Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HUMAN CELL SIGNALING PROTEINS (CSIG)
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human cell
signaling
proteins and to the use of these sequences in the diagnosis, treatment, and
prevention of neoplastic,
neurological, immunological, vesicle trafficking, and smooth muscle disorders.
BACKGROUND OF THE INVENTION
Intercellular communication is essential for the development and survival of
multicellular
organisms. This communication is achieved through the secretion of proteins by
signaling cells and
the internalization of these proteins by target cells. Signal transduction,
the general process by which
cells respond to extracelluiar signals {hormones, neurotransmitters, growth
and differentiation factors,
etc.) involves a cascade of biochemical reactions that begins with the binding
of the signaling
molecule to a cell membrane receptor and ends with the activation of an
intracellular target molecule.
Intermediate steps in this process involve the activation o:f various
cytoplasmic proteins by
phosphorylation via protein kinases and the eventual trap slocation of some of
these activated proteins
t5 to the cell nucleus where the transcription of specific genes is triggered.
Thus, the signal transduction
process regulates all types of cell functions including cell proliferation,
differentiation, and gene
transcription.
Growth factors are secreted proteins that mediate communication between
signaling and target
cells. Inside the signaling cell, growth factors are synthesized and
transported through the secretory
pathway. Entry into the secretory pathway is mediated by a signal peptide
sequence, a protein sorting
motif at the N-terminus of most secreted proteins. Growth factors can undergo
further post-
translational modifications within the secretory pathway. These modifications
include glycosylation,
phosphorylation, and intramolecular disulfide bond formation. Following
secretion into the
extracellular space, some growth factors self associate as dimers or
tetramers, while others associate
with extracellular matrix components. Secreted growth factors bind to specific
receptors on the
surface of target cells, and the growth factor-bound receptors trigger second
messenger signal
transduction pathways. These signal transduction pathways elicit specific
cellular responses in the
target cells. Such responses can include the modulation of gene expression and
the stimulation or
inhibition of cell division, cell differentiation, and cell motility.
Growth factors fall into three. broad and overlapping classes. The first class
of growth factors
includes hematopoietic growth factors, which have a narrow target specificity.
These factors stimulate
the proliferation and differentiation of blood cells such as I3-lyrnphocytes,
T-lymphocytes,
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erythrocytes, platelets, eosinophils, basophils, neutrophils, and macrophages,
and the stem cell
precursors of these cells, and include the colony-stimulating factors (e.g., G-
CSF, M-CSF, GM-CSF,
and CSF1-3), erythropoietin, and the cytokines.
The second class of growth factors includes smalfl peptide factors, which
primarily function as
hormones in the regulation of highly specialized processes other than cellular
proliferation. These
factors, typically less than 20 amino acids in length, are I;enerated by the
proteolytic processing of
larger precursor proteins. This class includes bombesin, vasopressin,
oxytocin, endothelin, transferrin,
angiotensin II, vasoactive intestinal peptide, bradykinin, and related
peptides.
The third and broadest class of growth factors includes large polypeptide
growth factors,
which are wide-ranging in their effects. Examples of these factors are
epidermal growth factor (EGF),
fibroblast growth factor (FGF), transforming growth factor-[3 (TGF-[3),
insulin-like growth factor
(IGF), nerve growth factor (NGF), and platelet-derived growth factor (PDGF),
each of which defines a
family containing numerous related factors. The large polypeptide growth
factors generally act as
mitogens on diverse cell types to stimulate wound heafinl;, bone synthesis and
remodeling,
I S extracellular matrix synthesis, and proliferation of epithelial,
epidermal, and connective tissues. Some
members of the EGF, FGF, and TGF-~ families are also involved in the
differentiation of embryonic
tissue. However, some of the large polypeptide growth factors carry out
specific functions on a
restricted set of target tissues. For example, mouse growt:hldifferentiation
factor 9 (GDF-9) is a TGF-
~i family member that is expressed solely in the ovary {McPherron, A. C. and
Lee, S.-J. (1993) J. Biol.
Chem. 268:3444-3449}. NGF functions specifically as a neurotrophic factor,
promoting neuronal
growth and differentiation. (See, e.g., Pimentel, E. ( 1994) Handbook of
Growth Factors, CRC Press,
Ann Arbor MI; McKay, I. and Leigh, I. (1993) Growth Factors: A Practical
Approach, Oxford
University Press, New York NY; and Habenicht, A. (1990)-Growth Factors.
Differentiation Factors
and Cvtokines, Springer-Verlag, New York NY.)
EGF is a growth factor that stimulates proliferation of several epithelial
tissues or cell lines. In
addition to this mitogenic effect, EGF produces non-mitol;enic effects in
certain tissues. For example,
in the stomach, EGF inhibits gastric acid secretion by parietal cells
(Massague, J. and Pandiella, A.
(1993) Annu. Rev. Biochem. 62:515-541). EGF is produced as a larger precursor
and contains an N-
terminal signal peptide sequence that is thought to aid in localization of EGF
to the plasma membrane.
EGF contains three repeats of the calcium-binding EGF-lilke domain signature
sequence. This
signature sequence is about forty amino acid residues in length and includes
six conserved cysteine
residues, and a calcium-binding site near the N-terminus of the signature
sequence. A number of
proteins that contain calcium-binding EGF-like domain sil;nature sequences are
involved in growth
and differentiation. Examples include bone morphogenic protein l, which
induces the formation of
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cartilage and bone; crumbs, which is a Drosophila epithelial development
protein; Notch and a number
of its homologs, which are involved in neural growth and differentiation; and
transforming growth
factor beta-1 binding protein (Expasy PROSITE document PDOC00913; Soler, C.
and Carpenter, G.,
in Nicola, N.A. (1994) The Cytokine Facts Book; Oxford University Press,
Oxford, UK, pp 193-197).
Growth and differentiation factors play critical roles in neoplastic
transformation of cells in
vitro, in tumor progression in viva, and in normal growth and development.
Overexpression of the
large polypeptide growth factors promotes the proliferatiion and
transformation of cells in culture.
