 genes are
nucleic
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16
acid molecules that contain insertions or deletions of the nucleotide
sequences
described herein. A variant Zsig67 gene can be identified by determining
whether the
gene hybridizes with a nucleic acid molecule having the nucleotide sequence of
SEQ
ID NO:1, or its complement, under stringent conditions.
Alternatively, variant Zsig67 genes can be identified by sequence
comparison. Two amino acid sequences have "100% amino acid sequence identity"
if
the amino acid residues of the two amino acid sequences are the same when
aligned for
maximal correspondence. Similarly, two nucleotide sequences have "100%
nucleotide
sequence identity" if the nucleotide residues of the two nucleotide sequences
are the
1 o same when aligned for maximal correspondence. Sequence comparisons can be
performed using standard software programs such as those included in the
LASERGENE bioinformatics computing suite, which is produced by DNASTAR
(Madison, Wisconsin). Other methods for comparing two nucleotide or amino acid
sequences by determining optimal alignment are well-known to those of skill in
the art
(see, for example, Peruski and Peruski, The Internet and the New Biology:
Tools for
Genomic and Molecular Research (ASM Press, Inc. 1997), Wu et al. (eds.),
"Information Superhighway and Computer Databases of Nucleic Acids and
Proteins,"
in Methods in Gene Biotechnology, pages 123-1 S 1 (CRC Press, Inc. 1997), and
Bishop
(ed.), Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.
1998)).
Particular methods for determining sequence identity are described below.
Regardless of the particular method used to identify a variant Zsig67
gene or variant Zsig67 polypeptide, a variant gene or polypeptide encoded by a
variant
gene may be characterized by its ability to bind specifically to an anti-
Zsig67 antibody.
The term "allelic variant" is used herein to denote any of two or more
alternative forms of a gene occupying the same chromosomal locus. Allelic
variation
arises naturally through mutation, and may result in phenotypic polymorphism
within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or
may encode polypeptides having altered amino acid sequence. The term allelic
variant
is also used herein to denote a protein encoded by an allelic variant of a
gene.
3o The term "ortholog" denotes a polypeptide or protein obtained from one
species that is the functional counterpart of a polypeptide or protein from a
different
species. Sequence differences among orthologs are the result of speciation.
"Paralogs" are distinct but structurally related proteins made by an
organism. Paralogs are believed to arise through gene duplication. For
example, a
globin, ~i-globin, and myoglobin are paralogs of each other.
The present invention includes functional fragments of Zsig67 genes.
Within the context of this invention, a "functional fragment" of a Zsig67 gene
refers to
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17
a nucleic acid molecule that encodes a portion of a Zsig67 polypeptide
specifically
binds with an anti-Zsig67 antibody.
As used herein, the term "Zsig67 component" includes both a Zsig67
polypeptide and a Zsig67 functional fragment.
The term, "cytotoxin," refers to a molecule or atom that is capable of
causing the death of a cell, or that is capable of inhibiting the growth of a
cell.
Examples of cytotoxins include drugs, ribosome-inactivating proteins,
immunomodulators, chelators, boron compounds, photoactive agents or dyes,
radioisotopes, and the like.
to A "Zsig67 targeting composition" comprises a Zsig67 component and a
cytotoxin. The association between the Zsig67 component and the cytotoxin can
be
covalent or noncovalent. For example, the Zsig67 component-cytotoxin
association in
Zsig67 conjugates and Zsig67 targeting fusion proteins is covalent, while
Zsig67
targeting liposomes illustrate a noncovalent association.
A "Zsig67 conjugate" is a type of Zsig67 targeting composition, which
is produced by covalently linking a Zsig67 component and a cytotoxin either
directly or
via a linking agent.
As used herein, the term "Zsig67 targeting fusion protein" refers to a
recombinant molecule that comprises a Zsig67 polypeptide component and a
cytotoxin.
Due to the imprecision of standard analytical methods, molecular
weights and lengths of polymers are understood to be approximate values. When
such
a value is expressed as "about" X or "approximately" X, the stated value of X
will be
understood to be accurate to ~ 10%.
2s 3. Production of the Human Zsig67Gene
Nucleic acid molecules encoding a human Zsig67 gene can be obtained
by screening a human cDNA or genomic library using polynucleotide probes based
upon SEQ ID NO:1. These techniques are standard and well-established.
As an illustration, a nucleic acid molecule that encodes a human Zsig67
3o gene can be isolated from a human cDNA library. In this case, the first
step would be to
prepare the cDNA library by isolating RNA from pancreatic tissue, such as
pancreatic
islet cells, using methods well-known to those of skill in the art. In
general, RNA
isolation techniques must provide a method for breaking cells, a means of
inhibiting
RNase-directed degradation of RNA, and a method of separating RNA from DNA,
35 protein, and polysaccharide contaminants. For example, total RNA can be
isolated by
freezing tissue in liquid nitrogen, grinding the frozen tissue with a mortar
and pestle to
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18
lyse the cells, extracting the ground tissue with a solution of
phenol/chloroform to remove
proteins, and separating RNA from the remaining impurities by selective
precipitation
with lithium chloride (see, for example, Ausubel et al. (eds.), Short
Protocols in
Molecular Biology, 3rd Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995)
["Ausubel
(1995)"]; Wu et al., Methods in Gene Biotechnology, pages 33-41 (CRC Press,
Inc. 1997)
["Wu (1997)"]).
Alternatively, total RNA can be isolated from pancreatic tissue by
extracting ground tissue with guanidinium isothiocyanate, extracting with
organic
solvents, and separating RNA from contaminants using differential
centrifugation (see,
1o for example, Chirgwin et al., Biochemistry 18:52 (1979); Ausubel (1995) at
pages 4-1 to
4-6; Wu (1997) at pages 33-41).
In order to construct a cDNA library, poly(A)+ RNA must be isolated from
a total RNA preparation. Poly(A)+ RNA can be isolated from total RNA using the
standard technique of oligo(dT)-cellulose chromatography (see, for example,
Aviv and
Leder, Proc. Nat'1 Acad. Sci. USA 69:1408 (1972); Ausubel (1995) at pages 4-11
to 4-
12).
Double-stranded cDNA molecules are synthesized from poly(A)+ RNA
using techniques well-known to those in the art. (see, for example, Wu (1997)
at pages
41-46). Moreover, commercially available kits can be used to synthesize double-
2o stranded cDNA molecules. For example, such kits are available from Life
Technologies, Inc. (Gaithersburg, MD), CLONTECH Laboratories, Inc. (Palo Alto,
CA), Promega Corporation (Madison, WI) and STRATAGENE (La Jolla, CA).
Various cloning vectors are appropriate for the construction of a cDNA
library. For example, a cDNA library can be prepared in a vector derived from
bacteriophage, such as a ~,gtl0 vector. See, for example, Huynh et al.,
"Constructing
and Screening cDNA Libraries in ~,gtl0 and ~,gtll," in DNA Cloning: A
Practical
Approach I~ol. I, Glover (ed.), page 49 (IRL Press, 1985); Wu (1997) at pages
47-52.
Alternatively, double-stranded cDNA molecules can be inserted into a
plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a
3o LAMDAGEM-4 (Promega Corp.) or other commercially available vectors.
Suitable
cloning vectors also can be obtained from the American Type Culture Collection
(Manassas, VA).
To amplify the cloned cDNA molecules, the cDNA library is inserted into
a prokaryotic host, using standard techniques. For example, a cDNA library can
be
introduced into competent E. coli DHS cells, which can be obtained, for
example, from
Life Technologies, Inc. (Gaithersburg, MD).
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19
A human genomic library can be prepared by means well-known in the art
(see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-
327).
Genomic DNA can be isolated by lysing tissue with the detergent Sarkosyl,
digesting the
lysate with proteinase K, clearing insoluble debris from the lysate by
centrifugation,
s precipitating nucleic acid from the lysate using isopropanol, and purifying
resuspended
DNA on a cesium chloride density gradient.
DNA fragments that are suitable for the production of a genomic library
can be obtained by the random shearing of genomic DNA or by the partial
digestion of
genomic DNA with restriction endonucleases. Genomic DNA fragments can be
inserted
1 o into a vector, such as a bacteriophage or cosmid vector, in accordance
with conventional
techniques, such as the use of restriction enzyme digestion to provide
appropriate termini,
the use of alkaline phosphatase treatment to avoid undesirable joining of DNA
molecules,
and ligation with appropriate ligases. Techniques for such manipulation are
well-known
in the art (see, for example, Ausubel (199s) at pages 5-1 to 5-6; Wu (1997) at
pages 307
1 s 327).
Nucleic acid molecules that encode a human Zsig67 gene can also be
obtained using the polymerase chain reaction (PCR) with oligonucleotide
primers
having nucleotide sequences that are based upon the nucleotide sequences of
the human
Zsig67 gene, as described herein. General methods for screening libraries with
PCR are
20 provided by, for example, Yu et al., "Use of the Polymerase Chain Reaction
to Screen
Phage Libraries," in Methods in Molecular Biology, Vol. I5: PCR Protocols:
Current
Methods and Applications, White (ed.), pages 211-215 (Humana Press, Inc.
1993).
Moreover, techniques for using PCR to isolate related genes are described by,
for
example, Preston, "Use of Degenerate Oligonucleotide Primers and the
Polymerase
2s Chain Reaction to Clone Gene Family Members," in Methods in Molecular
Biology,
Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages
317-
337 (Humana Press, Inc. 1993).
Alternatively, human genomic libraries can be obtained from commercial
sources such as Research Genetics (Huntsville, AL) and the American Type
Culture
3o Collection (Manassas, VA).
A library containing cDNA or genomic clones can be screened with one or
more polynucleotide probes based upon SEQ ID NO:1, using standard methods
(see, for
example, Ausubel (1995) at pages 6-1 to 6-11).
Anti-Zsig67 antibodies, produced as described below, can also be used
3s to isolate DNA sequences that encode human Zsig67 genes from cDNA
libraries. For
example, the antibodies can be used to screen ~,gtll expression libraries, or
the
antibodies can be used for immunoscreening following hybrid selection and
translation
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(see, for example, Ausubel (1995) at pages 6-12 to 6-16; Margolis et al.,
"Screening 7~
expression libraries with antibody and protein probes," in DNA Cloning 2:
Expression
Systems, 2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University
Press 1995)).
As an alternative, a Zsig67 gene can be obtained by synthesizing nucleic
5 acid molecules using mutually priming long oligonucleotides and the
nucleotide
sequences described herein (see, for example, Ausubel (1995) at pages 8-8 to 8-
9).
Established techniques using the polymerase chain reaction provide the ability
to
synthesize DNA molecules at least two kilobases in length (Adang et al., Plant
Molec.
Biol. 21:1131 (1993), Bambot et al., PCR Methods and Applications 2:266
(1993),
1 o Dillon et al., "Use of the Polymerase Chain Reaction for the Rapid
Construction of
Synthetic Genes," in Methods in Molecular Biology, Ilol. I5: PCR Protocols:
Current
Methods and Applications, White (ed.), pages 263-268, (Humana Press, Inc.
1993), and
Holowachuk et al., PCR Methods Appl. 4:299 (1995)).
The nucleic acid molecules of the present invention can also be
15 synthesized with "gene machines" using protocols such as the
phosphoramidite method.
If chemically-synthesized double stranded DNA is required for an application
such as
the synthesis of a gene or a gene fragment, then each complementary strand is
made
separately. The production of short genes (60 to 80 base pairs) is technically
straightforward and can be accomplished by synthesizing the complementary
strands
2o and then annealing them. For the production of longer genes (>300 base
pairs),
however, special strategies may be required, because the coupling efficiency
of each
cycle during chemical DNA synthesis is seldom 100%. To overcome this problem,
synthetic genes (double-stranded) are assembled in modular form from single-
stranded
fragments that are from 20 to 100 nucleotides in length. For reviews on
polynucleotide
synthesis, see, for example, Glick and Pasternak, Molecular Biotechnology,
Principles
and Applications of Recombinant DNA (ASM Press 1994), Itakura et al., Annu.
Rev.
Biochem. 53:323 (1984), and Climie et al., Proc. Nat'l Acad. Sci. USA 87:633
(1990).
The sequence of a Zsig67 cDNA or Zsig67 genomic fragment can be
determined using standard methods. Zsig67 polynucleotide sequences disclosed
herein
3o can also be used as probes or primers to clone 5' non-coding regions of a
Zsig67 gene.
Promoter elements from a Zsig67 gene can be used to direct the expression of
heterologous genes in, for example, pituitary tissue of transgenic animals or
patients
undergoing gene therapy. The identification of genomic fragments containing a
Zsig67
promoter or regulatory element can be achieved using well-established
techniques, such
as deletion analysis (see, generally, Ausubel (1995)).
Cloning of 5' flanking sequences also facilitates production of Zsig67
proteins by "gene activation," according to the general methods disclosed in
U.S. Patent
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21
No. 5,641,670. Briefly, expression of an endogenous Zsig67 gene in a cell is
altered by
introducing into the Zsig67 locus a DNA construct comprising at least a
targeting
sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
The
targeting sequence is a Zsig67 5' non-coding sequence that permits homologous
recombination of the construct with the endogenous Zsig67 locus, whereby the
sequences within the construct become operably linked with the endogenous
Zsig67
coding sequence. In this way, an endogenous Zsig67 promoter can be replaced or
supplemented with other regulatory sequences to provide enhanced, tissue-
specific, or
otherwise regulated expression.
4. Production of Zsig67 Gene Variants
The present invention provides a variety of nucleic acid molecules,
including DNA and RNA molecules, that encode the Zsig67 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 nucleotide sequence that
encompasses all nucleic acid molecules that encode the Zsig67 polypeptides 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
2o T. Thus, the present invention contemplates Zsig67 polypeptide-encoding
nucleic acid
molecules comprising nucleotide 111 to nucleotide 422 of SEQ ID NO:1, and
their
RNA equivalents.
Table 1 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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22
Table 1
NucleotideResolutionComplement 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
M 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 2.
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23
Table 2
One LetterCodons Degenerate
Amino 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 ~y
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 YT'N
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 'I'~
Asn~Asp B ~y
Glu~Gln Z SAR
Any X
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24
One of ordinary skill in the art will appreciate that some ambiguity is
introduced in determining a degenerate codon, representative of all possible
codons
encoding an amino acid. For example, the degenerate codon for serine (WSN)
can, in
some circumstances, encode arginine (AGR), and the degenerate codon for
arginine
(MGN) can, in some circumstances, encode serine (AGY). A similar relationship
exists
between codons encoding phenylalanine and leucine. Thus, some polynucleotides
encompassed by the degenerate sequence may encode variant amino acid
sequences,
but one of ordinary skill in the art can easily identify such variant
sequences by
reference to the amino acid sequence of SEQ ID N0:2. Variant sequences can be
1o readily tested for functionality as described herein.
Different species can exhibit "preferential codon usage." In general, see,
Grantham et al., Nuc. Acids Res. 8:1893 (1980), Haas et al. Curr. Biol. 6:315
(1996),
Wain-Hobson et al., Gene 13:355 (1981), Grosjean and Fiers, Gene 18:199
(1982),
Holm, Nuc. Acids Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982),
Sharp
1 s and Matassi, Curr. Opin. Genet. Dev. 4: 851 ( 1994), Kane, Curr. Opin.
Biotechnol.
6:494 (1995), and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the
term
"preferential codon usage" or "preferential codons" is a term of art referring
to protein
translation codons that are most frequently used in cells of a certain
species, thus
favoring one or a few representatives of the possible codons encoding each
amino acid
20 (See Table 2). For example, the amino acid Threonine (Thr) may be encoded
by ACA,
ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon;
in other species, for example, insect cells, yeast, viruses or bacteria,
different Thr
codons may be preferential. Preferential codons for a particular species can
be
introduced into the polynucleotides of the present invention by a variety of
methods
25 known in the art. Introduction of preferential codon sequences into
recombinant DNA
can, for example, enhance production of the protein by making protein
translation more
efficient within a particular cell type or species. Therefore, the degenerate
codon
sequence disclosed in SEQ ID N0:3 serves as a template for optimizing
expression of
polynucleotides in various cell types and species commonly used in the art and
3o disclosed herein. Sequences containing preferential codons can be tested
and optimized
for expression in various species, and tested for functionality as disclosed
herein.
The present invention further provides variant polypeptides and nucleic
acid molecules that represent counterparts from other species (orthologs).
These
species include, but are not limited to mammalian, avian, amphibian, reptile,
fish, insect
35 and other vertebrate and invertebrate species. Of particular interest are
Zsig67
polypeptides from other mammalian species, including marine, porcine, ovine,
bovine,
canine, feline, equine, and other primate polypeptides. Orthologs of human
Zsig67 can
CA 02358930 2001-07-17
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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 Zsig67 as
disclosed herein. Suitable sources of mRNA can be identified by probing
northern
5 blots with probes designed from the sequences disclosed herein. A library is
then
prepared from mRNA of a positive tissue or cell line.
A Zsig67-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
t o the polymerase chain reaction with primers designed from the
representative human
Zsig67 sequences disclosed herein. Within an additional method, the cDNA
library can
be used to transform or transfect host cells, and expression of the cDNA of
interest can
be detected with an antibody to Zsig67 polypeptide. Similar techniques can
also be
applied to the isolation of genomic clones.
15 Those skilled in the art will recognize that the sequence disclosed in
SEQ ID NO:l represents a single allele of human Zsig67, and that allelic
variation and
alternative splicing are expected to occur. Allelic variants of this sequence
can be
cloned by probing cDNA or genomic libraries from different individuals
according to
standard procedures. Allelic variants of the nucleotide sequence shown in SEQ
ID
2o 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. cDNA molecules generated
from
alternatively spliced mRNAs, which retain the properties of the Zsig67
polypeptide are
included within the scope of the present invention, as are polypeptides
encoded by such
25 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.
Within certain embodiments of the invention, the isolated nucleic acid
molecules can hybridize under stringent conditions to nucleic acid molecules
3o comprising nucleotide sequences disclosed herein. For example, such nucleic
acid
molecules can hybridize under stringent conditions to nucleic acid molecules
comprising the nucleotide sequence of SEQ ID NO:1, to nucleic acid molecules
having
the nucleotide sequence of nucleotides 111 to 422 of SEQ ID NO:1, or to
nucleic acid
molecules having a nucleotide sequence complementary to SEQ ID NO:1 or to
nucleotides 111 to 422 of SEQ ID NO:1. In general, stringent conditions are
selected to
be about 5°C lower than the thermal melting point (Tm) for the specific
sequence at a
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26
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.
A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and
DNA-RNA, can hybridize if the nucleotide sequences have some degree of
complementarity. Hybrids can tolerate mismatched base pairs in the double
helix, but
the stability of the hybrid is influenced by the degree of mismatch. The Tm of
the
mismatched hybrid decreases by 1 °C for every 1-1.5% base pair
mismatch. Varying
the stringency of the hybridization conditions allows control over the degree
of
mismatch that will be present in the hybrid. The degree of stringency
increases as the
1 o hybridization temperature increases and the ionic strength of the
hybridization buffer
decreases. Stringent hybridization conditions encompass temperatures of about
5-25°C
below the Tm of the hybrid and a hybridization buffer having up to 1 M Na+.
Higher
degrees of stringency at lower temperatures can be achieved with the addition
of
formamide which reduces the Tm of the hybrid about 1 °C for each 1 %
formamide in
the buffer solution. Generally, such stringent conditions include temperatures
of 20-
70°C and a hybridization buffer containing up to 6x SSC and 0-50%
formamide. A
higher degree of stringency can be achieved at temperatures of from 40-
70°C with a
hybridization buffer having up to 4x SSC and from 0-50% formamide. Highly
stringent conditions typically encompass temperatures of 42-70°C with a
hybridization
2o buffer having up to lx SSC and 0-50% formamide. Different degrees of
stringency can
be used during hybridization and washing to achieve maximum specific binding
to the
target sequence. Typically, the washes following hybridization are performed
at
increasing degrees of stringency to remove non-hybridized polynucleotide
probes from
hybridized complexes.
The above conditions are meant to serve as a guide and it is well within
the abilities of one skilled in the art to adapt these conditions for use with
a particular
polypeptide hybrid. The Tm for a specific target sequence is the temperature
(under
defined conditions) at which SO% of the target sequence will hybridize to a
perfectly
matched probe sequence. Those conditions which influence the Tm include, the
size
3o and base pair content of the polynucleotide probe, the ionic strength of
the
hybridization solution, and the presence of destabilizing agents in the
hybridization
solution. Numerous equations for calculating Tm 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, Inca 1987); Berger and
Kimmel
(eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987);
and
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27
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. D (Premier
Biosoft
International; Palo Alto, CA), as well as sites on the Internet, are available
tools for
analyzing a given sequence and calculating Tm 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
1o and DNA-RNA hybrids.
The length of the polynucleotide sequence influences the rate and
stability of hybrid formation. Smaller probe sequences, <50 base pairs, reach
equilibrium with complementary sequences rapidly, but may form less stable
hybrids.
Incubation times of anywhere from minutes to hours can be used to achieve
hybrid
formation. Longer probe sequences come to equilibrium more slowly, but form
more
stable complexes even at lower temperatures. Incubations are allowed to
proceed
overnight or longer. Generally, incubations axe carried out for a period equal
to three
times the calculated Cot time. Cot time, the time it takes for the
polynucleotide
sequences to reassociate, can be calculated for a particular sequence by
methods known
in the art.
The base pair composition of polynucleotide sequence will effect the
thermal stability of the hybrid complex, thereby influencing the choice of
hybridization
temperature and the ionic strength of the hybridization buffer. A-T pairs are
less stable
than G-C pairs in aqueous solutions containing sodium chloride. Therefore, the
higher
the G-C content, the more stable the hybrid. Even distribution of G and C
residues
within the sequence also contribute positively to hybrid stability. In
addition, the base
pair composition can be manipulated to alter the Tm of a given sequence. For
example,
5-methyldeoxycytidine can be substituted for deoxycytidine and 5-
bromodeoxuridine
can be substituted for thymidine to increase the Tm, whereas 7-deazz-2'-
3o deoxyguanosine can be substituted for guanosine to reduce dependence on Tm
.