Moreover, tumor cells and normal cells show differential expression of certain
growth factors. For
example, northern analysis shows the expression of human hepatoma-derived
growth factor messenger
RNA at various levels in hepatoma and other transformed cell lines and in
normal tissues (Nakamura,
H. et al. (1994) J. Biol. Chem. 269:25143-25149). Inappropriate expression of
large polypeptide
growth factors by tumor cells in vivo may contribute to vascularization and
metastasis of tumors.
Furthermore, some of the large polypeptide growth factors are both
structurally and functionally
related to oncoproteins, the cancer-correlated products ofoncogenes. Certain
FGF and PDGF family
1S members are themselves homologous to oncoproteins, wlhile receptors for
some members of the EGF,
NGF, and FGF families are encoded by proto-oncogenes.. Growth factors also
affect the
transcriptional regulation of both proto-oncogenes and oncosuppressor genes
(Pimentel, supra}.
Differentiation of tissues in metazoans appears tc~ be rooted in the synthesis
of critical
extracellular and intracellular proteins during oogenesis amd ernbryogenesis.
Oogenesis and
embryogenesis are regulated by interactions between environmental,
extracellular, and intracellular
signals. Changes in signaling pathways caused by geneti~~ mutation or
biochemical modification can
affect oogenesis and embryogenesis in a number of ways. Specifically, these
changes may result in
morphological changes during ontogeny which may result in sudden postnatal
death.
Tafazzins (TAZ) are skeletal muscle and heart proteins present in tissue in up
to ten isoforms.
Isoforms with hydrophobic N-termini are thought to be membrane-anchored.
Shorter isoforms
frequently start at methionine 25 and lack the hydrophobic stretch. These
isoforms are thought to be
cytosolic. Mutations which introduce premature stop codons in the TAZ gene are
the cause of Barth
Syndrome, a severe inherited disorder characterized by cardiac and skeletal
myopathy, short stature,
neutropenia, and abnormal mitochondria. Respiratory chain abnormalities have
been observed in
cultured fibroblasts from patients with Barth Syndrome which may be fatal in
childhood (Barth, P.G.
et al. (1996) J. Inher. Metab. Dis. 19:157-160).
The discovery of new human cell signaling proteins and the polynucleotides
encoding them
satisfies a need in the art by providing new compositions which are useful in
the diagnosis, prevention,
and treatment of neoplastie, neurological, immunological, vesicle trafficking,
and smooth muscle
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disorders.
SUMMARY OF THE (INVENTION
The invention is based on the discovery of new lhuman cell signaling proteins
(CSIG), referred
to collectively as "CSIG" and individually as "CSIG-I" .and "CSIG-2," the
polynucleotides encoding
CSIG, and the use of these compositions for the diagnosis, treatment, or
prevention of neoplastic,
neurological, immunological, vesicle trafficking, and smooth muscle disorders.
The invention features a substantially purified polypeptide comprising an
amino acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, a fragment of
SEQ ID NO:1, and
a fragment of SEQ ID N0:2.
The invention further provides a substantially purified variant having at
least 90% amino acid
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:I, SEQ
ID N0:2, a fragment of SEQ ID NO:1, and a fragment o:f SEQ ID N0:2. The
invention also provides
an isolated and purified polynucleotide encoding the polypeptide comprising an
amino acid sequence
selected from the group consisting of SEQ 1D NO:1, SEQ ID N0:2, a fragment of
SEQ ID NO: l, and
a fragment of SEQ ID N0:2. The invention also includes an isolated and
purified polynucleotide
variant having at least 70% polynucIeotide sequence identity to the
polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1,
SEQ ID N0:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID N0:2.
The invention further provides an isolated and purified polynucleotide which
hybridizes under
stringent conditions to the polynucleotide encoding the p~oiypeptide
comprising an amino acid
sequence selected, from the group consisting of SEQ ID TdO:1, SEQ ID N0:2, a
fragment of SEQ ID
NO: I, and a fragment of SEQ 1D N0:2 as well as an isolated and purified
polynucleotide having a
sequence which is complementary to the polynucleotide <,ncoding the
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
N0:2, a fragment
of SEQ ID NO:1, and a fragment of SEQ ID N0:2.
The invention also provides an isolated and purifiied poiynucleotide
comprising a
polynucleotide sequence selected from the group consisting of SEQ ID N0:3, SEQ
ID N0:4, a
fragment of SEQ ID N0:3, and a fragment of SEQ ID NO:4 and an isolated and
purified
polynucleotide variant having at least 70% polynucleotide sequence identity to
the polynucleotide
comprising a polynucIeotide sequence selected from the group consisting of SEQ
ID N0:3, SEQ ID
N0:4, a fragment of SEQ ID N0:3, and a fragment of SE~Q ID N0:4. Tlre
invention also provides an
isolated and purified poIynucleotide having a sequence complementary to the
polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:3, SEQ ID
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N0:4, a fragment of SEQ ID N0:3, and a fragment of SEQ ID N0:4.
The invention also provides a method for detecting a polynucleotide in a
sample containing
nucleic acids, the method comprising the steps of (a) hybridizing the
complement of the
polynucleotide sequence to at least one of the polynucleotides of the sample,
thereby forming a
hybridization complex; and (b) detecting the hybridization complex, wherein
the presence of the
hybridization complex correlates with the presence of a polynucleotide in the
sample. In one aspect,
the method further comprises amplifying the polynucleotide prior to
hybridization.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID NO:1, SEQ 1D N0:2; a fragment oiF SEQ ID NO: !, and a
fragment of SEQ ID
N0:2. In another aspect, the expression vector is contained within a host
cell.
The invention also provides a method for produciing a palypeptide, the method
comprising the
steps of: {a) culturing the host cell containing an expression vector
containing at least a fragment of a
polynucleotide under conditions suitable for the expression of the
polypeptide; and (b) recovering the
IS polypeptide from the host cell culture.
The invention also provides a pharmaceutical connposition comprising a
substantially purified
polypeptide having an amino acid sequence selected from the group consisting
of SEQ IDNO:1, SEQ
ID N0:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID N0:2 in
conjunction with a suitable
pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
N0:2, a fragment
of SEQ ID NO:1, and a fragment of SEQ ID N0:2; as well as a purified agonist
and a purified
antagonist of the polypeptide.