The ionic concentration of the hybridization buffer also affects the
stability of the hybrid. Hybridization buffers generally contain blocking
agents such as
Denhardt's solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon
sperm
DNA, tRNA, milk powders (BLOTTO), heparin or SDS, and a Na+ source, such as
SSC
(lx SSC: 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE: 1.8 M
NaCI, 10 mM NaH2P04, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration
of the buffer, the stability of the hybrid is increased. Typically,
hybridization buffers
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28
contain from between 10 mM - 1 M Na+. The addition of destabilizing or
denaturing
agents such as formamide, tetralkylammonium salts, guanidinium cations or
thiocyanate canons to the hybridization solution will alter the Tm of a
hybrid.
Typically, formamide is used at a concentration of up to 50% to allow
incubations to be
carried out at more convenient and lower temperatures. Formamide also acts to
reduce
non-specific background when using RNA probes.
As an illustration, a nucleic acid molecule encoding a variant Zsig67
polypeptide can be hybridized with a nucleic acid molecule having the
nucleotide
sequence of SEQ ID NO:1 (or its complement) at 42°C overnight in a
solution
1o comprising 50% formamide, SxSSC (lxSSC: 0.15 M sodium chloride and 15 mM
sodium citrate), 50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution (100x
Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and
2%
(w/v) bovine serum albumin), 10% dextran sulfate, and 20 ~,g/ml denatured,
sheared
salmon sperm DNA. One of skill in the art can devise variations of these
hybridization
conditions. For example, the hybridization mixture can be incubated at a
higher
temperature, such as about 65°C, in a solution that does not contain
formamide.
Moreover, premixed hybridization solutions are available (e.g., EXPRESSHYB
Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization
can be
performed according to the manufacturer's instructions.
2o Following hybridization, the nucleic acid molecules can be washed to
remove non-hybridized nucleic acid molecules under stringent conditions, or
under
highly stringent conditions. Typical stringent washing conditions include
washing in a
solution of O.Sx - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 -
65°C. That
is, nucleic acid molecules encoding a variant Zsig67 polypeptide remain
hybridized
following stringent washing conditions with a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1 (or its complement), in which the wash
stringency is equivalent to O.Sx - 2x SSC with 0.1% SDS at 55 - 65°C,
including O.Sx
SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C. One
of skill in the art
can readily devise equivalent conditions, for example, by substituting SSPE
for SSC in
3o the wash solution.
Typical highly stringent washing conditions include washing in a
solution of O.lx - 0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50 -
65°C. In
other words, nucleic acid molecules encoding a variant Zsig67 polypeptide
remain
hybridized following highly stringent washing conditions with a nucleic acid
molecule
having the nucleotide sequence of SEQ ID NO:1 (or its complement), in which
the
wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 -
65°C,
including O.lx SSC with 0.1% SDS at 50°C, or 0.2xSSC with 0.1% SDS at
65°C.
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29
The present invention also provides isolated Zsig67 polypeptides that
have a substantially similar sequence identity to the polypeptides of SEQ ID
N0:2, or
their orthologs. The term "substantially similar sequence identity" is used
herein to
denote polypeptides having 70%, 80%, 90%, 95% or greater than 95% sequence
identity to the sequences shown in SEQ ID N0:2, or their orthologs.
The present invention also contemplates Zsig67 variant nucleic acid
molecules that can be identified using two criteria: a determination of the
similarity
between the encoded polypeptide with the amino acid sequence of SEQ ID N0:2,
and a
hybridization assay, as described above. Such Zsig67 variants include nucleic
acid
i o molecules ( 1 ) that remain hybridized following stringent washing
conditions with a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its
complement), in which the wash stringency is equivalent to O.Sx - 2x SSC with
0.1%
SDS at 55 - 65°C, and (2) that encode a polypeptide having 70%, 80%,
90%, 95% or
greater than 95% sequence identity to the amino acid sequence of SEQ ID N0:2.
Alternatively, Zsig67 variants can be characterized as nucleic acid molecules
(1) that
remain hybridized following highly stringent washing conditions with a nucleic
acid
molecule having the nucleotide sequence of SEQ ID NO:1 (or its complement), in
which the wash stringency is equivalent to 0.1 x - 0.2x SSC with 0.1 % SDS at
50 -
65°C, and (2) that encode a polypeptide having 70%, 80%, 90%, 95% or
greater than
95% sequence identity to the amino acid sequence of SEQ ID N0:2.
Percent sequence identity is determined by conventional methods. See,
for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff and
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992). Briefly, two amino acid
sequences are aligned to optimize the alignment scores using a gap opening
penalty of
10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and
Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the
standard one-
letter codes). The percent identity is then calculated as: ([Total number of
identical
matches]/ [length of the longer sequence plus the number of gaps introduced
into the
longer sequence in order to align the two sequences])(100).
CA 02358930 2001-07-17
WO 00/43516 PCT/US00/00870
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CA 02358930 2001-07-17
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31
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 Zsig67 variant. The FASTA algorithm is
described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988),
and by
Pearson, Meth. Enzymol. 183:63 ( 1990). Briefly, FASTA first characterizes
sequence
similarity by identifying regions shared by the query sequence (e.g., SEQ ID
N0:2)
and a test sequence that have either the highest density of identities (if the
ktup
variable is 1) or pairs of identities (if ktup=2), without considering
conservative amino
acid substitutions, insertions, or deletions. The ten regions with the highest
density of
identities are then rescored by comparing the similarity of all paired amino
acids using
an amino acid substitution matrix, and the ends of the regions are "trimmed"
to
include only those residues that contribute to the highest score. If there are
several
regions with scores greater than the "cutofF' value (calculated by a
predetermined
formula based upon the length of the sequence and the ktup value), then the
trimmed
initial regions are examined to determine whether the regions can be joined to
form an
approximate alignment with gaps. Finally, the highest scoring regions of the
two
amino acid sequences are aligned using a modification of the Needleman-Wunsch-
2o Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970);
Sellers, SIAM
J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and
deletions.
Illustrative parameters for FASTA analysis are: ktup=l, gap opening
penalty=10, gap
extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be
introduced into ~ a FASTA program by modifying the scoring matrix file
("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63
(1990).
FASTA can also be used to determine the sequence identity of nucleic
acid molecules using a ratio as disclosed above. For nucleotide sequence
comparisons,
the ktup value can range between one to six, preferably from three to six,
most
3o preferably three, with other parameters set as described above.
The present invention includes nucleic acid molecules that encode a
polypeptide having a conservative amino acid change, compared with the amino
acid
sequence of SEQ ID N0:2. That is, variants can be obtained that contain one or
more
amino acid substitutions of SEQ ID N0:2, in which an alkyl amino acid is
substituted
for an alkyl amino acid in a Zsig67 amino acid sequence, an aromatic amino
acid is
substituted for an aromatic amino acid in a Zsig67 amino acid sequence, a
sulfur-
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32
containing amino acid is substituted for a sulfur-containing amino acid in a
Zsig67
amino acid sequence, a hydroxy-containing amino acid is substituted for a
hydroxy-
containing amino acid in a Zsig67 amino acid sequence, an acidic amino acid is
substituted for an acidic amino acid in a Zsig67 amino acid sequence, a basic
amino
acid is substituted for a basic amino acid in a Zsig67 amino acid sequence, or
a dibasic
monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino
acid in
a Zsig67 amino acid sequence.
Among the common amino acids, for example, a "conservative amino
acid substitution" is illustrated by a substitution among amino acids within
each of the
to following groups: (1) glycine, alanine, valine, leucine, and isoleucine,
(2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4)
aspartate and
glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and
histidine.
The BLOSUM62 table is an amino acid substitution matrix derived
from about 2,000 local multiple alignments of protein sequence segments,
representing highly conserved regions of more than 500 groups of related
proteins
(Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915 (1992)).
Accordingly,
the BLOSUM62 substitution frequencies can be used to define conservative amino
acid substitutions that may be introduced into the amino acid sequences of the
present
invention. Although it is possible to design amino acid substitutions based
solely
2o upon chemical properties (as discussed above), the language "conservative
amino acid
substitution" preferably refers to a substitution represented by a BLOSUM62
value of
greater than -1. For example, an amino acid substitution is conservative if
the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According
to
this system, certain conservative amino acid substitutions are characterized
by a
BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while other conservative amino
acid
substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or
3).
Particular variants of Zsig67 are characterized by having greater than
96%, at least 97%, at least 98%, or at least 99% sequence identity to the
corresponding
amino acid sequence (e.g., SEQ ID N0:2), wherein the variation in amino acid
3o sequence is due to one or more conservative amino acid substitutions.
Conservative amino acid changes in a Zsig67 gene can be introduced
by substituting nucleotides for the nucleotides recited in SEQ ID NO:1. Such
"conservative amino acid" variants can be obtained, for example, by
oligonucleotide-
directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the
polymerase
chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and
McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)).
A
CA 02358930 2001-07-17
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33
variant Zsig67 polypeptide can be identified by the ability to specifically
bind anti-
Zsig67 antibodies.
The proteins of the present invention can also comprise non-naturally
occurring amino acid residues. Non-naturally occurnng amino acids include,
without
limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4
hydroxyproline, N methylglycine, alto-threonine, methylthreonine,
hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine,
pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-
methylproline,
3,3-dimethylproline, tent-leucine, norvaline, 2-azaphenylalanine, 3-
azaphenylalanine,
l0 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in
the art
for incorporating non-naturally occurring amino acid residues into proteins.
For
example, an in vitro system can be employed wherein nonsense mutations are
suppressed using chemically aminoacylated suppressor tRNAs. Methods for
synthesizing amino acids and aminoacylating tRNA are known in the art.
Transcription and translation of plasmids containing nonsense mutations is
typically
carried out in, a cell-free system comprising an E coli S30 extract and
commercially
available enzymes and other reagents. Proteins are purified by chromatography.
See,
for example, Robertson et al., J. Am. Chem. Soc. 113:2722 ( 1991 ), Ellman et
al.,
Methods Enrymol. 202:301 (1991), Chung et al., Science 259:806 (1993), and
Chung
2o et al., Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
In a second method, translation is carried out in Xenopus oocytes by
microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs
(Turcatti et al., J. Biol. Chem. 271:19991 (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 occurnng amino
acids) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-
fluorophenylalanine). The non-naturally occurring amino acid is incorporated
into the
protein in place of its natural counterpart. See, Koide et al., Biochem.
33:7470 (1994).
Naturally occurnng amino acid residues can be converted to non-naturally
occurring
3o species by in vitro chemical modification. Chemical modification can be
combined
with site-directed mutagenesis to further expand the range of substitutions
(Wynn and
Richards, Protein Sci. 2:395 (1993)).
A limited number of non-conservative amino acids, amino acids that
are not encoded by the genetic code, non-naturally occurnng amino acids, and
unnatural amino acids may be substituted for Zsig67 amino acid residues.
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34
Essential amino acids in the polypeptides of the present invention can
be identified according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081 (1989), Bass et al., Proc. Nat'1 Acad. Sci. USA 88:4498 (1991),
Coombs and
Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis
and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In
the latter
technique, single alanine mutations are introduced at every residue in the
molecule,
and the resultant mutant molecules are tested for biological activity to
identify amino
acid residues that are critical to the activity of the molecule. See also,
Hilton et al., J.
1o Biol. Chem. 271:4699 (1996).
The location of Zsig67 receptor binding domains can also be
determined by physical analysis of structure, as determined by such techniques
as
nuclear magnetic resonance, crystallography, electron diffraction or
photoaffinity
labeling, in conjunction with mutation of putative contact site amino acids.
See, for
example, de Vos et al., Science 255:306 (1992), Smith et al., J. Mol. Biol.
224:899
(1992), and Wlodaver et al., FEBS Lett. 309:59 (1992). Moreover, Zsig67
labeled with
biotin or FITC can be used for expression cloning of Zsig67 receptors.
Multiple amino acid substitutions can be made and tested using known
methods of mutagenesis and screening, such as those disclosed by Reidhaar-
Olson and
2o Sauer (Science 241:53 (1988)) or Bowie and Sauer (Proc. Nat'l Acad. Sci.
USA
86:2152 (1989)). Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for functional
polypeptide, and then sequencing the mutagenized polypeptides to determine the
spectrum of allowable substitutions at each position. Other methods that can
be used
include phage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner
et al.,
U.S. Patent No. 5,223,409, Huse, international publication No. WO 92/06204,
and
region-directed mutagenesis (Derbyshire et al., Gene 46:145 (1986), and Ner et
al.,
DNA 7:127, (1988)).
Variants of the disclosed Zsig67 nucleotide and polypeptide sequences
3o can also be generated through DNA shuffling as disclosed by Stemmer, Nature
370:389 (1994), Stemmer, Proc. Nat'1 Acad Sci. USA 91:10747 (1994), and
international publication No. 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
CA 02358930 2001-07-17
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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.
5 Mutagenesis methods as disclosed herein can be combined with high-
throughput, automated screening methods to detect activity of cloned,
mutagenized
polypeptides in host cells. Mutagenized DNA molecules that encode biologically
active polypeptides, or polypeptides that bind with anti-Zsig67 antibodies,
can be
recovered from the host cells and rapidly sequenced using modern equipment.
These
1 o 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.
The present invention also includes "functional fragments" of Zsig67
polypeptides and nucleic acid molecules encoding such functional fragments.
Routine
15 deletion analyses of nucleic acid molecules can be performed to obtain
functional
fragments of a nucleic acid molecule that encodes a Zsig67 polypeptide. As an
illustration, DNA molecules having the nucleotide sequence of SEQ ID NO:1 can
be
digested with Ba131 nuclease to obtain a series of nested deletions. The
fragments are
then inserted into expression vectors in proper reading frame, and the
expressed
2o polypeptides are isolated and tested for the ability to bind anti-Zsig67
antibodies. One
alternative to exonuclease digestion is to use oligonucleotide-directed
mutagenesis to
introduce deletions or stop codons to specify production of a desired
fragment.
Alternatively, particular fragments of a Zsig67 gene can be synthesized using
the
polymerase chain reaction.
25 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 functional analysis of
proteins are
described by, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993),
Content
3o et al., "Expression and preliminary deletion analysis of the 42 kDa 2-SA
synthetase
induced by human interferon," in Biological Interferon Systems, Proceedings of
ISII~-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff
1987),
Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation, Tool.
I,
Boynton et al., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et
al., J.
35 Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291
(1995);
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36
Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al., Plant
Molec. Biol. 30:1 (1996).
In addition, sequence analysis can also identify functional fragments of
Zsig67. For example, comparison with other members of the secretin-glucagon-
VIP
peptide hormone family indicates that a polypeptide consisting of amino acid
residues
52 to 85 of SEQ ID N0:2 can effectively bind to the cognate Zsig67 receptor.
The present invention also contemplates functional fragments of a
Zsig67 gene that has amino acid changes, compared with the amino acid sequence
of
SEQ ID N0:2. A variant Zsig67 gene can be identified on the basis of structure
by
l0 determining the level of identity with nucleotide and amino acid sequences
of SEQ ID
NOs:l and 2, as discussed above. An alternative approach to identifying a
variant
gene on the basis of structure is to determine whether a nucleic acid molecule
encoding a potential variant Zsig67 gene can hybridize to a nucleic acid
molecule
having the nucleotide sequence of SEQ ID NO:1, as discussed above.
The present invention also provides polypeptide fragments or peptides
comprising an epitope-bearing portion of a Zsig67 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
2o methods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA
81:3998 (1983)).
In contrast, polypeptide fragments or peptides may comprise an
"antigenic epitope," which is a region of a protein molecule to which an
antibody can
specifically bind. Certain epitopes consist of a linear or contiguous stretch
of amino
acids, and the antigenicity of such an epitope is not disrupted by denaturing
agents. It
is known in the art that relatively short synthetic peptides that can mimic
epitopes of a
protein can be used to stimulate the production of antibodies against the
protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)). Accordingly, antigenic
epitope-
bearing peptides and polypeptides of the present invention are useful to raise
antibodies that bind with the polypeptides described herein.
Antigenic epitope-bearing peptides and polypeptides can contain at
least four to ten amino acids, at least ten to fifteen amino acids, or about
15 to about
30 amino acids of SEQ ID N0:2. Such epitope-bearing peptides and polypeptides
can
be produced by fragmenting a Zsig67 polypeptide, or by chemical peptide
synthesis,
as described herein. Moreover, epitopes can be selected by phage display of
random
peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol.
5:268
(1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)). Standard
methods
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37
for identifying epitopes and producing antibodies from small peptides that
comprise
an epitope are described, for example, by Mole, "Epitope Mapping," in Methods
in
Molecular Biology, Yol. 10, Manson (ed.), pages 105-116 (The Humana Press,
Inc.
1992), Price, "Production and Characterization of Synthetic Peptide-Derived
Antibodies," in Monoclonal Antibodies: Production, Engineering, and Clinical
Application, Ritter and Ladyman (eds.), pages 60-84 (Cambridge University
Press
1995), and Coligan et al. (eds.), Current Protocols in Immunology, pages 9.3.1
- 9.3.5
and pages 9.4.1 - 9.4.11 (John Wiley & Sons 1997).
Regardless of the particular nucleotide sequence of a variant Zsig67
1 o gene, the gene encodes a polypeptide that is characterized by its ability
to bind
specifically to an anti-Zsig67 antibody.
For any Zsig67 polypeptide, including variants and fusion proteins, one
of ordinary skill in the art can readily generate a fully degenerate
polynucleotide
sequence encoding that variant using the information set forth in Tables 1 and
2
above. Moreover, those of skill in the art can use standard software to devise
Zsig67
variants based upon the nucleotide and amino acid sequences described herein.
Accordingly, the present invention includes a computer-readable medium encoded
with a data structure that provides at least one of the following sequences:
SEQ ID
NO:1, SEQ ID N0:2, and SEQ ID N0:3. Suitable forms of computer-readable media
2o include magnetic media and optically-readable media. Examples of magnetic
media
include a hard or fixed drive, a random access memory (RAM) chip, a floppy
disk,
digital linear tape (DLT), a disk cache, and a ZIP disk. Optically readable
media are
exemplified by compact discs (e.g., CD-read only memory (ROM), CD-rewritable
(RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-
ROM,
DVD-RAM, and DVD+RW).
5. Production of Zsig67 Fusion Proteins
Fusion proteins of Zsig67 can be used to express Zsig67 in a
recombinant host, and to isolate expressed Zsig67. As described below,
particular
3o Zsig67 fusion proteins also have uses in diagnosis and therapy.
One type of fusion protein comprises a peptide that guides a Zsig67
polypeptide from a recombinant host cell. To direct a Zsig67 polypeptide into
the
secretory pathway of a eukaryotic host cell, a secretory signal sequence (also
known as
a signal peptide, a leader sequence, prepro sequence or pre sequence) is
provided in
the Zsig67 expression vector. While the secretory signal sequence may be
derived
from Zsig67, a suitable signal sequence may also be derived from another
secreted
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38
protein or synthesized de novo. The secretory signal sequence is operably
linked to a
Zsig67-encoding sequence such that the two sequences are joined in the correct
reading frame and positioned to direct the newly synthesized polypeptide into
the
secretory pathway of the host cell. Secretory signal sequences are commonly
positioned 5' to the nucleotide sequence encoding the polypeptide of interest,
although
certain secretory signal sequences may be positioned elsewhere in the
nucleotide
sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743;
Holland et al.,
U.S. Patent No. 5,143,830).
Although the secretory signal sequence of Zsig67 or another protein
io produced by mammalian cells (e.g., tissue-type plasminogen activator signal
sequence, as described, for example, in U.S. Patent No. 5,641,655) is useful
for
expression of Zsig67 in recombinant mammalian hosts, a yeast signal sequence
is
preferred for expression in yeast cells. Examples of suitable yeast signal
sequences
are those derived from yeast mating phermone a-factor (encoded by the MFal
gene),
invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PHOS
gene). See, for example, Romanos et al., "Expression of Cloned Genes in
Yeast," in
DNA Cloning 2: A Practical Approach, 2"a Edition, Glover and Hames (eds.),
pages
123-167 (Oxford University Press 1995).
In bacterial cells, it is often desirable to express a heterologous protein
2o as a fusion protein to decrease toxicity, increase stability, and to
enhance recovery of
the expressed protein. For example, Zsig67 can be expressed as a fusion
protein
comprising a glutathione S-transferase polypeptide. Glutathione S-transferease
fusion
proteins are typically soluble, and easily purifiable from E coli lysates on
immobilized glutathione columns. - In similar approaches, a Zsig67 fusion
protein
comprising a maltose binding protein polypeptide can be isolated with an
amylose
resin column, while a fusion protein comprising the C-terminal end of a
truncated
Protein A gene can be purified using IgG-Sepharose. Established techniques for
expressing a heterologous polypeptide as a fusion protein in a bacterial cell
are
described, for example, by Williams et al., "Expression of Foreign Proteins in
E. coli
3o Using Plasmid Vectors and Purification of Specific Polyclonal Antibodies,"
in DNA
Cloning 2: A Practical Approach, 2"d Edition, Glover and Hames (Eds.), pages
15-58
(Oxford University Press 1995). In addition, commercially available expression
systems are available. For example, the PINPOINT Xa protein purification
system
(Promega Corporation; Madison, WI) provides a method for isolating a fusion
protein
comprising a polypeptide that becomes biotinylated during expression with a
resin that
comprises avidin.
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39
Peptide tags that are useful for isolating heterologous polypeptides
expressed by either prokaryotic or eukaryotic cells include polyHistidine tags
(which
have an affinity for nickel-chelating resin), c-myc tags, calmodulin binding
protein
(isolated with calmodulin affinity chromatography), substance P, the RYIRS tag
(which binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag
(which
binds with anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.