The invention also provides a method for treating or preventing a disorder
associated with
decreased expression or activity of CSIG, the method comprising administering
to a subject in need of
such treatment an effective amount of a pharmaceutical composition comprising
a substantially
purified polypeptide having an amino acid sequence selecl;ed from the group
consisting of SEQ ID
NO:1, SEQ ID N0:2, a fragment of SEQ ID NO:1, and a fragment of SEQ ID N0:2,
in conjunction
with a suitable pharmaceutical carrier.
The invention also provides a method for treating .or preventing a disorder
associated with
increased expression or activiiy of CSIG, the method comprising administering
to a subject in need of
such treatment an effective amount of an antagonist of the polypeptide having
an amino acid sequence
selected from the group consisting of SEQ ID NO: I, SEQ ID NO:2, a fragment of
SEQ ID NO:1, and
a fragment of SEQ 1D N0:2.
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BRIEF DESCRIPTION OF THE IFIGURE AND TABLE
Figures lA and IB show the amino acid sequence; alignments between CSIG-1
(999661; SEQ
ID NO:1 ) and human tafazzin (GI 1263110; SEQ ID NO:.S); produced using the
multisequence
alignment program of LASERGENE software (DNASTAR Inc; Madison WI}.
Table 1 shows the programs, their descriptions, references, and threshold
parameters used to
analyze CSIG.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences,, and methods are described,
it is understood
IO that this invention is not limited to the particular machines, materials
and methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
I S and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example; a
reference to "a host cell" includes a plurality of such host cells, and a
reference to '.'an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
20 meanings as commonly understood by one of ordinary ski:fl in the art to
which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described: All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and wvhich might be
25 used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"CSIG" refers to the amino acid sequences of substantially purified CSIG
obtained from any
species, particularly a mammalian species; including bovine, ovine, porcine,
murine, equine; and
30 preferably the human species, from any source, whether natural, synthetic,
semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which, when bound to CSIG, increases
or prolongs the
duration of the effect of CSIG. Agonists may include proteins, nucleic acids,
carbohydrates, or any
other molecules which bind to and modulate the effect of C:SIG.
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An "allelic variant" is an alternative form of the gene encoding CSIG. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. Any given
natural or
recombinant gene may have none, one, or many allelic forms: Common mutational
changes which
give rise to allelic variants are generally ascribed to natural deletions,
additions, or substitutions of
nucleotides. Each of these types of changes may occur alone, or in combination
with the others, one
or more times in a given sequence.
'°Altered" nucleic acid sequences encoding CSIG include those sequences
with deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same. as CSIG or a
polypeptide with at least one functional characteristic of CSIG. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding CSIG, and improper or unexpected hybridization to
allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding CSIG.
The encoded protein may also be "altered," and may contain deletions,
insertions, or substitutions of
amino acid residues which produce a silent change and reault in a functionally
equivalent CSIG.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as tong as the
biological or immunologica) activity of CSIG is retained. For example,
negatively charged amino
acids may include aspartic acid and glutamic acid, positively charged amino
acids may include lysine
and arginine, and amino acids with uncharged polar head groups having similar
hydrophilicity values
may include leucine, isoleucine, and valine; glycine and a.lanine; asparagine
and glutamine; serine and
threonine; and phenylalanine and tyrosine.
The, terms "amino acid" or "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of 'these, and to
naturally occurring or synthetic
molecules. In this context, "fragments," "immunogenic fragments," or
"antigenic fragments" refer to
fragments of CSIG which are preferably at least 5 to about 15 amino acids in
length, most preferably
at least 14 amino acids, and which retain some biological activity or
immunological activity of CSIG.
Where "amino acid sequence" is recited to refer to an amino acid sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid sequence
to the complete native amino acid sequence associated with the recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which, when bound to CSIG,
decreases the amount
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or the duration of the effect of the biological or immunological activity of
CS1G. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any other
molecules which decrease the
effect of CSIG.
The term "antibody" refers to intact molecules as well as to fragments
thereof, such as Fab,
F(ab')2, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies that
bind CSIG polypeptides can be prepared using intact pollypeptides or using
fragments containing small
peptides of interest as the immunizing antigen. The pol~rpeptide or
oligopeptide used to immunize an
animal {e.g., a mouse, a rat, or a rabbit) can be derived from the translation
of RNA, or synthesized
chemically, and can be conjugated to a carrier protein if desired. Commonly
used carriers that are
chemically coupled to peptides include bovine serum albumin, thyroglobulin,
and keyhole limpet
hemocyanin (KLH). The coupled peptide is then used to~ immunize the animal.
The term "antigenic determinant" refers to that firagment of a molecule (i.e.,
an epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (given regions or three-
dimensional structures on the
protein). An antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to
elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid
sequence which is
complementary to the "sense" strand of a specific nucleic. acid sequence.
Antisense molecules may be
produced by any method including synthesis or transcription. Once introduced
into a cell, the
complementary nucleotides combine with natural sequences produced by the cell
to form duplexes and
to block either transcription or translation. The designation "negative" can
refer to the antisense
strand, and the designation "positive" can refer to the sense strand.
The term "biologically active," refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" refers to the
capability of the natural, recombinant, or synthetic CSIG, or of any
oligopeptide thereof, to induce a
specific immune response in appropriate animals or cells and to bind with
specific antibodies.
The terms "complementary" or "complementarity" refer to the natural binding of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-A 5'." Complementarit;y between two single-
stranded molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete," such that total
complementarity exists between the single stranded molecules. The degree
ofcomplementarity
between nucleic acid strands has significant effects on the efficiency and
strength of the hybridization
between the nucleic acid strands. This is of particular importance in
amplification reactions, which
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depend upon binding between nucleic acids strands, and in the design and use
of peptide nucleic acid
(PNA} molecules.
A "composition comprising a given polynucleotide sequence" or a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a .dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding CSIG or fragments of
CSIG may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., N<iCI), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, d;ry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
resequenced to
resolve uncalled bases, extended using XL-PCR kit (Perk.in-Elmer, Norwalk CT)
in the 5' and/or the 3'
direction, and resequenced, or which has been assembled from the overlapping
sequences of more
than one Incyte Clone using a computer program for fragiment assembly, such as
the GELVIEW
IS Fragment Assembly system (GCG, Madison WI). Some sequences have been both
extended and
assembled to produce the consensus sequence.
The term "correlates with expression of a polynuc;ieotide" indicates that the
detection of the
presence of nucleic acids, the same or related to a nucleic acid sequence
encoding CSIG, by northern
analysis is indicative of the presence of nucleic acids encoding CSIG in a
sample, and thereby
correlates with expression of the transcript from the polynucleotide encoding
CSIG.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to the chemical modification of a polypeptide
sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide sequence
can include, for
ZS example, replacement of hydrogen by an alkyl, acyl, or amino group. A
derivative polynucleotide
encodes a polypeptide which retains at least one biological or immunological
function ofthe natural
molecule. A derivative polypeptide is one modified by glycosylation,
pegylation, or any similar
process that retains at least one biological or immunological function of the
polypeptide from which it
was derived.
The term "similarity" refers to a degree of c4mple~mentarity. There may be
partial similarity
or complete similarity. The word "identity" may substitute; for the word
"similarity." A partially
complementary sequence that at least partially inhibits an identical sequence
from hybridizing to a
target nucleic acid is referred to as "substantially similar." The inhibition
of hybridization of the
completely complementary sequence to the target sequence may be examined using
a hybridization
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assay (Southern or northern blot, solution hybridization, and the like) under
conditions of reduced
stringency. A substantially similar sequence or hybridization probe will
compete for and inhibit the
binding of a completely similar (identical) sequence to the target sequence
under conditions of reduced
stringency. This is not to say that conditions of reduced stringency are such
that non-specific binding
is permitted, as reduced stringency conditions require that the binding of two
sequences to one another
be a specific (i.e., a selective) interaction. The absence of non-specific
binding may be tested by the
use of a second target sequence which lacks even a partial degree of
complementarity (e.g., less than
about 30% shmilarity or identity). In the absence of non, specific binding,
the substantially similar
sequence or probe will not hybridize to the second non-complementary target
sequence.
The phrases "percent identity" or "% identity" refer to the percentage of
sequence similarity
found in a comparison of two or more amino acid or nucleic acid sequences.
Percent identity can be
determined electronically, e.g., by using the MEGAI;IGN program (DNASTAR)
which creates
alignments between two or more sequences according to methods selected by the
user, e.g., the clustal
method. (See, e.g., Higgins, D.G. and P.M. Sharp (1988) Gene 73:237-244.) The
clustal algorithm
groups sequences into clusters by examining the distances between all pairs.
The clusters are aligned
pairwise and then in groups. The percentage similarity between two amino acid
sequences, e.g.,
sequence A and sequence B, is calculated by dividing the: length of sequence
A; minus the number of
gap residues in sequence A, minus the number of gap residues in sequence B,
into the sum of the
residue matches between sequence A and sequence B, tirnes one hundred. Gaps of
low or of no
similarity between the two amino acid sequences are not included in
determining percentage
similarity. Percent identity between nucleic acid sequences can also be
counted or calculated by other
methods known in the art, e.g., the Jotun Hein method. (.See, e.g., Hein, J.
(1990) Methods Enzymol.
183:626-645.) Identity between sequences can also be determined by other
methods known in the art,
e.g., by varying hybridization conditions.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino
acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to any process by which a atrand of nucleic acid binds
with a
complementary strand through base pairing.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
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hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or gllass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" or "addition" refer to changes in an amino acid or
nucleotide sequence
resulting in the addition of one or more amino acid residues or nucleotides,
respectively, to the
sequence found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression of
various factors, e.g., cytokines, chemokines, and other signaling molecules,
which may affect cellular
and systemic defense systems.
The term "microarray" refers to an arrangement of distinct poiynucleotides on
a substrate.
The terms "element" or "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of CSIG. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunoIogical properties of CSIG.
The phrases "nucleic acid" or "nucleic acid seque:nce," as used herein, refer
to a nucleotide,
oligonucleotide, polynucleotide, or any fragment thereof. These phrases also
refer to DNA or RNA of
genomic or synthetic origin which may be single-stranded or double-stranded
and may represent the
sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-
like or RNA-like material.
In this context, "fragments" refers to those nucleic acid sequences which
comprise a region of unique
polynucleotide sequence that specifically identifies SEQ ID N0:3 or SEQ ID
NO:4, for example, as
distinct from any other sequence in the same genome. For example, a fragment
of SEQ ID NO:3 or
SEQ ID N0:4 is useful in hybridization and ampliEcation technologies and in
analogous methods that
distinguish SEQ ID N0:3 or SEQ ID N0:4 from related polynucleotide sequences.
A fragment of
SEQ ID NO:3 or SEQ ID N0:4 is at least about 15-20 nucleotides in length. The
precise length of the
fragment of SEQ ID N0:3 or SEQ 1D N0:4 and the region of SEQ ID N0:3 or SEQ ID
N0:4 to which
the fragment corresponds are routinely determinable by one of ordinary skill
in the art based on the
intended purpose for the fragment. In some cases, a fragment, when translated,
would produce
polypeptides retaining some functional characteristic, e.g..,~ antigenicity,
or structural domain
characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms "operably associated" or "operably linked" refer to functionally
related nucleic acid
sequences. A promoter is operably associated or operably linked with a coding
sequence if the
CA 02339860 2001-02-20
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promoter controls the translation of the encoded polypeptide. While operably
associated or operably
linked nucleic acid sequences can be contiguous and in the same reading frame,
certain genetic
elements, e.g., repressor genes, are not contiguously linked to the sequence
encoding the polypeptide
but still bind to operator sequences that control expression of the
polypeptide.