Biophys. 329:215 ( 1996), Morganti et al., Biotechnol. Appl. Biochem. 23:67 (
1996),
and Zheng et al., Gene 186:55 (1997). Nucleic acid molecules encoding such
peptide
tags are available, for example, from Sigma-Aldrich Corporation (St. Louis,
MO).
1o The present invention also contemplates that the use of the secretory
signal sequence contained in the Zsig67 polypeptides of the present invention
to direct
other polypeptides into the secretory pathway. A signal fusion polypeptide can
be
made wherein a secretory signal sequence derived from amino acid residues 1 to
28 of
SEQ ID N0:2 is operably linked to another polypeptide using methods known in
the
art and disclosed herein. The secretory signal sequence contained in the
fusion
polypeptides of the present invention is preferably fused amino-terminally to
an
additional peptide to direct the additional peptide into the secretory
pathway. Such
constructs have numerous applications known in the art. For example, these
novel
secretory signal sequence fusion constructs can direct the secretion of an
active
component of a normally non-secreted protein, such as a receptor. Such fusions
may
be used in a transgenic animal or in a cultured recombinant host to direct
peptides
through the secretory pathway. With regard to the latter, exemplary
polypeptides
include pharmaceutically active molecules such as Factor VIIa, proinsulin,
insulin,
follicle stimulating hormone, tissue type plasminogen activator, tumor
necrosis factor,
interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-S, IL-6, IL-7,
IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15), colony stimulating factors
(e.g.,
granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-
colony
stimulating factor (GM-CSF)), interferons (e.g., interferons-a, -(3, -y, -w, -
S, and -i),
the stem cell growth factor designated "S 1 factor," erythropoietin, and
thrombopoietin. The Zsig67 secretory signal sequence contained in the fusion
polypeptides of the present invention is preferably fused amino-terminally to
an
additional peptide to direct the additional peptide into the secretory
pathway. Fusion
proteins comprising a Zsig67 secretory signal sequence can be constructed
using
standard techniques.
Another form of fusion protein comprises a Zsig67 polypeptide and an
immunoglobulin heavy chain constant region, typically an Fc fragment, which
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contains two constant region domains and a hinge region but lacks the variable
region.
As an illustration, Chang et al., U.S. Patent No. 5,723,125, describe a fusion
protein
comprising a human interferon and a human immunoglobulin Fc fragment. The C-
terminal of the interferon is linked to the N-terminal of the Fc fragment by a
peptide
5 linker moiety. An example of a peptide linker is a peptide comprising
primarily a T
cell inert sequence, which is immunologically inert. An exemplary peptide
linker has
the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ ID N0:4). In this fusion
protein, a preferred Fc moiety is a human y4 chain, which is stable in
solution and has
little or no complement activating activity. Accordingly, the present
invention
to contemplates a Zsig67 fusion protein that comprises a Zsig67 moiety and a
human Fc
fragment, wherein the C-terminus of the Zsig67 moiety is attached to the N-
terminus
of the Fc fragment via a peptide linker, such as a peptide consisting of the
amino acid
sequence of SEQ ID N0:4. The Zsig67 moiety can be a Zsig67 molecule or a
fragment thereof.
15 In another variation, a Zsig67 fusion protein comprises an IgG
sequence, a Zsig67 moiety covalently joined to the amino terminal end of the
IgG
sequence, and a signal peptide that is covalently joined to the amino terminal
of the
Zsig67 moiety, wherein the IgG sequence consists of the following elements in
the
following order: a hinge region, a CHZ domain, and a CH3 domain. Accordingly,
the
2o IgG sequence lacks a CH, domain. The Zsig67 moiety displays a Zsig67
activity,
such as the ability to bind with a Zsig67 receptor. This general approach to
producing
fusion proteins that comprise both antibody and nonantibody portions has been
described by LaRochelle et al., EP 742830 (WO 95/21258).
Fusion proteins comprising a Zsig67 moiety and an Fc moiety can be
25 used, for example, as an in vitro assay tool. For example, the presence of
a Zsig67
receptor in a biological sample can be. detected using a Zsig67-antibody
fusion
protein, in which the Zsig67 moiety is used to target the cognate receptor,
and a
macromolecule, such as Protein A or anti-Fc antibody, is used to detect the
bound
fusion protein-receptor complex. Moreover, such fusion proteins can be used to
3o identify agonists and antagonists that interfere with the binding of Zsig67
to its
receptor. In addition, antibody-Zsig67 fusion proteins, comprising antibody
variable
domains, are useful as therapeutic proteins, in which the antibody moiety
binds with a
target antigen, such as a tumor associated antigen.
Fusion proteins can be prepared by methods known to those skilled in
35 the art by preparing each component of the fusion protein and chemically
conjugating
them. Alternatively, a polynucleotide encoding both components of the fusion
protein
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41
in the proper reading frame can be generated using known techniques and
expressed
by the methods described herein. General methods for enzymatic and chemical
cleavage of fusion proteins are described, for example, by Ausubel (1995) at
pages 16-
19 to 16-25.
s
6. Production of Zsig67 Polypeptides in Cultured Cells
The polypeptides of the present invention, including full-length
polypeptides, functional fragments, and fusion proteins, can be produced in
recombinant
host cells following conventional techniques. To express a Zsig67 gene, a
nucleic acid
l0 molecule encoding the polypeptide must be operably linked to regulatory
sequences that
control transcriptional expression in an expression vector and then,
introduced into a
host cell. In addition to transcriptional regulatory sequences, such as
promoters and
enhancers, expression vectors can include translational regulatory sequences
and a
marker gene which is suitable for selection of cells that carry the expression
vector.
1 s Expression vectors that are suitable for production of a foreign protein
in eukaryotic cells typically contain (1) prokaryotic DNA elements coding for
a
bacterial replication origin and an antibiotic resistance marker to provide
for the
growth and selection of the expression vector in a bacterial host; (2)
eukaryotic DNA
elements that control initiation of transcription, such as a promoter; and (3)
DNA
20 elements that control the processing of transcripts, such as a
transcription
termination/polyadenylation sequence. As discussed above, expression vectors
can
also include nucleotide sequences encoding a secretory sequence that directs
the
heterologous polypeptide into the secretory pathway of a host cell. For
example, a
Zsig67 expression vector may comprise a Zsig67 gene and a secretory sequence
2s derived from a Zsig67 gene or another secreted gene.
Zsig67 proteins of the present invention may be expressed in
mammalian cells. Examples of suitable mammalian host cells include African
green
monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-
HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC
3o CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34),
Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 [Chasm et al.,
Som. Cell. Molec. Genet. 12:555 1986]), rat pituitary cells (GHl; ATCC CCL82),
HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548)
SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and marine
35 embryonic cells (NIH-3T3; ATCC CRL 1658).
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42
For a mammalian host, the transcriptional and translational regulatory
signals may be derived from viral sources, such as adenovirus, bovine
papilloma virus,
simian virus, or the like, in which the regulatory signals are associated with
a par-
ticular gene which has a high level of expression. Suitable transcriptional
and
translational regulatory sequences also can be obtained from mammalian genes,
such
as actin, collagen, myosin, and metallothionein genes.
Transcriptional regulatory sequences include a promoter region
sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic
promoters
include the promoter of the mouse metallothionein 1 gene (Hamer et al., J.
Molec.
1o Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell
31:355
(1982)), the SV40 early promoter (Benoist et al., Nature 290:304 (1981)), the
Rous
sarcoma virus promoter (Gorman et al., Proc. Nat'l Acad Sci. USA 79:6777
(1982)),
the cytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and the
mouse
mammary tumor virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein Engineering:
Principles
and Practice, Cleland et al. (eds.), pages 163-181 (John Wiley & Sons, Inc.
1996)).
Alternatively, a prokaryotic promoter, such as the bacteriophage T3
RNA polymerase promoter, can be used to control Zsig67 gene expression in
mammalian cells if the prokaryotic promoter is regulated by a eukaryotic
promoter
(Zhou et al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl. Acids
Res.
19:4485 (1991)).
An expression vector can be introduced into host cells using a variety of
standard techniques including calcium phosphate transfection, liposome-
mediated
transfection, microprojectile-mediated delivery, electroporation, and the
like.
Preferably, the transfected cells are selected and propagated to provide
recombinant host
cells that comprise the expression vector stably integrated in the host cell
genome.
Techniques for introducing vectors into eukaryotic cells and techniques for
selecting
such stable transformants using a dominant selectable marker are described,
for example,
by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expression Protocols
(Humana Press 1991 ).
For example, one suitable selectable marker is a gene that provides
resistance to the antibiotic neomycin. In this case, selection is carried out
in the
presence of a neomycin-type drug, such as G-418 or the like. Selection systems
can
also be used to increase the expression level of the gene of interest, a
process referred
to as "amplification." Amplification is carried out by culturing transfectants
in the
presence of a low level of the selective agent and then increasing the amount
of
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43
selective agent to select for cells that produce high levels of the products
of the
introduced genes. An illustrative amplifiable selectable marker is
dihydrofolate
reductase, which confers resistance to methotrexate. Other drug resistance
genes (e.g.,
hygromycin resistance, mufti-drug resistance, puromycin acetyltransferase) can
also
be used. Alternatively, markers that introduce an altered phenotype, such as
green
fluorescent protein, or cell surface proteins such as CD4, CDB, Class I MHC,
placental
alkaline phosphatase may be used to sort transfected cells from untransfected
cells by
such means as FACS sorting or magnetic bead separation technology.
Zsig67 polypeptides can also be produced by cultured mammalian cells
l0 using a viral delivery system. Exemplary viruses for this purpose include
adenovirus,
herpesvirus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a
double
stranded DNA virus, is currently the best studied gene transfer vector for
delivery of
heterologous nucleic acid (for a review, see Becker et al., Meth. Cell Biol.
43:161
(1994), and Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages of
the
adenovirus system include the accommodation of relatively large DNA inserts,
the
ability to grow to high-titer, the ability to infect a broad range of
mammalian cell
types, and flexibility that allows use with a large number of available
vectors
containing different promoters.
By deleting portions of the adenovirus genome, larger inserts (up to 7
kb) of heterologous DNA can be accommodated. These inserts can be incorporated
into the viral DNA by direct ligation or by homologous recombination with a co
transfected plasmid. An option is to delete the essential EI gene from the
viral vector,
which results in the inability to replicate unless the El gene is provided by
the host
cell. Adenovirus vector-infected human 293 cells (ATCC Nos. CRL-1573, 45504,
45505), for example, can be grown as adherent cells or in suspension culture
at
relatively high cell density to produce significant amounts of protein (see
Gamier et
al., Cytotechnol. 15:145 ( 1994)).
Zsig67 genes may also be expressed in other higher eukaryotic cells,
such as avian, fungal, insect, yeast, or plant cells. The baculovirus system
provides an
efficient means to introduce cloned Zsig67 genes into insect cells. Suitable
expression
vectors are based upon the Autographa californica multiple nuclear
polyhedrosis virus
(AcMNPV), and contain well-known promoters such as Drosophila heat shock
protein (hsp) 70 promoter, Autographa californica nuclear polyhedrosis virus
immediate-early gene promoter (ie-1 ) and the delayed early 39K promoter,
baculovirus p10 promoter, and the Drosophila metallothionein promoter. A
second
method of making recombinant baculovirus utilizes a transposon-based system
CA 02358930 2001-07-17
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44
described by Luckow (Luckow, et al., J. Virol. 67:4566 (1993)). This system,
which
utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life Technologies,
Rockville,
MD). This system utilizes a transfer vector, PFASTBAC (Life Technologies)
containing a Tn7 transposon to move the DNA encoding the Zsig67 polypeptide
into a
baculovirus genome maintained in E. coli as a large plasmid called a "bacmid."
See,
Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et al., J.
Gen. Virol.
75:1551 (1994), and Chazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (1995).
In
addition, transfer vectors can include an in-frame fusion with DNA encoding an
epitope tag at the C- or N-terminus of the expressed Zsig67 polypeptide, for
example,
1o a Glu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad Sci. 82:7952
(1985)).
Using a technique known in the art, a transfer vector containing a ZSig67 gene
is
transformed into E. coli, and screened for bacmids which contain an
interrupted lacZ
gene indicative of recombinant baculovirus. The bacmid DNA containing the
recombinant baculovirus genome is then isolated using common techniques.
The illustrative PFASTBAC vector can be modified to a considerable
degree. For example, the polyhedrin promoter can be removed and substituted
with
the baculovirus basic protein promoter (also known as Pcor, p6.9 or .MP
promoter)
which is expressed earlier in the baculovirus infection, and has been shown to
be
advantageous for expressing secreted proteins (see, for example, Hill-Perkins
and
Possee, J. Gen. Virol. 71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551
(1994),
and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In such transfer
vector constructs, a short or long version of the basic protein promoter can
be used.
Moreover, transfer vectors can be constructed which replace the native Zsig67
secretory signal sequences with secretory signal sequences derived from insect
proteins. For example, a secretory signal sequence from Ecdysteroid
Glucosyltransferase (EGT), honey bee Melittin (Invitrogen Corporation;
Carlsbad,
CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs
to
replace the native Zsig67 secretory signal sequence.
The recombinant virus or bacmid is used to transfect host cells.
3o Suitable insect host cells include cell lines derived from IPLB-Sf 21, a
Spodoptera
frugiperda pupal ovarian cell line, such as S~ (ATCC CRL 1711 ), Sf21 AE, and
Sf21
(Invitrogen Corporation; San Diego, CA), as well as Drosophila Schneider-2
cells,
and the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni (U.S.
Patent
No. 5,300,435). Commercially available serum-free media can be used to grow
and to
maintain the cells. Suitable media are 500 IIT"" (Life Technologies) or ESF
921T""
(Expression Systems) for the Sf~ cells; and Ex-ce11O405T"" (JRH Biosciences,
Lenexa,
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KS) or Express FiveOT"" (Life Technologies) for the T. ni cells. When
recombinant
virus is used, the cells are typically grown up from an inoculation density of
approximately 2-5 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
5 typically near 3.
Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus
Vectors," in Methods in Molecular Biology, Volume 7: Gene Transfer and
Expression
Protocols, Murray (ed.), pages 147-168 (The Humana Press, Inc. 1991), by Patel
et
l0 al., "The baculovirus expression system," in DNA Cloning 2: Expression
Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University Press 1995),
by
Ausubel (1995) at pages 16-37 to 16-57, by Richardson (ed.), Baculovirus
Expression
Protocols (The Humana Press, Inc. 1995), and by Lucknow, "Insect Cell
Expression
Technology," in Protein Engineering: Principles and Practice, Cleland et al.
(eds.),
15 pages 183-218 (John Wiley & Sons, Inc. 1996).
Fungal cells, including yeast cells, can also be used to express the
genes described herein. Yeast species of particular interest in this regard
include
Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Suitable
promoters for expression in yeast include promoters from GALL (galactose), PGK
20 (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOXI (alcohol
oxidase),
HIS4 (histidinol dehydrogenase), and the like. Many yeast cloning vectors have
been
designed and are readily available. These vectors include YIp-based vectors,
such as
YIpS, YRp vectors, such as YRp 17, YEp vectors such as YEp 13 and YCp vectors,
such as YCpl9. Methods for transforming S. cerevisiae cells with exogenous DNA
25 and producing recombinant polypeptides therefrom are disclosed by, for
example,
Kawasaki, U.S. Patent No. 4,599,311, Kawasaki et al., U.S. Patent No.
4,931,373,
Brake, U.S. Patent No. 4,870,008, Welch et al., U.S. Patent No. 5,037,743, and
Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly drug resistance or the
30 ability to grow in the absence of a particular nutrient (e.g., leucine). An
illustrative
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. Additional
suitable
promoters and terminators for use in yeast include those from glycolytic
enzyme genes
35 (see, e.g., Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al., U.S.
Patent No.
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46
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
to Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming
Neurospora are
disclosed by Lambowitz, U.S. Patent No. 4,486,533.
For example, the use of Pichia methanolica as host for the production
of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5,716,808,
Raymond, U.S. Patent No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998); and
in
international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO
98/02565. DNA molecules for use in transforming P. methanolica will commonly
be
prepared as double-stranded, circular plasmids, which .can be 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.
2o methanolica alcohol utilization gene (AUGl or AUG2). Other useful promoters
include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase
(FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the
host
chromosome, it is preferred to have the entire expression segment of the
plasmid
flanked at both ends by host DNA sequences. A suitable selectable marker for
use in
Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-
5-
aminoimidazole carboxylase (AIRC; EC 4.1.1.21), and which allows ade2 host
cells
to grow in the absence of adenine. For large-scale, industrial processes where
it is
desirable to minimize the use of methanol, it is preferred to use host cells
in which
both methanol utilization genes (AUGl and AUG2) are deleted. For production of
3o secreted proteins, host cells deficient in vacuolar protease genes (PEP4
and PRBI) are
preferred. Electroporation is used to facilitate the introduction of a plasmid
containing
DNA encoding a polypeptide of interest into P. methanolica cells. P.
methanolica
cells can be transformed by electroporation using an exponentially decaying,
pulsed
electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably
about 3.75
kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably
about 20
milliseconds.
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Expression vectors can also be introduced into plant protoplasts, intact
plant tissues, or isolated plant cells. Methods for introducing expression
vectors into
plant tissue include the direct infection or co-cultivation of plant tissue
with
Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA injection,
electroporation, and the like. See, for example, Horsch et al., Science
227:1229 (1985),
Klein et al., Biotechnology 10:268 (1992), and Miki et al., "Procedures for
Introducing
Foreign DNA into Plants," in Methods in Plant Molecular Biology and
Biotechnology,
Glick et al. (eds.), pages 67-88 (CR,C Press, 1993).
Alternatively, Zsig67 genes can be expressed in prokaryotic host cells.
1 o Suitable promoters that can be used to express Zsig67 polypeptides in a
prokaryotic
host are well-known to those of skill in the art and include promoters capable
of
recognizing the T4, T3, Sp6 and T7 polymerases, the PR and PL promoters of
bacteriophage lambda, the trp, recA, heat shock, lacUVS, tac, lpp-lacSpr,
phoA, and
lacZ promoters of E. coli, promoters of B. subtilis, the promoters of the
bacteriophages
of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda,
the
bla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl
trans-
ferase gene. Prokaryotic promoters have been reviewed by Glick, J. Ind.
Microbiol.
1:277 (1987), Watson et al., Molecular Biology of the Gene, 4th Ed. (Benjamin
Cummins 1987), and by Ausubel et al. (1995).
Useful prokaryotic hosts include E. coli and Bacillus subtilus. Suitable
strains of E coli include BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DHl,
DH4I, DHS, DHSI, DHSIF', DHSIMCR, DH10B, DHlOB/p3, DH11S, C600, HB101,
JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and
ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic
Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886, MI119,
MI120,
and B 170 (see, for example, Hardy, "Bacillus Cloning Methods," in DNA
Cloning: A
Practical Approach, Glover (ed.) (IRL Press 1985)).
When expressing a Zsig67 polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as insoluble granules,
or may
3o be directed to the periplasmic space by a bacterial secretion sequence. In
the former
case, the cells are lysed, and the granules are recovered and denatured using,
for
example, guanidine isothiocyanate or urea. The denatured polypeptide can then
be
refolded and dimerized by diluting the denaturant, such as by dialysis against
a
solution of urea and a combination of reduced and oxidized glutathione,
followed by
dialysis against a buffered saline solution. In the latter case, the
polypeptide can be
recovered from the periplasmic space in a soluble and functional form by
disrupting
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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.
Methods for expressing proteins in prokaryotic hosts are well-known to
those of skill in the art (see, for example, Williams et al., "Expression of
foreign
proteins in E. coli using plasmid vectors and purification of specific
polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
(eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic Manipulation and
Expression of Antibodies," in Monoclonal Antibodies: Principles and
Applications,
to page 137 (Wiley-Liss, Inc. 1995), and Georgiou, "Expression of Proteins in
Bacteria,"
in Protein Engineering: Principles and Practice, Cleland et al. (eds.), page
101 (John
Wiley & Sons, Inc. 1996)).
Standard methods for introducing expression vectors into bacterial, yeast,
insect, and plant cells are provided, for example, by Ausubel (1995).
General methods for expressing and recovering foreign protein produced
by a mammalian cell system are provided by, for example, Etcheverry,
"Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein Engineering:
Principles
and Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard
techniques for recovering protein produced by a bacterial system is provided
by, for
2o example, Grisshammer et al., "Purification of over-produced proteins from
E. coli
cells," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
(eds.), pages
59-92 (Oxford University Press 1995). Established methods for isolating
recombinant
proteins from a baculovirus system are described by Richardson (ed.),
Baculovirus
Expression Protocols (The Humana Press, Inc. 1995).
As an alternative, polypeptides of the present invention can be
synthesized by exclusive solid phase synthesis, partial solid phase methods,
fragment
condensation or classical solution synthesis. These synthesis methods are well-
known
to those of skill in the art (see, for example, Merrifield, J. Am. Chem. Soc.
85:2149
(1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce
3o Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton
et al.,
Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989), Fields
and
Colowick, "Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289
(Academic Press 1997), and Lloyd-Williams et al., Chemical Approaches to the
Synthesis of Peptides and Proteins (CRC Press, Inc. 1997)). Variations in
total
chemical synthesis strategies, such as "native chemical ligation" and
"expressed
protein ligation" are also standard (see, for example, Dawson et al., Science
266:776
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49
(1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,
Methods
Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA 95:6705
(1998), and
Severinov and Muir, J. Biol. Chem. 273:16205 (1998)).
Peptides and polypeptides of the present invention comprise at least
six, at least nine, or at least 15 contiguous amino acid residues of SEQ ID
N0:2. As
an illustration, polypeptides can comprise at least six, at least nine, or at
least 15
contiguous amino acid residues of any of the following amino acid sequences of
SEQ
ID N0:2: amino acid residues amino acid residues 29 to 104, amino acid
residues 52
to 104, amino acid residues 52 to 85, amino acid residues 68 to 104, and amino
acid
to residues 73 to 104. Within certain embodiments of the invention, the
polypeptides
comprise 20, 30, 40, 50, or more contiguous residues of these amino acid
sequences.