The term "oligonucleotide" refers to a nucleic acid sequence of at least about
6 nucleotides to
60 nucleotides, preferably about 1 S to 30 nucleotides, and most preferably
about 20 to 25 nucleotides,
which can be used in PCR amplification or in a hybridization assay or
microarray. "Oligonucleotide"
is substantially equivalent to the terms "arnplimer," "prinner," "oligomer,"
and "probe," as these terms
are commonly defined in the art.
'°Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-
gene agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysirne confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
1S The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding CSIG, or fragments thereof, or CSIG itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" or "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon the
presence of a particular structure of the protein, e.g., the antigenic
determinant or epitope, recognized
by the binding molecule. For example, if an antibody is specific for epitope
"A," the presence of a
polypeptide containing the epitope A, or the presence of free unlabeled A, in
a reaction containing free
labeled A and the antibody will reduce the amount of labeled A that binds to
the antibody.
2S The term "stringent conditions" refers to conditions which permit
hybridization between
polynucleotides and the claimed polynucIeotides. Stringent conditions can be
defined by salt
concentration, the concentration of organic solvent, e.g., formamide,
temperature, and other conditions
well known in the art. In particular, stringency can be increased by reducing
the concentration of salt,
increasing the concentration of formamide, or raising the hybridization
temperature.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated c>r separated, and are
at least about 60% free,
preferably about 7S% free, and most preferably about 90°ro free from
other components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
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different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or poiypeptides
are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
a
recipient cell. Transformation may occur under natural or artificial
conditions according to various
methods well known in the art, and may rely on any known method for the
insertion of foreign nucleic
acid sequences into a prokaryotic or eukaryatic host cell" The method for
transformation is selected
based on the type of host cell being transformed and may include, but is not
limited ta, viral infection,
electroporation, heat shock, lipofection, and particle bombardment. The term
"transformed" cells
includes stably transformed cells in which the inserted DNA is capable of
replication either as an
autonomously replicating plasmid or as part ofthe host chromosome, as well as
transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "variant" of CSIG polypeptides refers to an arr~ino acid sequence that is
altered by one or
more amino acid residues. The variant may have "conservative" changes, wherein
a substituted amino
acid has similar structural or chemical properties (e.g., replacement of
leucine with isoleucine). More
rarely, a variant may have "nonconservative" changes (e.g., replacement of
glycine with tryptophan).
Analogous minor variations may also include amino acid deletions or
insertions, or both. Guidance in
determining which amino acid residues may be substituted, inserted, or deleted
without abolishing
biological or immunological activity may be found using computer programs well
known in the art, for
example, LASERGENE software (DNASTAR).
The term "variant," when used in the context of a polynucleotide sequence,
may.encompass a
polynucleotide sequence related to CSIG. This definition. may also include;
for example, "allelic" (as
defined above), "splice," "species," or "polymorphic" variants. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides
due to alternate splicing of exons during mRNA processing. The corresponding
polypeptide may
possess additional functional domains or an absence of domains. Species
variants are polynucleotide
sequences that vary from one species to another. The resulting polypeptides
generally will have
significant amino acid identity relative to each other. A polymorphic variant
is a variation in the
polynucleotide sequence of a particular gene between individuals of a given
species. Polymorphic
variants also may encompass "single nucleotide poiymorphisms" (SNPs) in which
the polynucieotide
sequence varies by one base. The presence of SNPs may lbe indicative of, for
example, a certain
population, a disease state, or a propensity for a disease state.
13
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THE INVENTION
The invention is based on the discovery of new human cell signaling proteins
(CSIG), the
polynucleotides encoding CSIG, and the use of these compositions for the
diagnosis, treatment, or
prevention of neoplastic, neurological, immunological, vesicle trafficking,
and smooth muscle
disorders.
Nucleic acids encoding the CSIG-I of the present invention were identified in
Incyte Clone
999661 from the kidney cDNA library {KIDNTUTOI ) using a computer search,
e.g., BLAST, for
amino acid sequence alignments. A consensus sequence., SEQ ID N0:3, was
derived from the
following overlapping andlor extended nucleic acid sequences: Incyte Clones
99966186
l0 (KIDNTUT01), 805555H1 (BSTMNOT01), 4351062Ht {CONFTMT01), 1319852H1
(BLADNOT04), 875690H 1 (LUNGASTO1 ), and 89079rR6 (STOMTUTO 1 }.
In one embodiment, the invention encompasses a poiypeptide comprising the
amino acid
sequence of SEQ ID NO:1. CSIG-1 is 147 amino acids in length and has a
potential N-glycosylation
site at residue N 111; two potential casein kinase II phosphorylation sites at
residues SI 13 and T134;
one potential glycosaminoglycan attachment site at residue S43; a Wilms tumor
protein signature from
G46 through P60; and an inhibin ~i B chain signature from M41 through P60. As
shown in Figures l A
and 1 B, CSIG-1 has chemical and structural similarity with human tafazzin (GI
1263110; SEQ 1D
NO:S). In particular, CSIG-1 and human tafazzin share a4% identity, the
potential N-glycosylation
site, and the potential casein kinase Il phosphoryiation sites. A fragment of
SEQ ID N0:3 from about
nucleotide 739 to about nucleotide 771 is useful, for exarnple, for designing
oligonucleotides or as a
hybridization probe. Northern analysis shows the expression of SEQ ID N0:3 in
various libraries, at
least 53% of which are immortalized or cancerous and at least 38% of which
involve immune response
or inflammation. Of particular note is the expression of (:SIG-I in prostate,
breast, lung, brain,
bladder, fetal, and smooth muscle tissue; and in Wilms tumor kidney tissue.
Nucleic acids encoding the CSIG-2 of the present invention were identified in
lncyte Clone
1415354 from the brain tissue cDNA library (BRAINOTil2} using a computer
search, e.g., BLAST,
for amino acid sequence alignments. A consensus sequence, SEQ ID N0:4, was
derived from the
following overlapping andlor extended nucleic acid sequences: Incyte Clones
14I 5354F6
(BRAINOT12), 4782733H1 (BRATNOT12), and 659232H1 and 65923286 (BRAINOT03).