Nucleic acid molecules encoding such peptides and polypeptides are useful as
polymerase chain reaction primers and probes.
is 7. Isolation of Zsig67 Polypeptides
The polypeptides of the present invention can be purified to at least
about 80% purity, to at least about 90% purity, to at least about 95% purity,
or even
greater than 95% purity with respect to contaminating macromolecules,
particularly
other proteins and nucleic acids, and free of infectious and pyrogenic agents.
The
2o polypeptides of the present invention may also be purified to a
pharmaceutically pure
state, which is greater than 99.9% pure. In certain preparations, a purified
polypeptide
is substantially free of other polypeptides, particularly other polypeptides
of animal
origin.
Fractionation and/or conventional purification methods can be used to
25 obtain preparations of Zsig67 purified from natural sources (e.g.,
pancreatic tissue),
and recombinant Zsig67 polypeptides and fusion Zsig67 polypeptides purified
from
recombinant host cells. In general, ammonium sulfate precipitation and acid or
chaotrope extraction may be used for fractionation of samples. Exemplary
purification steps may include hydroxyapatite, size exclusion, FPLC and
reverse-
30 phase high performance liquid chromatography. Suitable chromatographic
media
include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty
silicas, and
the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary
chromatographic media include those media derivatized with phenyl, butyl, or
octyl
groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso
Haas,
35 Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic
resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid
supports
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include glass beads, silica-based resins, cellulosic resins, agarose beads,
cross-linked
agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the
like that
are insoluble under the conditions in which they are to be used. These
supports may
be modified with reactive groups that allow attachment of proteins by amino
groups,
5 carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate
moieties.
Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl
activation,
hydrazide activation, and carboxyl and amino derivatives for carbodiimide
coupling
chemistries. These and other solid media are well known and widely used in the
art,
1 o and are available from commercial suppliers. Selection of a particular
method for
polypeptide isolation and purification is a matter of routine design and is
determined
in part by the properties of the chosen support. See, for example, Affinity
Chromatography. Principles & Methods (Pharmacia LKB Biotechnology 1988), and
Doonan, Protein Purification Protocols (The Humana Press 1996).
15 Additional variations in Zsig67 isolation and purification can be
devised by those of skill in the art. For example, anti-Zsig67 antibodies,
obtained as
described below, can be used to isolate large quantities of protein by
immunoaffinity
purification. Moreover, methods for binding ligands, such as Zsig67, to
receptor
polypeptides bound to support media are well known in the art.
2o The polypeptides of the present invention can also be isolated by
exploitation of particular properties. For example, immobilized metal ion
adsorption
(IMAC) chromatography can be used to purify histidine-rich proteins, including
those
comprising polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions
to form a chelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-rich
proteins
25 will be adsorbed to this matrix with differing affinities, depending upon
the metal ion
used, and will be eluted by competitive elution, lowering the pH, or use of
strong
chelating agents. Other methods of purification include purification of
glycosylated
proteins by lectin affinity chromatography and ion exchange chromatography (M.
Deutscher, (ed.), Meth. Enzymol. 182:529 ( 1990)). Within additional
embodiments of
3o 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.
Zsig67 polypeptides or fragments thereof may also be prepared through
chemical synthesis, as described below. Zsig67 polypeptides may be monomers or
35 multimers; glycosylated or non-glycosylated; pegylated or non-pegylated;
and may or
may not include an initial methionine amino acid residue.
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51
8. Zsig67 Analogs and the Zsig67 Receptor
One general class of Zsig67 analogs are variants having an amino acid
sequence that is a mutation of the amino acid sequence disclosed herein.
Another
general class of Zsig67 analogs is provided by anti-idiotype antibodies, and
fragments
thereof, as described below. Moreover, recombinant antibodies comprising anti-
idiotype variable domains can be used as analogs (see, for example, Monfardini
et al.,
Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domains of
anti-
idiotype Zsig67 antibodies mimic Zsig67, these domains can provide either
Zsig67
agonist or antagonist activity. As an illustration, Lim and Larger, J.
Interferon Res.
13:295 (1993), describe anti-idiotypic interferon-a antibodies that have the
properties
of either interferon-a agonists or antagonists.
Another approach to identifying Zsig67 analogs is provided by the use
of combinatorial libraries. Methods for constructing and screening phage
display and
other combinatorial libraries are provided, for example, by Kay et al., Phage
Display
of Peptides and Proteins (Academic Press 1996), Verdine, U.S. Patent No.
5,783,384,
Kay, et. al., U.S. Patent No. 5,747,334, and Kauffman et al., U.S. Patent No.
5,723,323.
Zsig67 and its analogs can be used to identify and to isolate Zsig67
2o receptors. For example, proteins and peptides of the present invention can
be
immobilized on a column and used to bind receptor proteins from membrane
preparations that are run over the column (Hermanson et al. (eds.),
Immobilized
Amity Ligand Techniques, pages 195-202 (Academic Press 1992)). Radiolabeled or
affinity labeled Zsig67 polypeptides can also be used to identify or to
localize Zsig67
receptors in a biological sample (see, for example, Deutscher (ed.), Methods
in
Enzymol., vol. 182, pages 721-37 (Academic Press 1990); Brunner et al., Ann.
Rev.
Biochem: 62:483 (1993); Fedan et al., Biochem. Pharmacol. 33:1167 (1984)).
Also
see, Varthakavi and Minocha, J. Gen. Virol. 77:1875 (1996), who describe the
use of
anti-idiotype antibodies for receptor identification.
3o As a receptor ligand, the activity of Zsig67 can be measured by a
silicon-based biosensor microphysiometer which measures the extracellular
acidification rate or proton excretion associated with receptor binding and
subsequent
cellular responses. An exemplary device is the CYTOSENSOR Microphysiometer
manufactured by Molecular Devices Corp. (Sunnyvale, CA). A variety of cellular
responses, such as cell proliferation, ion transport, energy production,
inflammatory
response, regulatory and receptor activation, and the like, can be measured by
this
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52
method (see, for example, McConnell et al., Science 257:1906 (1992), Pitchford
et al.,
Meth. Enzymol. 228:84 (1997), Arimilli et al., J. Immunol. Meth. 212:49
(1998), and
Van Liefde et al., Eur. J. Pharmacol. 346:87 (1998)). Moreover, the
microphysiometer can be used for assaying adherent or non-adherent eukaryotic
cells.
Since energy metabolism is coupled with the use of cellular ATP, any
event which alters cellular ATP levels, such as receptor activation and the
initiation of
signal transduction, will cause a change in cellular acid section. By
measuring
extracellular acidification changes in cell media over time, therefore, the
microphysiometer directly measures cellular responses to various stimuli,
including
to Zsig67, its agonists, or antagonists. The microphysiometer can be used to
measure
responses of a Zsig67-responsive eukaryotic cell, compared to a control
eukaryotic
cell that does not respond to Zsig67 polypeptide. Zsig67 responsive eukaryotic
cells
comprise cells into which a receptor for Zsig67 has been transfected to create
a cell
that is responsive to Zsig67, or cells that are naturally responsive to
Zsig67. Zsig67
modulated cellular responses are measured by a change (e.g., an increase or
decrease
in extracellular acidification) in the response of cells exposed to Zsig67,
compared
with control cells that have not been exposed to Zsig67.
Accordingly, a microphysiometer can be used to identify cells, tissues,
or cell lines which respond to a Zsig67 stimulated pathway, and which express
a
functional Zsig67 receptor. As an illustration, cells that express a
functional Zsig67
receptor can be identified by (a) providing test cells, (b) incubating a first
portion of
the test cells in the absence of Zsig67, (c) incubating a second portion of
the test cells
in the presence of Zsig67, and (d) detecting a change (e.g., an increase or
decrease in
extracellular acidification rate, as measured by a microphysiometer) in a
cellular
response of the second portion of the test cells, as compared to the first
portion of the
test cells, wherein such a change in cellular response indicates that the test
cells
express a functional Zsig67 receptor. An additional negative control may be
included
in which a portion of the test cells is incubated with Zsig67 and an anti-
Zsig67
antibody to inhibit the binding of Zsig67 with its cognate receptor.
3o The microphysiometer also provides one means to identify Zsig67
agonists. For example, agonists of Zsig67 can be identified by a method,
comprising
the steps of (a) providing cells responsive to Zsig67, (b) incubating a first
portion of
the cells in the absence of a test compound, (c) incubating a second portion
of the cells
in the presence of a test compound, and (d) 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, wherein such a change in cellular
response
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53
indicates that the test compound is a Zsig67 agonist. An illustrative change
in cellular
response is a measurable change in extracellular acidification rate, as
measured by a
microphysiometer. Moreover, incubating a third portion of the cells in the
presence of
Zsig67 and in the absence of a test compound can be used as a positive control
for the
Zsig67 responsive cells, and as a control to compare the agonist activity of a
test
compound with that of Zsig67. An additional control may be included in which a
portion of the cells is incubated with a test compound (or Zsig67) and an anti-
Zsig67
antibody to inhibit the binding of the test compound (or Zsig67) with the
Zsig67
receptor.
l0 A Zsig67 variant gene product that lacks biological activity may be a
Zsig67 antagonist. These biologically-inactive Zsig67 variants can be
initially
identified on the basis of hybridization analysis, sequence identity
determination, or
by the ability to specifically bind anti-Zsig67 antibody. A Zsig67 antagonist
can be
further characterized by its ability to inhibit the biological response
induced by Zsig67
or by a Zsig67 agonist. This inhibitory effect may result, for example, from
the
competitive or non-competitive binding of the antagonist to the Zsig67
receptor.
The microphysiometer provides one means to identify Zsig67
antagonists. For example, Zsig67 antagonists can be identified by a method,
comprising the steps of (a) providing cells responsive to Zsig67, (b)
incubating a first
2o portion of the cells in the presence of Zsig67 and in the absence of a test
compound,
(c) incubating a second portion of the cells in the presence of both Zsig67
and the test
compound, and (d) comparing the cellular responses of the first and second
cell
portions, wherein a decreased response by the second portion, compared with
the
response of the first portion, indicates that the test compound is a Zsig67
antagonist.
An illustrative change in cellular response is a measurable change
extracellular
acidification rate, as measured by a microphysiometer.
Zsig67, its agonists and antagonists are valuable in both in vivo and in
vitro uses. For example, Zsig67 and its agonists may be used to supplement
serum-
free media, while Zsig67 antagonists are useful as research reagents for
characterizing
3o sites of interaction between Zsig67 and its receptor. In a therapeutic
setting,
pharmaceutical compositions comprising Zsig67 antagonists can be used to
inhibit
Zsig67 activity.
The present invention also contemplates chemically modified Zsig67
compositions, in which a Zsig67 polypeptide is linked with a polymer.
Illustrative
Zsig67 polypeptides include polypeptides comprising amino acid residues 52 to
85 of
SEQ ID N0:2. Typically, the polymer is water soluble so that the Zsig67
conjugate
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54
does not precipitate in an aqueous environment, such as a physiological
environment.
An example of a suitable polymer is one that has been modified to have a
single
reactive group, such as an active ester for acylation, or an aldehyde for
alkylation, In
this way, the degree of polymerization can be controlled. An example of a
reactive
aldehyde is polyethylene glycol propionaldehyde, or mono-(Cl-C10) alkoxy, or
aryloxy derivatives thereof (see, for example, Harns, et al., U.S. Patent No.
5,252,714). The polymer may be branched or unbranched. Moreover, a mixture of
polymers can be used to produce Zsig67 conjugates.
Zsig67 conjugates used for therapy can comprise pharmaceutically
to acceptable water-soluble polymer moieties. Suitable water-soluble polymers
include
polyethylene glycol (PEG), monomethoxy-PEG, mono-(C 1-C 10)alkoxy-PEG,
aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG
propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol
homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g.,
glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based
polymers.
Suitable PEG may have a molecular weight from about 600 to about 60,000,
including, for example, 5,000, 12,000, 20,000 and 25,000. A Zsig67 conjugate
can
also comprise a mixture of such water-soluble polymers.
One example of a Zsig67 conjugate comprises a Zsig67 moiety and a
2o polyalkyl oxide moiety attached to the N terminus of the Zsig67 moiety. PEG
is one
suitable polyalkyl oxide. As an illustration, Zsig67 can be modified with PEG,
a
process known as "PEGylation." PEGylation of Zsig67 can be carried out by any
of
the PEGylation reactions known in the art (see, for example, EP 0 154 316,
Delgado et
al., Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan
and
Spreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int
JHematol 68:1
(1998)). For example, PEGylation can be performed by an acylation reaction or
by an
alkylation reaction with a reactive polyethylene glycol molecule. In an
alternative
approach, Zsig67 conjugates are formed by condensing activated PEG, in which a
terminal hydroxy or amino group of PEG has been replaced by an activated
linker
(see, for example, Karasiewicz et al., U.S. Patent No. 5,382,657).
PEGylation by acylation typically requires reacting an active ester
derivative of PEG with a Zsig67 polypeptide. An example of an activated PEG
ester
is PEG esterified to N hydroxysuccinimide. As used herein, the term
"acylation"
includes the following types of linkages between Zsig67 and a water soluble
polymer:
amide, carbamate, urethane, and the like. Methods for preparing PEGylated
Zsig67 by
acylation will typically comprise the steps of (a) reacting a Zsig67
polypeptide with
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PEG (such as a reactive ester of an aldehyde derivative of PEG) under
conditions
whereby one or more PEG groups attach to Zsig67, and (b) obtaining the
reaction
product(s). Generally, the optimal reaction conditions for acylation reactions
will be
determined based upon known parameters and desired results. For example, the
larger
5 the ratio of PEG:Zsig67, the greater the percentage of polyPEGylated Zsig67
product.
The product of PEGylation by acylation is typically a polyPEGylated
Zsig67 product, wherein the lysine s-amino groups are PEGylated via an acyl
linking
group. An example of a connecting linkage is an amide. Typically, the
resulting
Zsig67 will be at least 95% mono-, di-, or tri-pegylated, although some
species with
to higher degrees of PEGylation may be formed depending upon the reaction
conditions.
PEGylated species can be separated from unconjugated Zsig67 polypeptides using
standard purification methods, such as dialysis, ultrafiltration, ion exchange
chromatography, affinity chromatography, and the like.
PEGylation by alkylation generally involves reacting a terminal
15 aldehyde derivative of PEG with Zsig67 in the presence of a reducing agent.
PEG
groups can be attached to the polypeptide via a -CHZ-NH group.
Derivatization via reductive alkylation to produce a monoPEGylated
product takes advantage of the differential reactivity of different types of
primary
amino groups available for derivatization. Typically, the reaction is
performed at a pH
2o that allows one to take advantage of the pKa differences between the s-
amino groups
of the lysine residues and the a-amino group of the N terminal residue of the
protein.
By such selective derivatization, attachment of a water-soluble polymer that
contains a
reactive group such as an aldehyde, to a protein is controlled. The
conjugation with
the polymer occurs predominantly at the N terminus of the protein without
significant
25 modification of other reactive groups such as the lysine side chain amino
groups. The
present invention provides a substantially homogenous preparation of Zsig67
monopolymer conjugates.
Reductive alkylation to produce a substantially homogenous population
of monopolymer Zsig67 conjugate molecule can comprise the steps of: (a)
reacting a
3o Zsig67 polypeptide with a reactive PEG under reductive alkylation
conditions at a pH
suitable to permit selective modification of the a-amino group at the amino
terminus
of the Zsig67, and (b) obtaining the reaction product(s). The reducing agent
used for
reductive alkylation should be stable in aqueous solution and able to reduce
only the
Schiff base formed in the initial process of reductive alkylation.
Illustrative reducing
35 agents include sodium borohydride, sodium cyanoborohydride, dimethylamine
borane,
trimethylamine borane, and pyridine borane.
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56
For a substantially homogenous population of monopolymer Zsig67
conjugates, the reductive alkylation reaction conditions are those which
permit the
selective attachment of the water soluble polymer moiety to the N terminus of
Zsig67.
Such reaction conditions generally provide for pKa differences between the
lysine
amino groups and the a-amino group at the N terminus. The pH also affects the
ratio
of polymer to protein to be used. In general, if the pH is lower, a larger
excess of
polymer to protein will be desired because the less reactive the N terminal a-
group,
the more polymer is needed to achieve optimal conditions. If the pH is higher,
the
polymer:Zsig67 need not be as large because more reactive groups are
available.
1 o Typically, the pH will fall within the range of 3 to 9, or 3 to 6.
Another factor to consider is the molecular weight of the water-soluble
polymer. Generally, the higher the molecular weight of the polymer, the fewer
number of polymer molecules which may be attached to the protein. For
PEGylation
reactions, the typical molecular weight is about 2 kDa to about 100 kDa, about
S kDa
to about 50 kDa, or about 12 kDa to about 25 kDa. The molar ratio of water-
soluble
polymer to Zsig67 will generally be in the range of 1:1 to 100:1. Typically,
the molar
ratio of water-soluble polymer to Zsig67 will be 1:1 to 20:1 for
polyPEGylation, and
1:1 to 5:1 for monoPEGylation.
General methods for producing conjugates comprising a polypeptide
2o and water-soluble polymer moieties are known in the art. See, for example,
Karasiewicz et al., U.S. Patent No. 5,382,657, Greenwald et al., U.S. Patent
No.
5,738, 846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996), Monkarsh et
al.,
Anal. Biochem. 247:434 (1997)).
The present invention contemplates compositions comprising a peptide
or polypeptide described herein. Such compositions can further comprise a
carrier.
The carrier can be a conventional organic or inorganic carrier. Examples of
carriers
include water, buffer solution, alcohol, propylene glycol, macrogol, sesame
oil, corn
oil, and the like.
9. Preparation of Zsig67 Targeting Compositions
A. Zsig67 Conjugates and Zsig67 Targeting Fusion Proteins
A Zsig67 targeting composition comprises a Zsig67 component and a
cytotoxin. Such targeting compositions can be used for tissue ablation, a
procedure
that is useful in both experimental and therapeutic settings. Either the
Zsig67
component or the cytotoxin, or both the Zsig67 component and the cytotoxin,
can be
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57
conjugated with a soluble polymer, as discussed above. For example,
polypeptide
cytotoxins can be conjugated with a soluble polymer either before or after
conjugation
to a Zsig67 component. Soluble polymers can also be conjugated with Zsig67
targeting fusion proteins.
s An example of a suitable polypeptide cytotoxin is a ribosome-
inactivating protein. Type I ribosome-inactivating proteins are single-chain
proteins,
while type II ribosome-inactivating proteins consist of two nonidentical
subunits (A
and B chains) joined by a disulfide bond (for a review, see Soria et al.,
Targeted
Diagn. Ther. 7:193 (1992)). Useful type I ribosome-inactivating proteins
include
to polypeptides from Saponaria o~cinalis (e.g., saporin-1, saporin-2, saporin-
3, saporin-
6), Momordica charantia (e.g, momordin), Byronia dioica (e.g., bryodin,
bryodin-2),
Trichosanthes kirilowii (e.g., trichosanthin, trichokirin), Gelonium
multiflorum (e.g.,
gelonin), Phytolacca americana (e.g., pokeweed antiviral protein, pokeweed
antiviral
protein-II, pokeweed antiviral protein-S), Phytolacca dodecandra (e.g.,
dodecandrin,
15 Mirabilis antiviral protein), and the like. Ribosome-inactivating proteins
are
described, for example, by Walsh et al., U.S. Patent No. 5,635,384.
Suitable type II ribosome-inactivating proteins include polypeptides
from Ricinus communis (e.g., ricin), Abrus precatorius (e.g., abrin), Adenia
digitata
(e.g., modeccin), and the like. Since type II ribosome-inactiving proteins
include a B
2o chain that binds galactosides and a toxic A chain that depurinates
adensoine, type II
ribosome-inactivating protein conjugates should include the A chain.
Additional
useful ribosome-inactivating proteins include bouganin, clavin, maize ribosome-
inactivating proteins, Vaccaria pyramidata ribosome-inactivating proteins,
nigrine b,
basic nigrine 1, ebuline, racemosine b, luffin-a, luffin-b, luffm-S, and other
ribosome-
2s inactivating proteins known to those of skill in the art. See, for example,
Bolognesi
and Stirpe, international publication No. W098/55623, Colnaghi et al.,
international
publication No. W097/49726, Hey et al., U.S. Patent No. 5,635,384, Bolognesi
and
Stirpe, international publication No. W095/07297, Arias et al., international
publication No. W094/20540, Watanabe et al., J. Biochem. 106:6 977 (1989);
Islam et
3o al., Agric. Biol. Chem. 55:229 (1991), and Gao etal., FEBSLett. 347:257
(1994).
Analogs and variants of naturally-occurring ribosome-inactivating
proteins are also suitable for the targeting compositions described herein,
and such
proteins are known to those of skill in the art. Ribosome-inactivating
proteins can be
produced using publicly available amino acid and nucleotide sequences. As an
3s illustration, a nucleotide sequence encoding saporin-6 is disclosed by
Lorenzetti et al.,
U.S. Patent No. 5,529,932, while Walsh et al., U.S. Patent No. 5,635,384,
describe
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58
maize and barley ribosome-inactivating protein nucleotide and amino acid
sequences.
Moreover, ribosome-inactivating proteins are also commercially available.
Another group of useful polypeptide cytotoxins include
immunomodulators. Zsig67 targeting compositions that include an
immunomodulator
provide a means to deliver an immunomodulator to a target cell and are
particularly
useful against tumor cells. The cytotoxic effects of immunomodulators are well
known to those of skill in the art. See, for example, Klegerman et al.,
"Lymphokines
and Monokines," in Biotechnology And Pharmacy, Pessuto et al. (eds.), pages 53-
70
(Chapman & Hall 1993). As an illustration, interferons can inhibit cell
proliferation
1o by inducing increased expression of class I histocompatibility antigens on
the surface
of various cells and thus, enhance the rate of destruction of cells by
cytotoxic T
lymphocytes. Furthermore, tumor necrosis factors, such as tumor necrosis
factor-a,
are believed to produce cytotoxic effects by inducing DNA fragmentation.
Additional polypeptide cytotoxins include ribonuclease, DNase I,
Staphylococcal enterotoxin-A, diphtheria toxin, Pseudomonas exotoxin, and
Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641 (1986),
and
Goldenberg, CA - A Cancer Journal for Clinicians 44:43 (1994). Other suitable
toxins are known to those of skill in the art.
Conjugates of cytotoxic polypeptides and Zsig67 components can be
prepared using standard techniques for conjugating polypeptides. As an
illustration of
the general approach, methods of conjugating FGF with saporin are described by
Lappi et al., Biochem. Biophys. Res. Commun. 160:917 (1989), Soria et al.,
Targeted
Diagn. Ther. 7:193 (1992), Buechler et al., Eur. J. Biochem. 234:706 (1995),
Behar
Cohen et al., Invest. Ophthalmol. Vis. Sci. 36:2434 (1995), Lappi and Baird,
U.S.
Patent No. 5,191,067, Calabresi et al., U.S. Patent No. 5,478,804, and Lappi
and
Baird, U.S. Patent No. 5,576,288. Additional approaches to conjugating
polypeptides
are known to those of skill in the art. For example, Lam and Kelleher, U.S.
Patent No.
5,055,291, describe the production of antibodies conjugated with either
diphtheria
toxin fragment A or ricin toxin.
3o Zsig67 targeting fusion proteins can also be produced using standard
techniques, as discussed above. As an illustration, FGF-saporin recombinant
proteins
are described by Lappi et al., J. Biol. Chem. 269:12552 (1994), Behar-Cohen et
al.,
Invest. Ophthalmol. Vis. Sci. 36:2434 (1995), McDonald et al., Protein Expr.
Purif.
8:97 (1996), and Lappi et al., U.S. Patent No. 5,916,772. In a similar manner,
Landgraf et al., Biochemistry 37:3220 (1998), produced a fusion protein
comprising
an epidermal growth factor moiety and diphtheria toxin. Methods of preparing
fusion
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59
proteins comprising a cytotoxic polypeptide moiety are well-known in the art
of
antibody-toxin fusion protein production. For example, antibody-Pseudomonas
exotoxin A fusion proteins have been described by Chaudhary et al., Nature
339:394
(1989), Brinkmann et al., Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et
al.,
Proc. Nat'l Acad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol.
150:3054
(1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J. Biol.
Chem.
271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996), and Schmidt et
al., Int.
J. Can. 65:538 (1996). Antibody-toxin fusion proteins containing a diphtheria
toxin
moiety have been described by Kreitman et al., Leukemia 7:553 (1993), Nicholls
et
to al., J. Biol. Chem. 268:5302 (1993), Thompson et al., J. Biol. Chem.
270:28037
(1995), and Vallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor
Targeting
1:177 (1995), have described an antibody-toxin fusion protein having an RNase
moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994), produced an
antibody-
toxin fusion protein comprising a DNase I component. Gelonin was used as the
toxin
moiety in the antibody-toxin fusion protein of Better et al., J. Biol. Chem.
270:14951
(1995). As a further example, Dohlsten et al., Proc. Nat'l Acad Sci. USA
91:8945
(1994), reported an antibody-toxin fusion protein comprising Staphylococcal
enterotoxin-A. In addition, antibody fusion proteins comprising an interleukin-
2
moiety are described by Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et
al., Cancer
2o Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad. Sci. USA 93:7826
(1996),
Hank et al., Clin. Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res.
56:4998
(1996), while Yang et al., Hum. Antibodies Hybridomas 6:129 (1995), describe a
fusion protein that includes an F(ab')z fragment and a tumor necrosis factor-a
moiety.
These approaches can be used to prepare the Zsig67 targeting fusion proteins
described herein.
As an alternative to a polypeptide cytotoxin, Zsig67 targeting
compositions can comprise a radioisotope as the cytotoxin moiety. For example,
a
Zsig67 targeting composition can comprise an a-emitting radioisotope, a (3-
emitting
radioisotope, a y-emitting radioisotope, an Auger electron emitter, a neutron
capturing
3o agent that emits a-particles or a radioisotope that decays by electron
capture. Suitable
radioisotopes include '98Au, '99Au, 3zP~ 33P~ tzsh ~3~I~ ~z3I~ 9oY~ ~a6Re~
lBgRe, 6'Cu, znAt,
a~Sc~ ~o3Pb~ ~o9Pd~ z~zPb~ ziGe~ zzAs~ ios~~ nsAg~ ~i9Sb~ iziSn~ i3iCs~ iasPr~
i6y.b~ nzLu~
'9'Os,'93MPt, 197Hg' ~d the like.
A radioisotope can be attached to a Zsig67 component directly or
indirectly, via a chelating agent. For example, 6'Cu, considered one of the
more
promising radioisotopes for radioimmunotherapy due to its 61.5 hour half life
and
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abundant supply of (3-particles and y-rays, can be conjugated to a Zsig67
component
using the chelating agent, p-bromoacetamido-benzyl-tetraethylaminetetraacetic
acid.
Chase and Shapiro, "Medical Applications of Radioisotopes," in Gennaro (ed.),
Remington's Pharmaceutical Sciences, 19th Edition, pages 843-865 (Mack
Publishing
5 Company 1995). As an alternative, 9°Y, which emits an energetic (3-
particle, can be
coupled to a Zsig67 component using diethylenetriaminepentaacetic acid.
Moreover,
an exemplary suitable method for the direct radiolabeling of a Zsig67
component with
'3'I is described by Stein et al., Antibody Immunoconj. Radiopharm. 4:703
(1991).
Alternatively, boron addends such as carboranes can be attached to Zsig67
1 o components, using standard techniques.
Another type of suitable cytotoxin for the preparation of Zsig67
conjugates is a chemotherapeutic drug. Illustrative chemotherapeutic drugs
include
nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid
analogs,
pyrimidine analogs, purine analogs, antibiotics, epipodophyllotoxins, platinum
15 coordination complexes, and the like. Specific examples of chemotherapeutic
drugs
include methotrexate, doxorubicin, daunorubicin, cytosinarabinoside, cis-
platin,
vindesine, mitomycin, bleomycin, melphalan, chlorambucil, and the like.
Suitable
chemotherapeutic agents are described in Remington's Pharmaceutical Sciences,
19th
Ed. (Mack Publishing Co. 1995), and in Goodman And Gilman's The
20 Pharmacological Basis Of Therapeutics, 7th Ed. (MacMillan Publishing Co.
1985).
Other suitable chemotherapeutic agents are known to those of skill in the art.
In another approach, Zsig67 conjugates are prepared conjugating
photoactive agents or dyes to a Zsig67 component. Fluorescent and other
chromogens, or dyes, such as porphyrins sensitive to visible light, have been
used to
25 detect and to treat lesions by directing the suitable light to the lesion.
This type of
"photoradiation," "phototherapy," or "photodynamic" therapy is described, for
example, by Mew et al., J. Immunol. 130:1473 (1983), Jori et al. (eds.),
Photodynamic
Therapy Of Tumors And Other Diseases (Libreria Progetto 1985), Oseroff et al.,
Proc.
Natl. Acad. Sci. USA 83:8744 (1986), van den Bergh, Chem. Britain 22:430
(1986),
3o Hasan et al., Prog. Clin. Biol. Res. 288:471 (1989), Tatsuta et al., Lasers
Surg. Med.
9:422 (1989), and Pelegrin et al., Cancer 67:2529 (1991).
Another general type of useful cytotoxin is a tyrosine kinase inhibitor.
Since the activation of proliferation by tyrosine kinases has been suggested
to play a
role in the development and progression of tumors, this activation can be
inhibited by
35 Zsig67 components that deliver tyrosine kinase inhibitors. Suitable
tyrosine kinase
inhibitors include isoflavones, such as genistein (5, 7, 4'-
trihydroxyisoflavone),
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61
daidzein (7,4'-dihydroxyisoflavone), and biochanin A (4-methoxygenistein), and
the
like. As an illustration of the general approach, methods of conjugating
tyrosine
inhibitors to a growth factor are described by Uckun, U.S. Patent No.
5,911,995.
The present invention also includes Zsig67 targeting compositions that
comprise a nucleic acid molecule encoding a cytotoxin. Such targeting
compositions
can comprise, for example, a Zsig67 polypeptide-polylysine conjugate condensed
with
an expression vector comprising a cytotoxin gene.
B. Zsig67 Targeting Liposomes
to Liposomes provide one means to deliver therapeutic polypeptides to a
subject intravenously, intraperitoneally, intrathecally, intramuscularly,
subcutaneously, or via oral administration, inhalation, or intranasal
administration.
Liposomes are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg et al.,
Eur. J.
Clin. Microbiol. Infect. Dis. IZ (Suppl. I):S61 (1993), Kim, Drugs 46:618
(1993), and
Ranade, "Site-Specific Drug Delivery Using Liposomes as Carriers," in Drug
Delivery
Systems, Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)). Liposomes
are
similar in composition to cellular membranes and as a result, liposomes can be
administered safely and are biodegradable. Depending on the method of
preparation,
liposomes may be unilamellar or multilamellar, and liposomes can vary in size
with
diameters ranging from 0.02 ~,m to greater than 10 p.m. A variety of agents
can be
encapsulated in liposomes: hydrophobic agents partition in the bilayers and
hydrophilic agents partition within the inner aqueous spaces) (see, for
example,
Machy et al., Liposomes In Cell Biology And Pharmacology (John Libbey 1987),
and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover, it is
possible to
control the therapeutic availability of the encapsulated agent by varying
liposome size,
the number of bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
Liposomes can adsorb to virtually any type of cell and then slowly
3o release the encapsulated agent. Alternatively, an absorbed liposome may be
endocytosed by cells that are phagocytic. Endocytosis is followed by
intralysosomal
degradation of liposomal lipids and release of the encapsulated agents
(Scherphof et
al., Ann. N. Y. Acad. Sci. 446:368 (1985)). After intravenous administration,
small
liposomes (0.1 to 1.0 Vim) are typically taken up by cells of the
reticuloendothelial
system, located principally in the liver and spleen, whereas liposomes larger
than 3.0
~,m are deposited in the lung. This preferential uptake of smaller liposomes
by the
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62
cells of the reticuloendothelial system has been used to deliver
chemotherapeutic
agents to macrophages and to tumors of the liver.
The reticuloendothelial system can be circumvented by several
methods including saturation with large doses of liposome particles, or
selective
macrophage inactivation by pharmacological means (Claassen et al., Biochim.
Biophys. Acta 802:428 (1984)). In addition, incorporation of glycolipid- or
polyethelene glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the reticuloendothelial
system
(Allen et al., Biochim. Biophys. Acta 1068:133 (1991); Allen et al., Biochim.
Biophys.
l0 Acta 1150:9 (1993)).
Liposomes can also be prepared to target particular cells or organs by
varying phospholipid composition or by inserting receptors or ligands into the
liposomes. For example, liposomes, prepared with a high content of a nonionic
surfactant, have been used to target the liver (Hayakawa et al., Japanese
Patent 04-
244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)). These formulations
were
prepared by mixing soybean phospatidylcholine, a,-tocopherol, and ethoxylated
hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under
vacuum, and then reconstituting the mixture with water. A liposomal
formulation of
dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived sterylglucoside
2o mixture (SG) and cholesterol (Ch) has also been shown to target the liver
(Shimizu et
al., Biol. Pharm. Bull. 20:881 (1997)).
Alternatively, various targeting ligands can be bound to the surface of
the liposome, such as antibodies, antibody fragments, carbohydrates, vitamins,
and
transport proteins. For example, liposomes can be modified with branched type
galactosyllipid derivatives to target asialoglycoprotein (galactose)
receptors, which are
exclusively expressed on the surface of liver cells (Kato and Sugiyama, Crit.
Rev.
Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.
Bull.20:259
(1997)). Similarly, Wu et al., Hepatology 27:772 (1998), have shown that
labeling
liposomes with asialofetuin led to a shortened liposome plasma half life and
greatly
3o enhanced uptake of asialofetuin-labeled liposome by hepatocytes. On the
other hand,
hepatic accumulation of liposomes comprising branched type galactosyllipid
derivatives can be inhibited by preinjection of asialofetuin (Murahashi et
al., Biol.
Pharm. Bull.20:259 (1997)). Polyaconitylated human serum albumin liposomes
provide another approach for targeting liposomes to liver cells (Kamps et al.,
Proc.
Nat'l Acad. Sci. USA 94:11681 (1997)). Moreover, Geho, et al. U.S. Patent No.
4,603,044, describe a hepatocyte-directed liposome vesicle delivery system,
which has
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63
specificity for hepatobiliary receptors associated with the specialized
metabolic cells
of the liver.
Zsig67 targeting compositions can be encapsulated within liposomes
using standard techniques of protein microencapsulation (see, for example,
Anderson
et al., Infect. Immun. 31:1099 (1981), Anderson et al., Cancer Res. 50:1853
(1990),
and Cohen et al., Biochim. Biophys. Acta 1063:95 (1991), Alving et al.
"Preparation
and Use of Liposomes in Immunological Studies," in Liposome Technology, 2nd
Edition, Vol. III, Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et
al., Meth.
Enzymol. 149:124 (1987)). Suitable liposomes can contain a variety of
moieties, such
to as lipid derivatives of polyethylene glycol) (Allen et al., Biochim.
Biophys. Acta
1150:9 (1993)).
In addition to providing a means of delivering Zsig67 targeting
compositions, liposomes can be produced to target cells expressing a Zsig67
receptor.
Accordingly, the present invention includes liposomes comprising a Zsig67
component bound to the surface to effect delivery of encapsulated cytotoxins.
10. Production of Antibodies to Zsig67 Proteins
Antibodies to Zsig67 can be obtained, for example, using the product
of a Zsig67 expression vector or Zsig67 isolated from a natural source as an
antigen.
2o Particularly useful anti-Zsig67 antibodies "bind specifically" with Zsig67.
Antibodies
are considered to be specifically binding if the antibodies exhibit at least
one of the
following two properties: (1) antibodies bind to Zsig67 with a threshold level
of
binding activity, and (2) antibodies do not significantly cross-react with
polypeptides
related to Zsig67.
With regard to the first characteristic, antibodies specifically bind if
they bind to a Zsig67 polypeptide, peptide or epitope with a binding affinity
(Ka) of
1 O6 M-' or greater, preferably 10' M'' or greater, more preferably 1 Og M-'
or greater, and
most preferably 109 M-' or greater. The binding affinity of an antibody can be
readily
determined by one of ordinary skill in the art, for example, by Scatchard
analysis
(Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). With regard to the second
characteristic, antibodies do not significantly cross-react with related
polypeptide
molecules, for example, if they detect Zsig67, but not known related
polypeptides
using a standard Western blot analysis. Examples of known related polypeptides
are
orthologs and proteins from the same species that are members of a protein
family.
For example, specifically-binding anti-Zsig67 antibodies bind with Zsig67, but
not
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64
with polypeptides such human secretin, human glucagon, human vasoactive
intestinal
peptide, or the human glucagon-like peptides.
Anti-Zsig67 antibodies can be produced using antigenic Zsig67
epitope-bearing peptides and polypeptides. Antigenic epitope-bearing peptides
and
s polypeptides of the present invention contain a sequence of at least four,
or between
15 to about 30 amino acids contained within SEQ ID N0:2. 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
to antibodies that bind with Zsig67. It is desirable that the amino acid
sequence of the
epitope-bearing peptide is selected to provide substantial solubility in
aqueous
solvents (i. e. , the sequence includes relatively hydrophilic residues, while
hydrophobic
residues are preferably avoided). Moreover, amino acid sequences containing
proline
residues may be also be desirable for antibody production.
15 As an illustration, potential antigenic sites in Zsig67 were identified
using the Jameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988), as
implemented by the PROTEAN program (version 3.14) of LASERGENE
(DNASTAR; Madison, WI). Default parameters were used in this analysis.
The Jameson-Wolf method predicts potential antigenic determinants by
20 combining six major subroutines for protein structural prediction. Briefly,
the Hopp
Woods method, Hopp et al., Proc. Nat'1 Acad. Sci. USA 78:3824 (1981), was
first
used to identify amino acid sequences representing areas of greatest local
hydrophilicity (parameter: seven residues averaged). In the second step,
Emini's
method, Emini et al., J. Virology 55:836 (1985), was used to calculate surface
25 probabilities (parameter: surface decision threshold (0.6) = 1). Third, the
Karplus-
Schultz method, Karplus and Schultz, Naturwissenschaften 72:212 (1985), was
used
to predict backbone chain flexibility (parameter: flexibility threshold (0.2)
= 1 ). In the
fourth and fifth steps of the analysis, secondary structure predictions were
applied to
the data using the methods of Chou-Fasman, Chou, "Prediction of Protein
Structural
3o Classes from Amino Acid Composition," in Prediction of Protein Structure
and the
Principles of Protein Conformation, Fasman (ed.), pages 549-586 (Plenum Press
1990), and Gamier-Robson, Gamier et al., J. Mol. Biol. 120:97 (1978) (Chou-
Fasman
parameters: conformation table = 64 proteins; a region threshold = 103; (3
region
threshold = 105; Gamier-Robson parameters: a and ~3 decision constants = 0).
In the
35 sixth subroutine, flexibility parameters and hydropathy/solvent
accessibility factors
were combined to determine a surface contour value, designated as the
"antigenic
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index." Finally, a peak broadening function was applied to the antigenic
index, which
broadens major surface peaks by adding 20, 40, 60, or 80% of the respective
peak
value to account for additional free energy derived from the mobility of
surface
regions relative to interior regions. This calculation was not applied,
however, to any
5 major peak that resides in a helical region, since helical regions tend to
be less
flexible.
The results of this analysis indicated that a peptide consisting of amino
acids 45 to 72 of SEQ ID N0:2 ("antigenic peptide 1"), two major subfragments
(amino acids 45 to 54 of SEQ ID N0:2 ("antigenic peptide 2") and 61 to 72 of
SEQ
1o ID N0:2 ("antigenic peptide 3")), and their subfragments (amino acids 45 to
50
("antigenic peptide 4"), amino acids 46 to 51 ("antigenic peptide 5"), amino
acids 47
to 52 ("antigenic peptide 6"), amino acids 48 to 53 ("antigenic peptide 7"),
amino
acids 49 to 54 ("antigenic peptide 8"), amino acids 61 to 66 ("antigenic
peptide 9"),
amino acids 62 to 67 ("antigenic peptide 10"), amino acids 63 to 68
("antigenic
15 peptide 11 "), amino acids 64 to 69 ("antigenic peptide 12"), amino acids
65 to 70
("antigenic peptide 13"), amino acids 66 to 71 ("antigenic peptide 14"), and
amino
acids 67 to 72 ("antigenic peptide 15")) would provide suitable antigenic
peptides.
The analysis also indicated that amino acid residues 87 to 92 of SEQ ID N0:2
("antigenic peptide 16") would provide a suitable antigenic peptide. The
present
20 invention contemplates the use of any one of antigenic peptides 1 to 16 to
generate
antibodies to Zsig67. The present invention also contemplates polypeptides
comprising at least one of antigenic peptides 1 to 16.
Polyclonal antibodies to recombinant Zsig67 protein or to Zsig67
isolated from natural sources can be prepared using methods well-known to
those of
25 skill in the art. See, for example, Green et al., "Production of Polyclonal
Antisera," in
Immunochemical Protocols (Manson, ed.), pages 1-5 (Hurriana Press 1992), and
Williams et al., "Expression of foreign proteins in E. coli using plasmid
vectors and
purification of specific polyclonal antibodies," in DNA Cloning 2: Expression
Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press
1995).
3o The immunogenicity of a Zsig67 polypeptide can be increased through the use
of an
adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete
adjuvant. Polypeptides useful for immunization also include fusion
polypeptides,
such as fusions of Zsig67 or a portion thereof with an immunoglobulin
polypeptide or
with maltose binding protein. The polypeptide immunogen may be a full-length
35 molecule or a portion thereof. If the polypeptide portion is "hapten-like,"
such portion
may be advantageously joined or linked to a macromolecular carrier (such as
keyhole
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66
limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
Although polyclonal antibodies are typically raised in animals such as
horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, or
sheep, an anti-
s Zsig67 antibody of the present invention may also be derived from a subhuman
primate antibody. General techniques for raising diagnostically and
therapeutically
useful antibodies in baboons may be found, for example, in Goldenberg et al.,
international patent publication No. WO 91/11465, and in Losman et al., Int.
J.
Cancer 46:310 ( 1990).
1 o Alternatively, monoclonal anti-Zsig67 antibodies can be generated.
Rodent monoclonal antibodies to specific antigens may be obtained by methods
known to those skilled in the art (see, for example, Kohler et al., Nature
256:495
(1975), Coligan et al. (eds.), Current Protocols in Immunology, I~ol. 1, pages
2.5.1-
2.6.7 (John Wiley & Sons 1991) ["Coligan"], Picksley et al., "Production of
15 monoclonal antibodies against proteins expressed in E. coli," in DNA
Cloning 2:
Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford
University
Press 1995)).
Briefly, monoclonal antibodies can be obtained by injecting mice with
a composition comprising a Zsig67 gene product, verifying the presence of
antibody
2o production by removing a serum sample, removing the spleen to obtain B
lymphocytes, fusing the B-lymphocytes with myeloma cells to produce
hybridomas,
cloning the hybridomas, selecting positive clones which produce antibodies to
the
antigen, culturing the clones that produce antibodies to the antigen, and
isolating the
antibodies from the hybridoma cultures.