In one embodiment, the invention encompasses a polypeptide comprising the
amino acid
sequence of SEQ ID N0:2. CSIG-2 is 133 amino acids in length and has two
potential casein kinase
II phosphorylation sites at residues S83 and T109, and three potential protein
kinase C
phosphorylation sites at residues T64, T77; and T126. BLIMPS analysis using
the PRINTS database
identified a Type II EGF-like signature, which the algorithm characterizes
using four segments,
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CA 02339860 2001-02-20
WO OOI11169 PCT/US99/19072
designated PROOOlOA, PR00010B, PROOOI OC, and PROOOI OD. The region of CSIG-2
from amino
acid Q63 through K73, corresponding to segment PR00010C, received a score of
1013 on a strength
of 1190, and was supported by the presence of segments PR0001 OA and PR0001 OB
with a P value of
less than 3.9 x 10''. HMM analysis identified a signal peptide in CSIG-2, from
amino acid MI
through G25. A fragment of SEQ ID N0:4 from about nucleotide A330 to about
nucleotide A390 is
useful in hybridization or amplification technologies to identify SEQ ID N0:4
and to distinguish
between SEQ ID N0:4 and similar poiynucleotide sequences. Northern analysis
shows the expression
of this sequence exclusively in brain-derived libraries, at least 40% of which
are associated with
cancer. Of particular note is the expression of CSIG-2 in brain tumor
libraries.
The invention also encompasses CSIG variants. A preferred CSIG variant is one
which has at
least about 80%, more preferably at least about 90%, and. most preferably at
least about 95% amino
acid sequence identity to the CSIG amino acid sequence, and which contains at
least one functional or
structural characteristic of CSIG.
The invention also encompasses polynucleotides which encode CS1G. In a
particular
embodiment, the invention encompasses a polynucleotide; sequence comprising
the sequence of SEQ
ID N0:3, which encodes a CSIG. In a further embodiment, the invention
encompasses the
polynucleotide sequence comprising the sequence of SEQ ID N0:4, which encodes
a CSIG.
The invention also encompasses a variant of a poIynucleotide sequence encoding
CSIG. In
particular, such a variant polynucleotide sequence will have at least about
70%, more preferably at
least about 85%, and most preferably at least about 95% polynucleotide
sequence identity to the
polynucleotide sequence encoding CSIG. A particular aspect of the invention
encompasses a variant
of SEQ ID N0:3 which has at least about 70%, more preferably at least about
85%, and most
preferably at least about 95% polynucleotide sequence identity to SEQ ID N0:3.
The invention
further encompasses a poiynucleotide variant of SEQ ID N0:4 having at least
about 70%, more
preferably at least about 85%, and most preferably at least about 95%
polynucleotide sequence identity
to SEQ ID N0:4. Any one of the polynucleotide variants described above can
encode an amino acid
sequence which contains at least one functional or structural characteristic
of CSIG.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the genetic
code, a multitude of polynucleotide sequences encoding CSIG, some bearing
minimal similarity to the
polynucleotide sequences of any known and naturally occurring gene, may be
produced. Thus, the
invention contemplates each and every possible variation of polynucleotide
sequence that could be
made by selecting combinations based on possible codon choices. These
combinations are made in
accordance with the standard triplet genetic code as applied to the
polynucleotide sequence of
naturally occurring CSIG, and all such variations are to be considered as
being specifically disclosed.
CA 02339860 2001-02-20
WO 00/11169 PCT/US99/19072
Although nucleotide sequences which encode C~>IG and its variants are
preferably capable of
hybridizing to the nucleotide sequence of the naturally occurring CSIG under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding CSIG or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate atwhich
expression of the peptide
occurs in a particular prokaryotic or cukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding CSIG and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater half Life,
than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode CSIG
and CSIG
derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the synthetic
sequence may be inserted into any of the many available expression vectors and
cell systems using
reagents well known in the art. Moreover, synthetic chemistry may be used to
introduce mutations
into a sequence encoding CSIG or any fragment thereof.
Also encompassed by the invention are polynucle:otide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:3, SEQ ID N0:4, a fragment of SEQ ID N0:3, or a fragment of SEQ ID N0:4,
under various
conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987)
Methods Enzymol. 152:399-
407; Kimmei, A.R. ( 1987) Methods Enzymol. I 52:507-S ll I .) For example;
stringent salt concentration
will ordinarily be less than about 750 mM NaCI and 75 mM trisodium citrate,
preferably less than
about S00 mM NaCI and 50 mM trisodium citrate, and most preferably less than
about 250 mM NaCI
and 25 mM trisodium citrate. Low stringency hybridization can be obtained in
the absence of organic
solvent, e.g., formamide, while high stringency hybridization can be obtained
in the presence of at
least about 35% formamide, and most preferably at least about SO% formamide.
Stringent temperature
conditions will ordinarily include temperatures of at least about 30°C,
more preferably of at least about
37°C, and most preferably of at least about 42°C. Varying;
additional parameters, such as
hybridization time, the concentration of detergent, e.g., sodium dodecyl
sulfate (SDS), and the
inclusion or exclusion of carrier DNA, are well known to those skilled in the
art. Various levels of
stringency are accomplished by combining these various conditions as needed.
In a preferred
embodiment, hybridization will occur at 30°C in 750 mM NaCI, 75 mM
trisodium citrate, and 1
SDS. In a more preferred embodiment, hybridization will occur at 37°C
in 500 mM NaCI, 50 mM
trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm
DNA (ssDNA).
In a most preferred embodiment, hybridization will occur .at 42°C in
250 mM NaCI, 25 mM trisodium
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CA 02339860 2001-02-20
WO 00/11169 PCT/US99/I9072
citrate, 1 % SDS, 50 % formamide, and 200 pg/ml ssDNA. Useful variations on
these conditions will
be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency
conditions can be defined by salt concentration and by temperature. As above,
wash stringency can be
increased by decreasing salt concentration or by increasing temperature. For
example, stringent salt
concentration for the wash steps will preferably be less than about 30 mM NaCI
and 3 mM trisodium
citrate, and most preferably less than about 1S mM NaCI and 1.5 mM trisodium
citrate. Stringent
temperature conditions for the wash steps will ordinarily include temperature
of at least about 25°C,
more preferably of at least about 42°C, and most preferably of at least
about 68°C. In a preferred
embodiment, wash steps will occur at 25°C in 30 mM NiCI, 3 mM trisodium
citrate, and 0.1 % SDS.