25 In addition, an anti-Zsig67 antibody of the present invention may be
derived from a human monoclonal antibody. Human monoclonal antibodies are
obtained from transgenic mice that have been engineered to produce specific
human
antibodies in response to antigenic challenge. In this technique, elements of
the human
heavy and light chain locus are introduced into strains of mice derived from
embryonic
30 stem cell lines that contain targeted disruptions of the endogenous heavy
chain and light
chain loci. The transgenic mice can synthesize human antibodies specific for
human
antigens, and the mice can be used to produce human antibody-secreting
hybridomas.
Methods for obtaining human antibodies from transgenic mice are described, for
example, by Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature
368:856
35 (1994), and Taylor et al., Int. Immun. 6:579 (1994).
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67
Monoclonal antibodies can be isolated and purified from hybridoma
cultures by a variety of well-established techniques. Such isolation
techniques include
affinity chromatography with Protein-A Sepharose, size-exclusion
chromatography,
and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-
2.7.12
and pages 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G
(IgG)," in
Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc.
1992)).
For particular uses, it may be desirable to prepare fragments of anti
Zsig67 antibodies. Such antibody fragments can be obtained, for example, by
to proteolytic hydrolysis of the antibody. Antibody fragments can be obtained
by pepsin
or papain digestion of whole antibodies by conventional methods. As an
illustration,
antibody fragments can be produced by enzymatic cleavage of antibodies with
pepsin
to provide a 5S fragment denoted F(ab')z. This fragment can be further cleaved
using
a thiol reducing agent to produce 3.SS Fab' monovalent fragments. Optionally,
the
cleavage reaction can be performed using a blocking group for the sulfllydryl
groups
that result from cleavage of disulfide linkages. As an alternative, an
enzymatic
cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg, U.S. patent
No.
4,331,647, Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter,
Biochem. J.
73:119 (1959), Edelman et al., in Methods in Enzymology Vol. l, page 422
(Academic
Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
Other methods of cleaving antibodies, such as separation of heavy
chains to form monovalent light-heavy chain fragments, further cleavage of
fragments,
or other enzymatic, chemical or genetic techniques may also be used, so long
as the
fragments bind to the antigen that is recognized by the intact antibody.
For example, Fv fragments comprise an association of VH and VL
chains. This association can be noncovalent, as described by mbar et al.,
Proc. Nat'l
Acad. Sci. USA 69:2659 (1972). Alternatively, the variable chains can be
linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde
(see, for example, Sandhu, Crit. Rev. Biotech. 12:437 (1992)).
The Fv fragments may comprise VH and VL chains which are connected
by a peptide linker. These single-chain antigen binding proteins (scFv) are
prepared
by constructing a structural gene comprising DNA sequences encoding the VH and
VL
domains which are connected by an oligonucleotide. The structural gene is
inserted
into an expression vector which is subsequently introduced into a host cell,
such as E
coli. The recombinant host cells synthesize a single polypeptide chain with a
linker
CA 02358930 2001-07-17
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68
peptide bridging the two V domains. Methods for producing scFvs are described,
for
example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97
(1991) (also see, Bird et al., Science 242:423 (1988), Ladner et al., U.S.
Patent No.
4,946,778, Pack et al., BiolTechnology 11:1271 (1993), and Sandhu, supra).
As an illustration, a scFV can be obtained by exposing lymphocytes to
Zsig67 polypeptide in vitro, and selecting antibody display libraries in phage
or
similar vectors (for instance, through use of immobilized or labeled Zsig67
protein or
peptide). Genes encoding polypeptides having potential Zsig67 polypeptide
binding
domains can be obtained by screening random peptide libraries displayed on
phage
1o (phage display) or on bacteria, such as E. coli. Nucleotide sequences
encoding the
polypeptides can be obtained in a number of ways, such as through random
mutagenesis and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides which interact with a known
target which
can be a protein or polypeptide, such as a ligand or receptor, a biological or
synthetic
macromolecule, or organic or inorganic substances. Techniques for creating and
screening such random peptide display libraries are known in the art (Ladner
et al.,
U.S. Patent No. 5,223,409, Ladner et al., U.S. Patent No. 4,946,778, Ladner et
al.,
U.S. Patent No. 5,403,484, Ladner et al., U.S. Patent No. 5,571,698, and Kay
et al.,
Phage Display of Peptides and Proteins (Academic Press, Inc. 1996)) and random
peptide display libraries and kits for screening such libraries are available
commercially, for instance from CLONTECH Laboratories, Inc. (Palo Alto, 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 Zsig67 sequences disclosed herein to
identify
proteins which bind to Zsig67.
Another form of an antibody fragment is a peptide coding for a single
complementarity-determining region (CDR). CDR peptides ("minimal recognition
units") can be obtained by constructing genes encoding the CDR of an antibody
of
interest. Such genes are prepared, for example, by using the polymerase chain
reaction to synthesize the variable region from RNA of antibody-producing
cells (see,
for example, Larrick et al., Methods: A Companion to Methods in Enzymology
2:106
(1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in
Monoclonal Antibodies: Production, Engineering and Clinical Application,
Ritter et
al. (eds.), page 166 (Cambridge University Press 1995), and Ward et al.,
"Genetic
Manipulation and Expression of Antibodies," in Monoclonal Antibodies:
Principles
and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).
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69
Alternatively, an anti-Zsig67 antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies are produced
by transferring mouse complementary determining regions from heavy and light
variable chains of the mouse immunoglobulin into a human variable domain.
Typical
residues of human antibodies are then substituted in the framework regions of
the
marine counterparts. The use of antibody components derived from humanized
monoclonal antibodies obviates potential problems associated with the
immunogenicity of marine constant regions. General techniques for cloning
marine
immunoglobulin variable domains are described, for example, by Orlandi et al.,
Proc.
to Nat'1 Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al., Nature
321:522
(1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992), Sandhu,
Crit. Rev.
Biotech. 12:437 (1992), Singer et al., J. Immun. 150:2844 (1993), Sudhir
(ed.),
Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, "Engineering
Therapeutic Antibodies," in Protein Engineering: Principles and Practice,
Cleland et
al. (eds.), pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al.,
U.S.
Patent No. 5,693,762 (1997).
Polyclonal anti-idiotype antibodies can be prepared by immunizing
animals with anti-Zsig67 antibodies or antibody fragments, using standard
techniques.
2o See, for example, Green et al., "Production of Polyclonal Antisera," in
Methods In
Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively, monoclonal
anti-
idiotype antibodies can be prepared using anti-Zsig67 antibodies or antibody
fragments as immunogens with the techniques, described above. As another
alternative, humanized anti-idiotype antibodies or subhuman primate anti-
idiotype
antibodies can be prepared using the above-described techniques. Methods for
producing anti-idiotype antibodies are described, for example, by Irie, U.S.
Patent No.
5,208,146, Greene, et. al., U.S. Patent No. 5,637,677, and Varthakavi and
Minocha, J.
Gen. Virol. 77:1875 (1996).
11. Detection of Zsig67 Gene Expression and Examination of the
Zsig67 Chromosomal Locus
Nucleic acid molecules can be used to detect the expression of a Zsig67
gene in a biological sample. Such probe molecules include double-stranded
nucleic
acid molecules comprising the nucleotide sequence of SEQ ID NO:I, or a
fragment
thereof, as well as single-stranded nucleic acid molecules having the
complement of
CA 02358930 2001-07-17
WO 00/43516 PCT/US00/00870
the nucleotide sequence of SEQ ID NO:1, or a fragment thereof. Probe molecules
may be DNA, RNA, oligonucleotides, and the like.
Particular probes can comprise a portion of the nucleotide sequence of
nucleotides 111 to 422 of SEQ ID NO:1, or complement thereof, and bind with
5 regions of the Zsig67 gene that have a low sequence similarity to comparable
regions
in other members of the secretin-glucagon-VIP family. As used herein, the term
"portion" refers to at least eight nucleotides to at least 20 or more
nucleotides.
In a basic assay, a single-stranded probe molecule is incubated with
RNA, isolated from a biological sample, under conditions of temperature and
ionic
0 strength that promote base pairing between the probe and target Zsig67 RNA
species.
After separating unbound probe from hybridized molecules, the amount of
hybrids is
detected.
Well-established hybridization methods of RNA detection include
northern analysis and dot/slot blot hybridization (see, for example, Ausubel
(1995) at
15 pages 4-1 to 4-27, and Wu et al. (eds.), "Analysis of Gene Expression at
the RNA
Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc.
1997)).
Nucleic acid probes can be detectably labeled with radioisotopes such as 3zP
or 355.
Alternatively, Zsig67 RNA can be detected with a nonradioactive hybridization
method
(see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis by
Nonradioactive
2o Probes (Humana Press, Inc. 1993)). Typically, nonradioactive detection is
achieved by
enzymatic conversion of chromogenic or chemiluminescent substrates.
Illustrative
nonradioactive moieties include biotin, fluorescein, and digoxigenin.
Zsig67 oligonucleotide probes are also useful for in vivo diagnosis. As
an illustration, '8F-labeled oligonucleotides can be administered to a subject
and
25 visualized by positron emission tomography (Tavitian et al., Nature
Medicine 4:467
( 1998)).
Numerous diagnostic procedures take advantage of the polymerase
chain reaction (PCR) to increase sensitivity of detection methods. Standard
techniques for performing PCR are well-known (see, generally, Mathew (ed.),
3o Protocols in Human Molecular Genetics (Humana Press, Inc. 1991 ), White
(ed.), PCR
Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter
(ed.),
Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek
(eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical
Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in
35 Bioanalysis (Humana Press, Inc. 1998)).
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71
PCR primers can be designed to amplify a portion of the Zsig67 gene
that has a low sequence similarity to a comparable region in other members of
the
secretin-glucagon-VIP family.
One variation of PCR for diagnostic assays is reverse transcriptase
PCR (RT-PCR). In the RT-PCR technique, RNA is isolated from a biological
sample,
reverse transcribed to cDNA, and the cDNA is incubated with Zsig67 primers
(see, for
example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by
PCR," in
Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)). PCR is
then
performed and the products are analyzed using standard techniques.
to As an illustration, RNA is isolated from biological sample using, for
example, the gunadinium-thiocyanate cell lysis procedure described above.
Alternatively, a solid-phase technique can be used to isolate mRNA from a cell
lysate.
A reverse transcription reaction can be primed with the isolated RNA using
random
oligonucleotides, short homopolymers of dT, or Zsig67 anti-sense oligomers.
Oligo-
dT primers offer the advantage that various mRNA nucleotide sequences are
amplified
that can provide control target sequences. Zsig67 sequences are amplified by
the
polymerase chain reaction using two flanking oligonucleotide primers that are
typically 20 bases in length.
PCR amplification products can be detected using a variety of
2o approaches. For example, PCR products can be fractionated by gel
electrophoresis,
and visualized by ethidium bromide staining. Alternatively, fractionated PCR
products can be transferred to a membrane, hybridized with a detectably-
labeled
Zsig67 probe, and examined by autoradiography. Additional alternative
approaches
include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to
provide
chemiluminescence detection; and the C-TRAK colorimetric assay.
Another approach for detection of Zsig67 expression is cycling probe
technology (CPT), in which a single-stranded DNA target binds with an excess
of
DNA-RNA-DNA chimeric probe to form a complex, the RNA portion is cleaved with
RNAase H, and the presence of cleaved chimeric probe is detected (see, for
example,
Beggs et al., J. Clin. Microbiol. 34:2985 (1996), Bekkaoui et al.,
Biotechniques
20:240 (1996)). Alternative methods for detection of Zsig67 sequences can
utilize
approaches such as nucleic acid sequence-based amplification (NASBA),
cooperative
amplification of templates by cross-hybridization (CATCH), and the ligase
chain
reaction (LCR) (see, for example, Marshall et al., U.S. Patent No. 5,686,272
(1997),
Dyer et al., J. Virol. Methods 60:161 (1996), Ehricht et al., Eur. J. Biochem.
243:358
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72
(1997), and Chadwick et al., J. Virol. Methods 70:59 (1998)). Other standard
methods
are known to those of skill in the art.
Zsig67 probes and primers can also be used to detect and to localize
Zsig67 gene expression in tissue samples. Methods for such in situ
hybridization are
well-known to those of skill in the art (see, for example, Choo (ed.), In Situ
Hybridization Protocols (Humana Press, Inc. 1994), Wu et al. (eds.), "Analysis
of
Cellular DNA or Abundance of mRNA by Radioactive In Situ Hybridization
IRISH),"
in Methods in Gene Biotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu
et
al. (eds.), "Localization of DNA or Abundance of mRNA by Fluorescence In Situ
to Hybridization IRISH)," in Methods in Gene Biotechnology, pages 279-289 (CRC
Press,
Inc. 1997)). Various additional diagnostic approaches are well-known to those
of skill
in the art (see, for example, Mathew (ed.), Protocols in Human Molecular
Genetics
(Humana Press, Inc. 1991 ), Coleman and Tsongalis, Molecular Diagnostics
(Humana
Press, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases (Humana
Press,
Inc., 1996)).
The Zsig67 gene resides in chromosome 8q24. This region is
associated with various diseases and disorders, including Larger-Giedion
Syndrome,
Type I Trichorhinophalangeal Syndrome, renal cell carcinoma, Burkitt lymphoma,
idiopathic epilepsy, neonatal epilepsy, macular dystrophy, nephroblastoma,
Stargardt
2o Disease, and Pendred Syndrome. Thus, Zsig67 nucleotide sequences can be
used in
linkage-based testing for various diseases, and to determine whether a
subject's
chromosomes contain a mutation in the Zsig67 gene. Detectable chromosomal
aberrations at the Zsig67 gene locus include, but are not limited to,
aneuploidy, gene
copy number changes, insertions, deletions, restriction site changes and
rearrangements. Of particular interest are genetic alterations that inactivate
the Zsig67
gene.
Aberrations associated with the Zsig67 locus can be detected using
nucleic acid molecules of the present invention by employing molecular genetic
techniques, such as restriction fragment length polymorphism analysis, short
tandem
3o repeat analysis employing PCR techniques, amplification-refractory mutation
system
analysis, single-strand conformation polymorphism detection, RNase cleavage
methods, denaturing gradient gel electrophoresis, fluorescence-assisted
mismatch
analysis, and other genetic analysis techniques known in the art (see, for
example,
Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics
(Human Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases
CA 02358930 2001-07-17
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73
(Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protocols for Mutation
Detection (Oxford University Press 1996), Birren et al. (eds.), Genome
Analysis, Vol.
2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et
al.
(eds.), Current Protocols in Human Genetics (John Wiley & Sons 1998), and
Richards
and Ward, "Molecular Diagnostic Testing," in Principles of Molecular Medicine,
pages 83-88 (Humana Press, Inc. 1998)).
The protein truncation test is also useful for detecting the inactivation
of a gene in which translation-terminating mutations produce only portions of
the
encoded protein (see, for example, Stoppa-Lyonnet et al., Blood 91:3920
(1998)).
1 o According to this approach, RNA is isolated from a biological sample, and
used to
synthesize cDNA. PCR is then used to amplify the Zsig67 target sequence and to
introduce an RNA polymerase promoter, a translation initiation sequence, and
an in-
frame ATG triplet. PCR products are transcribed using an RNA polymerase, and
the
transcripts are translated in vitro with a T7-coupled reticulocyte lysate
system. The
translation products are then fractionated by SDS-PAGE to determine the
lengths of
the translation products. The protein truncation test is described, for
example, by
Dracopoli et al. (eds.), Current Protocols in Human Genetics, pages 9.11.1 -
9.11.18
(John Wiley & Sons 1998).
The present invention also contemplates kits for performing a diagnostic
assay for Zsig67 gene expression or to examine the Zsig67 locus. Such kits
comprise
nucleic acid probes, such as double-stranded nucleic acid molecules comprising
the
nucleotide sequence of SEQ ID NO:1, or a fragment thereof, as well as single-
stranded
nucleic acid molecules having the complement of the nucleotide sequence of SEQ
ID
NO:I, or a fragment thereof. Probe molecules may be DNA, RNA,
oligonucleotides,
and the like. Kits may comprise nucleic acid primers for performing PCR.
Such a kit can contain all the necessary elements to perform a nucleic
acid diagnostic assay described above. A kit will comprise at least one
container
comprising a Zsig67 probe or primer. The kit may also comprise a second
container
comprising one or more reagents capable of indicating the presence of Zsig67
3o sequences. Examples of such indicator reagents include detectable labels
such as
radioactive labels, fluorochromes, chemiluminescent agents, and the like. A
kit may
also comprise a means for conveying to the user that the Zsig67 probes and
primers
are used to detect Zsig67 gene expression. For example, written instructions
may state
that the enclosed nucleic acid molecules can be used to detect either a
nucleic acid
molecule that encodes Zsig67, or a nucleic acid molecule having a nucleotide
sequence that is complementary to a Zsig67-encoding nucleotide sequence. The
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74
written material can be applied directly to a container, or the written
material can be
provided in the form of a packaging insert.
12. Use of Anti Zsig67 Antibodies to Detect Zsig67 Protein
The present invention contemplates the use of anti-Zsig67 antibodies to
screen biological samples in vitro for the presence of Zsig67. In one type of
in vitro
assay, anti-Zsig67 antibodies are used in liquid phase. For example, the
presence of
Zsig67 in a biological sample can be tested by mixing the biological sample
with a trace
amount of labeled Zsig67 and an anti-Zsig67 antibody under conditions that
promote
1 o binding between Zsig67 and its antibody. Complexes of Zsig67 and anti-
Zsig67 in the
sample can be separated from the reaction mixture by contacting the complex
with an
immobilized protein which binds with the antibody, such as an Fc antibody or
Staphylococcus protein A. The concentration of Zsig67 in the biological sample
will be
inversely proportional to the amount of labeled Zsig67 bound to the antibody
and
directly related to the amount of free labeled Zsig67.
Alternatively, in vitro assays can be performed in which anti-Zsig67
antibody is bound to a solid-phase carrier. For example, antibody can be
attached to a
polymer, such as aminodextran, in order to link the antibody to an insoluble
support
such as a polymer-coated bead, a plate or a tube. Other suitable in vitro
assays will be
2o readily apparent to those of skill in the art.
In another approach, anti-Zsig67 antibodies can be used to detect Zsig67
in tissue sections prepared from a biopsy specimen. Such immunochemical
detection
can be ~ used to determine the relative abundance of Zsig67 and to determine
the
distribution of Zsig67 in the examined tissue. General immunochemistry
techniques are
well established (see, for example, Ponder, "Cell Marking Techniques and Their
Application," in Mammalian Development.' A Practical Approach, Monk (ed.),
pages
115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages
14.6.1
to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods In Molecular
Biology,
Vo1.10.~ Immunochemical Protocols (The Humana Press, Inc. 1992)).
Immunochemical detection can be performed by contacting a biological
sample with an anti-Zsig67 antibody, and then contacting the biological sample
with a
detectably labeled molecule which binds to the antibody. For example, the
detectably
labeled molecule can comprise an antibody moiety that binds to anti-Zsig67
antibody.
Alternatively, the anti-Zsig67 antibody can be conjugated with
avidin/streptavidin (or
biotin) and the detectably labeled molecule can comprise biotin (or
avidin/streptavidin).
Numerous variations of this basic technique are well-known to those of skill
in the art.
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Alternatively, an anti-Zsig67 antibody can be conjugated with a
detectable label to form an anti-Zsig67 immunoconjugate. Suitable detectable
labels
include, for example, a radioisotope, a fluorescent label, a chemiluminescent
label, an
enzyme label, a bioluminescent label or colloidal gold. Methods of making and
detect-
s ing such detectably-labeled immunoconjugates are well-known to those of
ordinary skill
in the art, and are described in more detail below.
The detectable label can be a radioisotope that is detected by
autoradiography. Isotopes that are particularly useful for the purpose of the
present
invention are 3H,'zSI,'3'I,'SS and'4C.
l0 Anti-Zsig67 immunoconjugates can also be labeled with a fluorescent
compound. The presence of a fluorescently-labeled antibody is determined by
exposing
the immunoconjugate to light of the proper wavelength and detecting the
resultant
fluorescence. Fluorescent labeling compounds include fluorescein
isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and
15 fluorescamine.
Alternatively, anti-Zsig67 immunoconjugates can be detectably labeled
by coupling an antibody component to a chemiluminescent compound. The presence
of
the chemiluminescent-tagged immunoconjugate is determined by detecting the
presence
of luminescence that arises during the course of a chemical reaction. Examples
of
20 chemiluminescent labeling compounds include luminol, isoluminol, an
aromatic
acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
Similarly, a bioluminescent compound can be used to label anti-Zsig67
immunoconjugates of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic protein
increases
25 the efFciency of the chemiluminescent reaction. The presence of a
bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent
compounds that are useful for labeling include luciferin, luciferase and
aequorin.
Alternatively, anti-Zsig67 immunoconjugates can be detectably labeled
by linking an anti-Zsig67 antibody component to an enzyme. When the anti-
Zsig67
3o enzyme conjugate is incubated in the presence of the appropriate substrate,
the enzyme
moiety reacts with the substrate to produce a chemical moiety which can be
detected, for
example, by spectrophotometric, fluorometric or visual means. Examples of
enzymes
that can be used to detectably label polyspecific immunoconjugates include ~3-
galac-
tosidase, glucose oxidase, peroxidase and alkaline phosphatase.
35 Those of skill in the art will know of other suitable labels which can be
employed in accordance with the present invention. The binding of marker
moieties to
CA 02358930 2001-07-17
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76
anti-Zsig67 antibodies can be accomplished using standard techniques known to
the art.
Typical methodology in this regard is described by Kennedy et al., Clin. Chim.
Acta
70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1 (1977), Shih et al., Int'l
J. Cancer
46:1101 (1990), Stein et al., Cancer Res. 50:1330 (1990), and Coligan, supra.
Moreover, the convenience and versatility of immunochemical detection
can be enhanced by using anti-Zsig67 antibodies that have been conjugated with
avidin,
streptavidin, and biotin (see, for example, Wilchek et al. (eds.), "Avidin-
Biotin
Technology," Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer
et
al., "Immunochemical Applications of Avidin-Biotin Technology," in Methods In
l0 Molecular Biology, Yol. 10, Manson (ed.), pages 149-162 (The Humana Press,
Inc.
1992).
Methods for performing immunoassays are well-established. See, for
example, Cook and Self, "Monoclonal Antibodies in Diagnostic Immunoassays," in
Monoclonal Antibodies: Production, Engineering, and Clinical Application,
Ritter and
Ladyman (eds.), pages 180-208, (Cambridge University Press, 1995), Perry, "The
Role
of Monoclonal Antibodies in the Advancement of Immunoassay Technology," in
Monoclonal Antibodies: Principles and Applications, Birch and Lennox (eds.),
pages
107-120 (Whey-Liss, Inc. 1995), and Diamandis, Immunoassay (Academic Press,
Inc.
1996).
In a related approach, biotin- or FITC-labeled Zsig67 can be used to
identify cells that bind Zsig67. Such can binding can be detected, for
example, using
flow cytometry.
The present invention also contemplates kits for performing an
immunological diagnostic assay for Zsig67 gene expression. Such kits comprise
at least
one container comprising an anti-Zsig67 antibody, or antibody fragment. A kit
may
also comprise a second container comprising one or more reagents capable of
indicating the presence of Zsig67 antibody or antibody fragments. Examples of
such
indicator reagents include detectable labels such as a radioactive label, a
fluorescent
label, a chemiluminescent label, an enzyme label, a bioluminescent label,
colloidal gold,
3o and the like. A kit may also comprise a means for conveying to the user
that Zsig67
antibodies or antibody fragments are used to detect Zsig67 protein. For
example,
written instructions may state that the enclosed antibody or antibody fragment
can be
used to detect Zsig67. The written material can be applied directly to a
container, or
the written material can be provided in the form of a packaging insert.
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77
13. Therapeutic Uses of Polypeptides Having Zsig67 Activity
The present invention includes the use of proteins, polypeptides, and
peptides described herein that have Zsig67 activity (such as Zsig67
polypeptides, anti
idiotype anti-Zsig67 antibodies, and Zsig67 fusion proteins) to a subject who
lacks an
adequate amount of this polypeptide.
Standard methods can be used to prepare pharmaceutically useful
compositions comprising a protein, polypeptide, or peptide having Zsig67
activity, in
which the therapeutic proteins are combined in a mixture with a
pharmaceutically
acceptable carrier. A composition is said to be a "pharmaceutically acceptable
carrier"
to if its administration can be tolerated by a recipient subject. Sterile
phosphate-buffered
saline is one example of a pharmaceutically acceptable carrier. Other suitable
carriers
are well-known to those in the art. See, for example, Gennaro (ed.),
Remington's
Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
A pharmaceutical composition comprising molecules having Zsig67
activity can be furnished in liquid form, in an aerosol, or in solid form.
Liquid forms,
are illustrated by injectable solutions and oral suspensions. Exemplary solid
forms
include capsules, tablets, and controlled-release forms. The latter form is
illustrated
by miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239
(1997); Ranade, "Implants in Drug Delivery," in Drug Delivery Systems, Ranade
and
2o Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al., "Protein
Delivery
with Infusion Pumps," in Protein Delivery. Physical Systems, Sanders and
Hendren
(eds.), pages 239-254 (Plenum Press 1997); Yewey et al., "Delivery of Proteins
from a
Controlled Release Injectable Implant," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).
~ Degradable polymer microspheres have been designed to maintain high
systemic levels of therapeutic proteins. Microspheres are prepared from
degradable
polymers such as poly(lactide-co-glycolide), polyanhydrides, poly (ortho
esters),
nonbiodegradable ethylvinyl acetate polymers, in which proteins are entrapped
in the
polymer (Gombotz and Pettit, Bioconjugate Chem. 6:332 (1995); Ranade, "Role of
3o Polymers in Drug Delivery," in Drug Delivery Systems, Ranade and Hollinger
(eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable Controlled
Release Systems Useful for Protein Delivery," in Protein Delivery: Physical
Systems,
Sanders and Hendren (eds.), pages 45-92 (Plenum Press 1997); Bartus et al.,
Science
281:1161 (1998); Putney and Burke, Nature Biotechnology 16:153 (1998); Putney,
Curr. Opin. Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
CA 02358930 2001-07-17
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78
nanospheres can also provide carriers for intravenous administration of
therapeutic
proteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167 (1997)).
Administration of a molecule having Zsig67 activity to a subject can be
intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous,
intrapleural,
intrathecal, by perfusion through a regional catheter, or by direct
intralesional
injection. When administering therapeutic proteins by injection, the
administration
may be by continuous infusion or by single or multiple boluses.
Additional routes of administration include oral, dermal, mucosal
membrane, pulmonary, and transcutaneous. Oral delivery is suitable for
polyester
l0 microspheres, zero microspheres, proteinoid microspheres, polycyanoacrylate
microspheres, and lipid-based systems (see, for example, DiBase and Morrel,
"Oral
Delivery of Microencapsulated Proteins," in Protein Delivery: Physical
Systems,
Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The
feasibility of
an intranasal delivery is exemplified by such a mode of insulin administration
(see, for
example, Hinchcliffe and Illum, Adv. Drug Deliv. Rev. 35:199 (1999)). Dry or
liquid
particles comprising a Zsig67 polypeptide, functional fragment, can be
prepared and
inhaled with the aid of dry-powder dispersers, liquid aerosol generators, or
nebulizers
(e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al., Adv. Drug
Deliv.
Rev. 35:235 (1999)). This approach is illustrated by the AERX diabetes
management
2o system, which is a hand-held electronic inhaler that delivers aerosolized
insulin into
the lungs. Studies have shown that proteins as large as 48,000 kDa have been
delivered across skin at therapeutic concentrations with the aid of low-
frequency
ultrasound, which illustrates the feasibility of trascutaneous administration
(Mitragotri
et al., Science 269:850 (1995)). Transdermal delivery using electroporation
provides
another means to administer Zsig67 molecules (Potts et al., Pharm. Biotechnol.
10:213 ( 1997)).
Generally, the dosage of administered polypeptide, protein or peptide
will vary depending upon such factors as the subject's age, weight, height,
sex, general
medical condition and previous medical history. Typically, it is desirable to
provide
3o the subject with a dosage of a molecule having Zsig67 activity which is in
the range of
from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of subject),
although a
lower or higher dosage also may be administered as circumstances dictate.
For purposes of therapy, molecules having Zsig67 activity and a
pharmaceutically acceptable carrier are administered to a subject in a
therapeutically
effective amount. A combination of a protein, polypeptide, or peptide having
Zsig67
activity and a pharmaceutically acceptable carrier is said to be administered
in a
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79
"therapeutically effective amount" if the amount administered is
physiologically
significant. An agent is physiologically significant if its presence results
in a
detectable change in the physiology of a recipient subject. Suitable subjects
include a
mammalian subject, such as a human.
s Zsig67 pharmaceutical compositions may be supplied as a kit
comprising a container that comprises Zsig67. Zsig67 can be provided in the
form of
an injectable solution for single or multiple doses, or as a sterile powder
that will be
reconstituted before injection. Such a kit may further comprise written
information on
indications and usage of the pharmaceutical composition. Moreover, such
information
to may include a statement that the Zsig67 composition is contraindicated in
subjects
with known hypersensitivity to Zsig67.
14. Therapeutic Uses of Zsig67 Nucleotide Sequences
The present invention includes the use of Zsig67 nucleotide sequences
15 to provide Zsig67 to a subject in need of such treatment. In addition, a
therapeutic
expression vector can be provided that inhibits Zsig67 gene expression, such
as an
anti-sense molecule, a ribozyme, or an external guide sequence molecule.
There are numerous approaches to introduce a Zsig67 gene to a subject,
including the use of recombinant host cells that express Zsig67, delivery of
naked
2o nucleic acid encoding Zsig67, use of a cationic lipid carrier with a
nucleic acid
molecule that encodes Zsig67, and the use of viruses that express Zsig67, such
as
recombinant retroviruses, recombinant adeno-associated viruses, recombinant
adenoviruses, and recombinant Herpes simplex viruses [HSV] (see, for example,
Mulligan, Science 260:926 (1993), Rosenberg et al., Science 242:1575 (1988),
LaSalle
25 et al., Science 259:988 (1993), Wolff et al., Science 247:1465 (1990),
Breakfield and
Deluca, The New Biologist 3:203 (1991)). In an ex vivo approach, for example,
cells
are isolated from a subject, transfected with a vector that expresses a Zsig67
gene, and
then transplanted into the subject.
In order to effect expression of a Zsig67 gene; an expression vector is
3o constructed in which a nucleotide sequence encoding a Zsig67 gene is
operably linked to
a core promoter, and optionally a regulatory element, to control gene
transcription. The
general requirements of an expression vector are described above.
Alternatively, a Zsig67 gene can be delivered using recombinant viral
vectors, including for example, adenoviral vectors (e.g., Kass-Eisler et al.,
Proc. Nat'l
35 Acad. Sci. USA 90:11498 (1993), Kolls et al., Proc. Nat'l Acad. Sci. USA
91:215
(1994), Li et al.; Hum. Gene Ther. 4:403 (1993), Vincent et al., Nat. Genet.
5:130
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(1993), and Zabner et al., Cell 75:207 (1993)), adenovirus-associated viral
vectors
(Flotte et al., Proc. Nat'l Acad. Sci. USA 90:10613 (1993)), alphaviruses such
as
Semliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857
(1992), Raju
and Huang, J. Vir. 65:2501 (1991), and Xiong et al., Science 243:1188 (1989)),
herpes
5 viral vectors (e.g., U.S. Patent Nos. 4,769,331, 4,859,587, 5,288,641 and
5,328,688),
parvovirus vectors (Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus
vectors (Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali
and
Paoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)), pox viruses, such as
canary pox
virus or vaccinia virus (Fisher-Hoch et al., Proc. Nat'l Acad. Sci. USA 86:317
( 1989),
to and Flexner et al., Ann. N Y. Acad. Sci. 569:86 (1989)), and retroviruses
(e.g., Baba et
al., J. Neurosurg 79:729 (1993), Ram et al., Cancer Res. 53:83 (1993),
Takamiya et
al., J. Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993),
Vile
and Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S. Patent No.
5,399,346). Within various embodiments, either the viral vector itself, or a
viral
15 particle which contains the viral vector may be utilized in the methods and
compositions described below.
As an illustration of one system, adenovirus, a double-stranded DNA
virus, is a well-characterized gene transfer vector for delivery of a
heterologous
nucleic acid molecule (for a review, see Becker et al., Meth. Cell Biol.
43:161 (1994);
20 Douglas and Curiel, Science & Medicine 4:44 ( 1997)). The adenovirus system
offers
several advantages including: (i) the ability to accommodate relatively large
DNA
inserts, (ii) the ability to be grown to high-titer, (iii) the ability to
infect a broad range
of mammalian cell types, and (iv) the ability to be used with many different
promoters
including ubiquitous, tissue sneciflc. and re~ulatahle nrnmntPrc Tn a~~;t;n"
25 adenoviruses can be administered by intravenous injection, because the
viruses are
stable in the bloodstream.
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,
3o the essential E1 gene is deleted from the viral vector, and the virus will
not replicate
unless the E1 gene is provided by the host cell. When intravenously
administered to
intact animals, adenovirus primarily targets the liver. Although an adenoviral
delivery
system with an E 1 gene deletion cannot replicate in the host cells, the
host's tissue
will express and process an encoded heterologous protein. Host cells will also
secrete
35 the heterologous protein if the corresponding gene includes a secretory
signal
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81
sequence. Secreted proteins will enter the circulation from tissue that
expresses the
heterologous gene (e.g., the highly vascularized liver).
Moreover, adenoviral vectors containing various deletions of viral
genes can be used to reduce or eliminate immune responses to the vector. Such
adenoviruses are E1-deleted, and in addition, contain deletions of E2A or E4
(Lusky et
al., .I. Virol. 72:2022 (1998); Raper et al., Human Gene Therapy 9:671
(1998)). The
deletion of E2b has also been reported to reduce immune responses (Amalfitano
et al.,
J. Virol. 72:926 (1998)). By deleting the entire adenovirus genome, very large
inserts
of heterologous DNA can be accommodated. Generation of so called "gutless"
1 o adenoviruses, where all viral genes are deleted, are particularly
advantageous for
insertion of large inserts of heterologous DNA (for a review, see Yeh. and
Perricaudet,
FASEB J. 11:615 (1997)).
High titer stocks of recombinant viruses capable of expressing a
therapeutic gene can be obtained from infected mammalian cells using standard
methods. For example, recombinant HSV can be prepared in Vero cells, as
described
by Brandt et al., J. Gen. Virol. 72:2043 ( 1991 ), Herold et al., J. Gen.
Virol. 75:1211
(1994), Visalli and Brandt, Virology 185:419 (1991), Grau et al., Invest.
Ophthalmol.
Vis. Sci. 30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992), and by
Brown
and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).
Alternatively, an expression vector comprising a Zsig67 gene can be
introduced into a subject's cells by lipofection in vivo using liposomes.
Synthetic
cationic lipids can be used to prepare liposomes for in vivo transfection of a
gene
encoding a marker (Felgner et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987);
Mackey et al., Proc. Nat'l Acad. Sci. USA 85:8027 (1988)). The use of
lipofection to
introduce exogenous genes into specific organs in vivo has certain practical
advantages. Liposomes can be used to direct transfection to particular cell
types,
which is particularly advantageous in a tissue with cellular heterogeneity,
such as the
pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other
molecules for the purpose of targeting. Targeted peptides (e.g., hormones or
neurotransmitters), proteins such as antibodies, or non-peptide molecules can
be
coupled to liposomes chemically.
Electroporation is another alternative mode of administration of a
Zsig67 nucleic acid molecules. For example, Aihara and Miyazaki, Nature
Biotechnology 16:867 (1998), have demonstrated the use of in vivo
electroporation for
gene transfer into muscle.
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In an alternative approach to gene therapy, a therapeutic gene may
encode a Zsig67 anti-sense RNA that inhibits the expression of Zsig67.
Suitable
sequences for Zsig67 anti-sense molecules can be derived from the nucleotide
sequences of Zsig67 disclosed herein.
s Alternatively, an expression vector can be constructed in which a
regulatory element is operably linked to a nucleotide sequence that encodes a
ribozyme. Ribozymes can be designed to express endonuclease activity that is
directed to a certain target sequence in a mRNA molecule (see, for example,
Draper
and Macejak, U.S. Patent No. 5,496,698, McSwiggen, U.S. Patent No. 5,525,468,
to Chowrira and McSwiggen, U.S. Patent No. 5,631,359, and Robertson and
Goldberg,
U.S. Patent No. 5,225,337). In the context of the present invention, ribozymes
include
nucleotide sequences that bind with Zsig67 mRNA.
In another approach, expression vectors can be constructed in which a
regulatory element directs the production of RNA transcripts capable of
promoting
15 RNase P-mediated cleavage of mRNA molecules that encode a Zsig67 gene.
According
to this approach, an external guide sequence can be constructed for directing
the
endogenous ribozyme, RNase P, to a particular species of intracellular mRNA,
which is
subsequently cleaved by the cellular ribozyme (see, for example, Altman et
al., U.S.
Patent No. 5,168,053, Yuan et al., Science 263:1269 (1994), Pace et al.,
international
20 publication No. WO 96/18733, George et al., international publication No.
WO
96/21731, and Werner et al., international publication No. WO 97/33991).
Preferably,
the external guide sequence comprises a ten to fifteen nucleotide sequence
complementary to Zsig67 mRNA, and a 3'-NCCA nucleotide sequence, wherein N is
preferably a purine. The external guide sequence transcripts bind to the
targeted mRNA
25 species by the formation of base pairs between the mRNA and the
complementary
external guide sequences, thus promoting cleavage of mRNA by RNase P at the
nucleotide located at the 5'-side of the base-paired region.
In general, the dosage of a composition comprising a therapeutic vector
having a Zsig67 nucleotide acid sequence, such as a recombinant virus, will
vary
30 depending upon such factors as the subject's age, weight, height, sex,
general medical
condition and previous medical history. Suitable routes of administration of
therapeutic vectors include intravenous injection, intraarterial injection,
intraperitoneal
injection, intramuscular injection, intratumoral injection, and injection into
a cavity
that contains a tumor.
35 A composition comprising viral vectors, non-viral vectors, or a
combination of viral and non-viral vectors of the present invention can be
formulated
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according to known methods to prepare pharmaceutically useful compositions,
whereby vectors or viruses are combined in a mixture with a pharmaceutically
acceptable carrier. As noted above, a composition, such as phosphate-buffered
saline
is said to be a "pharmaceutically acceptable carrier" if its administration
can be
tolerated by a recipient subject. Other suitable carriers are well-known to
those in the
art (see, for example, Remington's Pharmaceutical Sciences, 19th Ed. (Mack
Publishing Co. 1995), and Gilman's the Pharmacological Basis of Therapeutics,
7th
Ed. (MacMillan Publishing Co. 1985)).
For purposes of therapy, a therapeutic gene expression vector, or a
1o recombinant virus comprising such a vector, and a pharmaceutically
acceptable carrier
are administered to a subject in a therapeutically effective amount. A
combination of
an expression vector (or virus) and a pharmaceutically acceptable carrier is
said to be
administered in a "therapeutically effective amount" if the amount
administered is
physiologically significant. An agent is physiologically significant if its
presence
results in a detectable change in the physiology of a recipient subject.
When the subject treated with a therapeutic gene expression vector or a
recombinant virus is a human, then the therapy is preferably somatic cell gene
therapy.
That is, the preferred treatment of a human with a therapeutic gene expression
vector
or a recombinant virus does not entail introducing into cells a nucleic acid
molecule
that can form part of a human germ line and be passed onto successive
generations
(i. e., human germ line gene therapy).
15. Production of Transgenic Mice
Transgenic mice can be engineered to over-express the Zsig67 gene in
all tissues or under the control of a tissue-specific or tissue-preferred
regulatory
element. These over-producers of Zsig67 can be used to characterize the
phenotype
that results from over-expression, and the transgenic animals can serve as
models for
human disease caused by excess Zsig67. Transgenic mice that over-express
Zsig67
also provide model bioreactors for production of Zsig67, such as soluble
Zsig67, in
3o the milk or blood of larger animals. Methods for producing transgenic mice
are well-
known to those of skill in the art (see, for example, Jacob, "Expression and
Knockout
of Interferons in Transgenic Mice," in Overexpression and Knockout of
Cytokines in
Transgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994),
Monastersky and Robl (eds.), Strategies in Transgenic Animal Science (ASM
Press
1995), and Abbud and Nilson, "Recombinant Protein Expression in Transgenic
Mice,"
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84
in Gene Expression Systems: Using Nature for the Art of Expression, Fernandez
and
Hoeffler (eds.), pages 367-397 (Academic Press, Inc. 1999)).
For example, a method for producing a transgenic mouse that expresses
a Zsig67 gene can begin with adult, fertile males (studs) (B6C3fl, 2-8 months
of age
(Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl, 2-8
months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5
weeks,
(laconic Farms)) and adult fertile females (recipients) (B6D2f1, 2-4 months,
(laconic
Farms)). The donors are acclimated for one week and then injected with
approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma
to Chemical Company; St. Louis, MO) LP., and 46-47 hours later, 8 IU/mouse of
human
Chorionic Gonadotropin (hCG (Sigma)) LP. to induce superovulation. Donors are
mated with studs subsequent to hormone injections. Ovulation generally occurs
within 13 hours of hCG injection. Copulation is confirmed by the presence of a
vaginal plug the morning following mating.
Fertilized eggs are collected under a surgical scope. The oviducts are
collected and eggs are released into urinanalysis slides containing
hyaluronidase
(Sigma). Eggs are washed once in hyaluronidase, and twice in Whitten's W640
medium (described, for example, by Menino and O'Claray, Biol. Reprod. 77:159
(1986), and Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated
with
5% CO2, 5% OZ, and 90% Nz at 37°C. The eggs are then stored in a
37°C/5% COZ
incubator until microinjection.
Ten to twenty micrograms of plasmid DNA containing a Zsig67
encoding sequence is linearized, gel-purified, and resuspended in 10 mM Tris-
HCl
(pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10 nanograms
per
microliter for microinjection. For example, the Zsig67 encoding sequences can
encode a polypeptide comprising amino acid resides 111 to 422 of SEQ ID N0:2.
Plasmid DNA is microinjected into harvested eggs contained in a drop
of W640 medium overlaid by warm, COZ-equilibrated mineral oil. The DNA is
drawn
into an injection needle (pulled from a 0.75mm ID, lmm OD borosilicate glass
capillary), and injected into individual eggs. Each egg is penetrated with the
injection
needle, into one or both of the haploid pronuclei.
Picoliters of DNA are injected into the pronuclei, and the injection
needle withdrawn without coming into contact with the nucleoli. The procedure
is
repeated until all the eggs are injected. Successfully microinjected eggs are
transferred into an organ tissue-culture dish with pre-gassed W640 medium for
storage
overnight in a 37°C/5% COZ incubator.
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The following day, two-cell embryos are transferred into
pseudopregnant recipients. The recipients are identified by the presence of
copulation
plugs, after copulating with vasectomized duds. Recipients are anesthetized
and
shaved on the dorsal left side and transferred to a surgical microscope. A
small
5 incision is made in the skin and through the muscle wall in the middle of
the
abdominal area outlined by the ribcage, the saddle, and the hind leg, midway
between
knee and spleen. The reproductive organs are exteriorized onto a small
surgical drape.
The fat pad is stretched out over the surgical drape, and a baby serrefine
(Roboz,
Rockville, MD) is attached to the fat pad and left hanging over the back of
the mouse,
1 o preventing the organs from sliding back in.