In a more preferred embodiment, wash steps will occur at 42°C in 15 mM
NaCI, 1.5 mM trisodium
citrate, and 0.1 % SDS. In a most preferred embodiment, wash steps will occur
at 68°C in 15 mM
NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these
conditions will be
readily apparent to those skilled in the art:
Methods for DNA sequencing are well known in the art and maybe used to
practice any of the
embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment of
DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Perkin-Elmer),
thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway NJ), or
combinations of
polymerises and proofreading exonucleases such as those found in the ELONGASE
amplification
system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation
is automated with
machines such as the HYDRA microdispenser (Robbins Scientific, Sunnyvale CA)
or MICROLAB
2200 (Hamilton, Reno NV) systems in combination with the PTC-200 thermal
cyclers (MJ Research,
Watertown MA) and the ABI CATALYST 800 (Perkin-E',lmer). Sequencing is then
carried out using
either ABI 373 or 377 DNA sequencing systems (Perkin-:Elmer) or the MEGABACE
1000 DNA
sequencing system (Molecular Dynamics, Sunnyvale CA;). The resulting sequences
are analyzed
using a variety of algorithms which are welt known in the: art. {See, e.g.,
Ausubel, F:M. ( 1997) Short
Protocols in Molecular Biolo~v, John Wiley & Sons, Nev~r York NY, unit 7.7;
Meyers, R.A. (1995)
Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding CSIG may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
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CA 02339860 2001-02-20
WO 00/11169 PCT/US99/19072
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (Se:e, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
(1991) PCR Methods Applic. l:l I 1-i 19:) In this method, multiple restriction
enzyme digestions and
ligations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art: (See, e.g., Parker, J.D. et al. ( 1991 ) Nucleic Acids
Res. 19:3055-306).
Additionally, one may use PCR, nested primers, and PR~OMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids. the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucIeotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the
entire process
from loading of samples to computer analysis and electronic data display may
be computer controlled.
Capillary electrophoresis is especially preferable for sequencing small DNA
fragments which may be
present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode CSIG may be cloned in recombinant DNA molecules that direct expression
of CSIG, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy
of the genetic code, other DNA sequences which encode :>ubstantially the same
or a functionally
equivalent amino acid sequence may be produced and used to express CSIG.
18
CA 02339860 2001-02-20
WO OOI11169 PCTIUS99l190'72
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter CSIG-encoding sequences for a variety of
purposes including, but not
limited to, modification of the cloning, processing, and/or expression of the
gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change colon preference, produce splice
variants, and so forth.
In another embodiment, sequences encoding CSIG may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g;., Caruthers, M.H. et
ai. ( 1980) Nucl. Acids
Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp.
Ser. 225-232.)
Alternatively, CSIG itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solid-phase
techniques. (See, e.g.,
Roberge, J.Y. et al. ( 1995) Science 269:202-204.) Automated synthesis may be
achieved using the
ABI 431 A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of CSIG, or
IS any part thereof, may be altered during direct synthesis and/or combined
with sequences from other
proteins, or any part thereof, to produce a variant polypeptide.
The peptide may be substantially purifZed by preparative high performance
liquid
chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnie;r (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, T: { 1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY.)
In order to express a biologically active CSIG, the nucleotide sequences
encoding CSIG or
derivatives thereof may be inserted into an appropriate e:!cpression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in a
suitable host. These elements include regulatory sequences, such as enhancers,
constitutive and
inducible promoters, and 5° and 3' untranslated regions in the vector
and in polynucleotide sequences
encoding CSIG. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
CSIG. Such signals
include the ATG initiation colon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding CSIG and its initiation colon and upstream regulatory
sequences are inserted into
the appropriate expression vector, no additional transcriptional or
translational control signals may be
needed. However, in cases where only coding sequence, or a fragment thereof,
is inserted, exogenous
translational control signals including an in-frame ATG initiation colon
should be provided by the
vector. Exogenous translational elements and initiation colons may be of
various origins, both natural
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CA 02339860 2001-02-20
WO 00/11169 PCT/US99119072
and synthetic. The efficiency of expression may be enhanced by the inclusion
of enhancers
appropriate for the particular host cell system used. (See;, e.g., Scharf, D.
et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
Methods which~are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding CSIG and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et a.l. ( 1989) Molecular
Cloning. A Laboratory
Manual, Cold Spring Harbor Press, Plaiinview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Bioloav, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding CSIG. These include, but are not limited to, miicroorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DN,A expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected witlh viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
IS tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding CSIG. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding CSIG can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or pSPORTI plasmid
(Life Technologies). Ligation of sequences encoding CSIG into the vector's
multiple cloning site
disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of transformed
bacteria containing recombinant molecules. In addition, these vectors may be
useful for in vitro
transcription, dideoxy sequencing, single strand rescue wiith helper phage,
and creation of nested
deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster
( 1989) J. Biol. Chem.
264:5503-5509.) When large quantities of CSIG are needled, e.g. for the
production of antibodies,
vectors which direct high level expression of CS1G may b~e used. For example,
vectors containing the
strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of CSIG. A number of
vectors
containing constitutive or induciible promoters, such as alpha factor, alcohol
oxidase; and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Grant et al. ( 1987) Methods Enzymol. 153:516-54; and Scorer, C. A. et al. ( i
994) Bio/Technology
CA 02339860 2001-02-20
WO 00/11 lb9 PCTIUS99/19072
12:181-184.)
Plant systems may also be used for expression o:f CSIG.. Transcription of
sequences encoding
CSIG may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. ( 1987)
EMBO J. 6:307-311 ).