With a fine transfer pipette containing mineral oil followed by
alternating W640 and air bubbles, 12-17 healthy two-cell embryos from the
previous
day's injection are transferred into the recipient. The swollen ampulla is
located and
holding the oviduct between the ampulla and the bursa, a nick in the oviduct
is made
15 with a 28 g needle close to the bursa, making sure not to tear the ampulla
or the bursa.
The pipette is transferred into the nick in the oviduct, and the embryos
are blown in, allowing the first air bubble to escape the pipette. The fat pad
is gently
pushed into the peritoneum, and the reproductive organs allowed to slide in.
The
peritoneal wall is closed with one suture and the skin closed with a wound
clip. The
20 mice recuperate on a 37°C slide warmer for a minimum of four hours.
The recipients are returned to cages in pairs, and allowed 19-21 days
gestation. After birth, 19-21 days postpartum is allowed before weaning. The
weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy
(used for
genotyping) is snipped off the tail with clean scissors.
25 Genomic DNA is prepared from the tail snips using, for example, a
QIAGEN DNEASY kit following the manufacturer's instructions. Genomic DNA is
analyzed by PCR using primers designed to amplify a Zsig67 gene or a
selectable
marker gene that was introduced in the same plasmid. After animals are
confirmed to
be transgenic, they are back-crossed into an inbred strain by placing a
transgenic
30 female with a wild-type male, or a transgenic male with one or two wild-
type
female(s). As pups are born and weaned, the sexes are separated, and their
tails
snipped for genotyping.
To check for expression of a transgene in a live animal, a partial
hepatectomy is performed. A surgical prep is made of the upper abdomen
directly
35 below the zyphoid process. Using sterile technique, a small 1.5-2 cm
incision is made
below the sternum and the left lateral lobe of the liver exteriorized. Using 4-
0 silk, a
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86
tie is made around the lower lobe securing it outside the body cavity. An
atraumatic
clamp is used to hold the tie while a second loop of absorbable Dexon
(American
Cyanamid; Wayne, N.J.) is placed proximal to the first tie. A distal cut is
made from
the Dexon tie and approximately 100 mg of the excised liver tissue is placed
in a
sterile petri dish. The excised liver section is transferred to a 14 ml
polypropylene
round bottom tube and snap frozen in liquid nitrogen and then stored on dry
ice. The
surgical site is closed with suture and wound clips, and the animal's cage
placed on a
37°C heating pad for 24 hours post operatively. The animal is checked
daily post
operatively and the wound clips removed 7-10 days after surgery. The
expression
to level of Zsig67 mRNA is examined for each transgenic mouse using an RNA
solution
hybridization assay or polymerase chain reaction.
In addition to producing transgenic mice that over-express Zsig67, it is
useful to engineer transgenic mice with either abnormally low or no expression
of the
gene. Such transgenic mice provide useful models for diseases associated with
a lack
of Zsig67. As discussed above, Zsig67 gene expression can be inhibited using
anti-
sense genes, ribozyme genes, or external guide sequence genes. To produce
transgenic mice that under-express the Zsig67 gene, such inhibitory sequences
are
targeted to Zsig67 mRNA. Methods for producing transgenic mice that have
abnormally low expression of a particular gene are known to those in the art
(see, for
2o example, Wu et al., "Gene Underexpression in Cultured Cells and Animals by
Antisense DNA and RNA Strategies," in Methods in Gene Biotechnology, pages 205-
224 (CRC Press 1997)).
An alternative approach to producing transgenic mice that have little or
no Zsig67 gene expression is to generate mice having at least one normal
Zsig67 allele
replaced by a nonfunctional Zsig67 gene. One method of designing a
nonfunctional
Zsig67 gene is to insert another gene, such as a selectable marker gene,
within a
nucleic acid molecule that encodes Zsig67. Standard methods for producing
these so-
called "knockout mice" are known to those skilled in the art (see, for
example, Jacob,
"Expression and Knockout of Interferons in Transgenic Mice," in Overexpression
and
3o Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124
(Academic
Press, Ltd. 1994), and Wu et al., "New Strategies for Gene Knockout," in
Methods in
Gene Biotechnology, pages 339-365 (CRC Press 1997)).
The present invention, thus generally described, will be understood more
readily by reference to the following examples, which are provided by way of
illustration and is not intended to be limiting of the present invention.
CA 02358930 2001-07-17
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87
EXAMPLE 1
Construction of a Nucleic Acid Molecule Encoding Zsig67
Pituitary gland RNA was purchased from CLONTECH Laboratories,
Inc. (Palo Alto, CA) and reversed transcribed in the following manner. The
first strand
cDNA reaction contained 10 ~1 of human pituitary poly (A)+ mRNA (CLONTECH),
which had been twice selected with poly d(T), at a concentration of 1.0 mg/ml,
and 2
~.1 of 20 pmole/~l first strand primer ZC6191 (GTCTG GGTTC GCTAC TCGAG
GCGGC CGCTA TTTTT TTTTT TTTTT TTT; SEQ ID NO:S) containing a XhoI
1o restriction site. The mixture was heated at 70°C for 2.0 minutes and
cooled by
chilling on ice. First strand cDNA synthesis was initiated by the addition of
8 ~.l of
first strand buffer (Sx SUPERSCRIPT buffer; Life Technologies, Inc.;
Gaithersburg,
MD), 4 ~l of 100 mM dithiothreitol, and 2 ~,l of a deoxynucleotide
triphosphate
(dNTP) solution containing 10 mM each of dTTP, dATP, dGTP and 5-methyl-dCTP
(Pharmacia LKB Biotechnology; Piscataway, NJ) to the RNA-primer mixture. The
reaction mixture was incubated at 37°C for 2 minutes, followed by the
addition of 10
ql of 200 U/~l RNase H reverse transcriptase (SUPERSCRIPT II; Life
Technologies).
The efficiency of the first strand synthesis was analyzed in a parallel
reaction by the
addition of 10 ~,Ci of 32P-adCTP to a 5 ~1 aliquot from one of the reaction
mixtures
to label the reaction for analysis. The reactions were incubated at
37°C for 5 minutes,
45°C for 45 minutes, then incubated at 50°C for 10 minutes.
Unincorporated 32p_
adCTP in the labeled reaction was removed by chromatography on a 400 pore size
gel
filtration column (CLONTECH). The unincorporated nucleotides and primers in
the
unlabeled first strand reactions were removed by chromatography on 400 pore
size gel
filtration column (CLONTECH). The length of labeled first strand cDNA was
determined by agarose gel electrophoresis.
The second strand reaction contained 100 ~1 of the unlabeled first
strand cDNA, 30 ~l of Sx polymerase I buffer (125 mM Tris-HCI, pH 7.5, 500 mM
KCI, 25 mM MgCl2, SOmM (NH4)2504)), 2.0 ~l of 100 mM dithiothreitol, 3.0 q.l
of
a solution containing 10 mM of each deoxynucleotide triphosphate, 7 ~,l of 5
mM (3-
NAD, 2.0 ~,l of 10 U/~l E. coli DNA ligase (NEW ENGLAND BIOLABS; Beverly,
MA), 5 ~,l of 10 U/~,l E. coli DNA polymerase I (NEW ENGLAND BIOLABS), and
1.0 ~,1 of 2 U/~,1 RNase H (Life Technologies). A 10 ~l aliquot from one of
the second
strand synthesis reactions was labeled by the addition of 10 ~,Ci 32P-adCTP to
monitor the efficiency of second strand synthesis. The reactions were
incubated at
16°C for two hours, followed by the addition of 1 ~,1 of a 10 mM dNTP
solution and
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88
5.0 p.l T4 DNA polymerase (10 U/p,l, Boehringer Mannheim Corporation;
Indianapolis, IN) and incubated for an additional 10 minutes at 16°C.
Unincorporated
32p_adCTP in the labeled reaction was removed by chromatography through a 400
pore size gel filtration column (CLONTECH) before analysis by agarose gel
electrophoresis. The reaction was terminated by the addition of 10.0 pl 0.5 M
EDTA
and extraction with phenol/chloroform and chloroform followed by ethanol
precipitation in the presence of 3.0 M sodium acetate and 2 ~l of Pellet Paint
carrier
(Novagen, Inc.; Madison, WI). The yield of cDNA was estimated to be
approximately
2 pg from starting mRNA template of 10 p.g.
1 o EcoRI adapters were ligated onto the 5' ends of the cDNA described
above to enable cloning into an expression vector. A 12.5 p,l aliquot of cDNA
(about
2.0 p,g) and 3 p,l of 69 pmole/~.l of EcoRI adapter (Pharmacia LKB
Biotechnology,
Inc.) were mixed with 2.5 p,l lOx ligase buffer (660 mM Tris-HCl pH 7.5, 100
mM
MgCl2), 2.5 p,l of 10 mM ATP, 3.5 pl 0.1 M DTT and 1 pl of 15 U/p,l T4 DNA
ligase
(Promega Corp.; Madison, WI). The reaction was incubated 1 hour at S°C,
2 hours at
7.5°C, 2 hours at 10°C, 2 hours at 12.5°C, and 16 hours
at 10°C. The reaction was
terminated by the addition of 65 pl Hz0 and 10 ~1 lOx H buffer (Boehringer
Mannheim) and incubation at 70°C for 20 minutes.
To facilitate the directional cloning of the cDNA into an expression
2o vector, the cDNA was digested with XhoI, resulting in cDNA molecules having
a 5'
EcoRI cohesive end and a 3' XhoI cohesive end. The XhoI restriction site at
the 3' end
of the cDNA had been previously introduced. Restriction enzyme digestion was
carried out in a reaction mixture by the addition of 1.0 pl of 40 U/~.1 XhoI
(Boehringer
Mannheim) at 37°C for 45 minutes. The reaction was terminated by
incubation at
70°C for 20 minutes and chromatography through a 400 pore size gel
filtration column
(CLONTECH).
The cDNA was precipitated with ethanol, washed with 70% ethanol,
air-dried and resuspended in 13.5 ~.1 water, 2 p,l of lOx kinase buffer (660
mM Tris-
HCI, pH 7.5, 100 mM MgClz), 0.5 pl 0.1 M DTT, 2 p,l 10 mM ATP, 2 p,l T4
3o polynucleotide kinase (10 U/p,l, Life Technologies). Following incubation
at 37° C
for 30 minutes, the cDNA was precipitated with ethanol in the presence of 2.5
M
ammonium acetate, and fractionated on a 0.8% low melt agarose electrophoresis
gel.
The contaminating adapters and cDNA below 0.6 kb in length were excised from
the
gel. The electrodes were reversed, and the cDNA was fractionated until
concentrated
near the lane origin. The area of the gel containing the concentrated cDNA was
excised and placed in a microfuge tube, and the approximate volume of the gel
slice
CA 02358930 2001-07-17
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89
was determined. An aliquot of water approximately three times the volume of
the gel
slice (300 ~1) and 35 ~.1 of lOx (3-agarose I buffer (NEW ENGLAND BIOLABS)
were
added to the tube, and the agarose was melted by heating to 65°C for 15
minutes.
Following equilibration of the sample to 45°C, 3 ~1 of 1 U/~,1 (3-
agarose I (NEW
ENGLAND BIOLABS) were added, and the mixture was incubated for 60 minutes at
45°C to digest the agarose. After incubation, 40 ~,1 of 3 M sodium
acetate were added
to the sample, and the mixture was incubated on ice for 15 minutes. The sample
was
centrifuged at 14,000 x g for 15 minutes at room temperature to remove
undigested
agarose. The cDNA was precipitated with ethanol, washed in 70% ethanol, air-
dried
to and resuspended in 20 p.l water.
Following recovery from low-melt agarose gel, the cDNA was cloned
into the EcoRI and XhoI sites of pBLUESCRIPT SK+ vector (Life Technologies,
Inc.)
and electroporated into DH10B cells. Bacterial colonies containing ESTs of
known
genes were identif ed and eliminated from sequence analysis by reiterative
cycles of
probe hybridization to hi-density colony filter arrays (Genome Systems, Inc.;
St.
Louis, MO). cDNAs of known genes were pooled in groups of 50 - 100 inserts and
were labeled with 3zP using a MEGAPRIME labeling kit (AMERSHAM
PHARMACIA BIOTECH, Inc.; Piscataway, NJ). Colonies that did not hybridize to
the probe mixture were selected for sequencing. Sequencing was performed with
an
ABI 377 sequencer using either the T3 or the reverse primer. The resulting
data were
analyzed which resulted in the identification of novel ESTs, including an EST
with the
following sequence: GCACGAGGGCT ATGTTACCCC ATGACAGTTG
ACCTCTTCTG GGTGAATATT ATCTGATTGA TTTTGCAAAT TCCACACCTG
TATGGATGAA ' GAGAATATAT ATTGTAGATG CTAGATCATG
AGGACTCCCA ACACCTCATT TCTAGTCCTG GCTTCACAGC CTCTTCTGGT
CCTCATCTCA CTATCAGCAT TGATCCTGGC ATCATATTCA AGTCCCCTCC
TAACCCGGGT CTCTCTTGAG ACAGTGAGAA CTAAAGAAGA
TGGAAGACAC AATGATTTCA ACAAGATTAA GGATAAGGAT
GCAAGTAGAG CAGGCAGGGA ACGAGGTTAT AGAAACTTTT
3o TATTCCATTT CCATCTGTCT CTCTTTCCTC ACAACAAGTC (SEQ ID N0:6).
This EST was used to obtain the complete Zsig67 coding region.
EXAMPLE 2
Southern Analysis of the Zsig67 Gene
Southern analysis was performed using a commercially prepared
Interspecies Zoo-Blot from CLONTECH Laboratories, Inc. The hybridization probe
CA 02358930 2001-07-17
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was generated from a gel-purified PCR amplification product of about 140 base
pairs.
The probe was made using ZC20,973 (5' ATGCT AGATC ATGAG GACTC CCAAC
3'; SEQ ID N0:7) and ZC20,974 (5' CCATC TTCTT TAGTT CTCAC TGTCT 3';
SEQ ID N0:8) as primers and pituitary gland cDNA as template. The probe was a
5 radioactively labeled using the REDIPRIME II labeling kit (AMERSHAM
PHARMACIA BIOTECH, Inc.; Piscataway, NJ) according to the manufacturer's
protocol. The probe was purified using a NUCTRAP push column (STRATAGENE,
La Jolla, CA). EXPRESSHYB (CLONTECH) solution was used for the
prehybridization and hybridization solutions for the Southern blots.
Hybridization
1o took place overnight at 65°C. Following hybridization, the blots
were washed four
times in 2x SSC, 0.05% SDS at room temperature, followed by two washes in O.lx
SSC and 0.1% SDS at 50°C. The blots were then exposed to Kodak BioMax
film at -
80°C. Hybridizing fragments were observed in genomic DNA samples from
rabbit,
cow, dog, monkey, and human.
CA 02358930 2001-07-17
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1
SEQUENCE LISTING
<110> ZymoGenetics. Inc.
<120> ZSIG67: A MEMBER OF THE HUMAN SECRETIN-GLUCAGON-UIP FAMILY
<130> 98-70PC
<160> 8
<170> FastSEQ for Windows Uersion 3.0
<210>1
<211>1682
<212>DNA
<213>Homo Sapiens
<220>
<221> CDS
<222> (111)...(422)
<400> 1
gctatgttac cccatgacag ttgacctctt ctgggtgaat attatctgat tgattttgca 60
aattccacac ctgtatggat gaagagaata tatattgtag atgctagatc atg agg 116
Met Arg
1
act ccc aac acc tca ttt cta gtc ctg get tca cag cct ctt ctg gtc 164
Thr Pro Asn Thr Ser Phe Leu Ual Leu Ala Ser Gln Pro Leu Leu Val
10 15
ctc atc tca cta tca gca ttg atc ctg gca tca tat tca agt ccc ctc 212
Leu Ile Ser Leu Ser Ala Leu Ile Leu Ala Ser Tyr Ser Ser Pro Leu
20 25 30
cta acc cgg gtc tct ctt gag aca gtg aga act aaa gaa gat gga aga 260
Leu Thr Arg Val Ser Leu Glu Thr Val Arg Thr Lys Glu Asp Gly Arg
35 40 45 50
cac aat gat ttc aac aag att aag gat aag gat gca agt aga gca ggc 308
His Asn Asp Phe Asn Lys Ile Lys Asp Lys Asp Ala Ser Arg Ala Gly
55 60 65
CA 02358930 2001-07-17
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2
agg gaa cga ggt tat aga aac ttt tta ttc cat ttc cat ctg tct ctc 356
Arg Glu Arg Gly Tyr Arg Asn Phe Leu Phe His Phe His Leu Ser Leu
70 75 80
ttt cct cac aac agt ccc aat agc att tca aag ggt ttc aag ttc cat 404
Phe Pro His Asn Ser Pro Asn Ser Ile Ser Lys Gly Phe Lys Phe His
85 90 g5
gta agt tat aga aaa aaa taaagcacaa gttctctgtg acatgttcag 452
Ual Ser Tyr Arg Lys Lys
100
cttatctatgaatataacggaactgaaaccatatttcaataaattattaacagcaatccc512
ttgccaagaaggatatcagcacagataacagggcaaatccattataactcttaagcaaat572
cctggaattttaaaaaaattgaatagtgtatcaaataagtgcactgttagctgggacttc632
attaaacagcattttttttcctttctttcctgttcctccccctttcccaccagcttccag692
ccagaataattgataactaaatctcaccatgaatttgttgggaatttacccacaatagtt752
ctcaatttattttaagatttaaaataatatatttttaaatagtagattaagactttacag812
aaatttcagataggataatacagaaccttgaaaagacttattttttacaaacttccttaa872
aaattcttaattgttctaacagtcacagttttgtttatgtatacaattggagttctgcag932
taagacactgtggggttattagtacatatatcaaattcatgaggctcctattcctgtata992
ttgttacagagctgttcttgtatattgatgtttaatgatatggggccgaattgttattca1052
aataattacagcccattcctgattatttagatgctgattaactagtcaatgacttattcc1112
ctttgacaaaatttcccaccctctggctttcctttgatgttaatgaggaagaggatccag1172
ttttcatcccatttggcaaggttggtttcatggactgagccactctggatgtaagatcag1232
tagctactctgtagactttatggtaatggaagttaggttaggttgcatcagaacactcaa1292
aagtcaagcctatcaccatatttgagaagaaactcacagtatttgatataccttagtgga1352
tttgaaacaacatcttatgttacaaaaaaatagcattttaaaaattgggtggggcactgt1412
ggatcactcctgtaatcccaacacttggggaggccagggtgggcagatcacttgaggccg1472
ggatttcgaggctagcctggccaacgtggtgaagccccatctctattagaactacaggaa1532
ttagctgggcatggtggtgcacgcctgtggtgccagccactcgggtggttgagacatgag1592
aattgctttgacccgggaggcggaggttgcagtgagccaagatcatgccactgcactcta1652
gcctgggcgacagagcaagactctgtctca 1682
<210>2
<211>104
<212>PRT
<213>Homo Sapiens
<400> 2
Met Arg Thr Pro Asn Thr Ser Phe Leu Val Leu Ala Ser Gln Pro Leu
1 5 10 15
Leu Ual Leu Ile Ser Leu Ser Ala Leu Ile Leu Ala Ser Tyr Ser Ser
20 25 30
CA 02358930 2001-07-17
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3
Pro Leu Leu Thr Arg Val Ser Leu Glu Thr Val Arg Thr Lys Glu Asp
35 40 45
Gly Arg His Asn Asp Phe Asn Lys Ile Lys Asp Lys Asp Ala Ser Arg
50 55 60
Ala Gly Arg Glu Arg Gly Tyr Arg Asn Phe Leu Phe His Phe His Leu
65 70 75 80
Ser Leu Phe Pro His Asn Ser Pro Asn Ser Ile Ser Lys Gly Phe Lys
85 90 95
Phe His Val Ser Tyr Arg Lys Lys
100
<210> 3
<211> 312
<212> DNA
<213> Artificial Sequence
<220>
<223> This degenerate sequence encodes the amino acid
sequence of SEQ ID N0:2.
<221> variation
<222> (1)...(312)
<223> N is any nucleotide.
<400>
3
atgmgnacnccnaayacnwsnttyytngtnytngcnwsncarccnytnytngtnytnath 60
wsnytnwsngcnytnathytngcnwsntaywsnwsnccnytnytnacnmgngtnwsnytn 120
garacngtnmgnacnaargargayggnmgncayaaygayttyaayaarathaargayaar 180
gaygcnwsnmgngcnggnmgngarmgnggntaymgnaayt,tyytnttycayttycayytn 240
wsnytnttyccncayaaywsnccnaaywsnathwsnaarggnttyaarttycaygtnwsn 300
taymgnaaraar 312
<210> 4
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Peptide linker
<400> 4
Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 5
CA 02358930 2001-07-17
WO 00/43516 PCT/US00/00870
4
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer
<400> 5
gtctgggttc gctactcgag gcggccgcta tttttttttt tttttttt 48
<210> 6
<211> 381
<212> DNA
<213> Artificial Sequence
<220>
<223> EST
<400>
6
gcacgagggctatgttaccccatgacagttgacctcttctgggtgaatattatctgattg 60
attttgcaaattccacacctgtatggatgaagagaatatatattgtagatgctagatcat 120
gaggactcccaacacctcatttctagtcctggcttcacagcctcttctggtcctcatctc 180
actatcagcattgatcctggcatcatattcaagtcccctcctaacccgggtctctcttga 240
gacagtgagaactaaagaagatggaagacacaatgatttcaacaagattaaggataagga 300
tgcaagtagagcaggcagggaacgaggttatagaaactttttattccatttccatctgtc 360
tctctttcctcacaacaagtc 381
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 7
atgctagatc atgaggactc ccaac 25
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
CA 02358930 2001-07-17
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<400> 8
ccatcttctt tagttctcac tgtct 25