Alternatively, plant promoters such as the small subunit ~of RUBISCO or heat
shock promoters may be
used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:16'71-1680; Broglie, R.
et al. (1984) Science
224:838-843; and Winter, J. et al. (1991) Results Probl. tell Differ. 17:85-
105.) These constructs can
be introduced into plant cells by direct DNA transformation or pathogen-
mediated transfection. (See,
e.g., The McGraw Hill Yearbook of Science and Techno'l~ (1992} McGraw Hill,
New York NY, pp.
191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding CSIG
may be ligated into an
adenovirus transcriptionltransiation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential El or E3 region of the viral genome may
be used to obtain
infective virus which expresses CSIG in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as
the Rous sarcoma virus
(RSV) enhancer, may be used to increase expression in mammalian host cells.
SV40 or EBV-based
vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to I 0 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
CSIG in cell lines is preferred. For example, sequences encoding CSIG can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
andlor endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow fir about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the sei~ectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transforrr~ed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
21
CA 02339860 2001-02-20
WO OO/111G9 PCT/US99/19072
phosphoribosyltransferase genes, for use in tk or ap>' cells, respectively.
(See, e.g., WigIer, M. et aI.
(1977) Cell 11:223-232; Lowy, I. et al. (1980} Cell 22:8:17-823.) Also,
antimetaliolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For example, dhfr
confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als or pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin,
F. et al. (1981) J. MoI.
Biol. 150:1-14) Additional selectable genes have been described, e.g., trpB
and hisD, which alter
cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C.
Mulligan (1988} Proc. Natl.
Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins (GFP;
Clontech), f3 glucuronidase and its substrate 13-glucuronide, or luciferase
and its substrate luciferin may
be used. These markers can be used not only to identify transformants, but
also to quantify the amount
of transient or stable protein expression attributable to a specific vector
system. (See, e.g., Rhodes,
C.A. (1995) Methods Mol. Biol. SS:I21-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
I S also present, the presence and expression of the gene may need to be
confirmed. Far example, if the
sequence encoding CSIG is inserted within a marker gene sequence, transformed
cells containing
sequences encoding CSIG can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding CSIG under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually. indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding CSIG
and that express
CS1G may be identified by a variety of procedures known to those of skill in
the art. These procedures
include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and
protein bioassay or immunoassay techniques which include membrane, solution,
or chip based
technologies for the detection and/or quantification of nucleic acid or
protein sequences.
Immunological methods for detecting and measuring the expression of CSIG using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FRCS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on CS1G is
preferred, but a competitive
binding assay may be employed. These and other assays are well known in the
art. (See, e.g.,
Hampton, R. et al. (1990) Serological MethodsLa Laboratory Manual, APS Press,
St Paul MN, Sect.
IV; Coligan, J. E. et al. (1997) Current Protocols in Immunolotyv, Greene Pub.
Associates and Wiley-
Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols,
Humana Press,
22
CA 02339860 2001-02-20
WO 00/11169 PCT/US99/I9072
Totowa NJ).
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding CSIG
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding CSIG, or any fragments thereof, maybe
cloned into a vector for
the production of an mRNA probe. Such vectors are known in the art; are
commercially available, and
may be used to synthesize RNA probes in vitro by addition of an appropriate
RNA polymerase such as
T7, T3, or SP6 and labeled nucleotides. These procedurEa may be conducted
using a variety of
commercially available kits, such as those provided by A,mersham Pharmacia
Biotech, Promega
(Madison WI}, and US Biochemical. Suitable reporter molecules or labels which
may be used for ease
of detection include radionuclides, enzymes, fluorescent., chemiluminescent,
or chromogenic agents, as
well as substrates, cofactors, inhibitors, magnetic particlca, and the like.
1-lost cells transformed with nucleotide sequences encoding CSIG may be
cultured under
conditions suitable for the expression and recovery of thE; protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode CSIG may be designed to contain signal
sequences which
direct secretion of CSIG through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain maybe chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include; but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation: Post-translati~onal processing
which cleaves a "prepro"
form of the protein may also be used to specify protein targeting, folding,
and/or activity. Different
host cells which have specific cellular machinery and characteristic
mechanisms for post-translational
activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38}, are available from the
American Type
Culture Collection (ATCC, Mantissas, VA) and may be chosen to ensure the
correct modification and
processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding CSIG may be ligated to a heterologous sequence resulting in
translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric CSIG protein
containing a heterologous moiety that can be recognized lby a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of CSIG activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
23
CA 02339860 2001-02-20
WO 00111169 PCT/US99l19072
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate
fusion proteins on immobilized glutathione, maltose, phe;nylarsiine oxide,
calmodulin, and metal-
s chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies that
specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the CSIG encoding sequence and the
heterologous protein
sequence, so that CSIG may be cleaved away from the h~eterologous moiety
following purification.
Methods for fusion protein expression and~purification are discussed in
Ausubel (1995, supra, ch 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled CSIG may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems
(Promega). These
IS systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the: presence of a
radiolabeled amino acid
precursor, preferably 35S-methionine.
Fragments of CSIG may be produced not only b:y recombinant production; but
also by direct
peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra
pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation. Automated
synthesis may be
achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin-Elmer).
Various fragments of
CS1G may be synthesized separately and then combined to produce the full
length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of CSIG and cell signaling proteins. In addition, the expression of
CSIG is closely associated
with cancerous, immunologic, and inflamed tissues; neurological disorders;
prostate, breast, lung,
brain, bladder, fetal, and smooth muscle tissues; and Wilim's tumor kidney
tissue. Therefore, CSIG
appears to play a role in neoplastic, neurological, immunological, vesicle
trafficking, and smooth
muscle disorders. Therefore, in the treatment of these conditions associated
with increased expression
or activity of CSIG, it is desirable to decrease the expression or activity of
CSIG. In the treatment of
the above conditions associated with expression or activiity CSIG activity, it
is desirable to provide the
protein or to increase the expression of CSIG.
Therefore, in one embodiment, CSiG or a fragment or derivative thereof may be
administered
to a subject to treat or prevent a disorder associated with decreased
expression or activity of CSIG.
24
CA 02339860 2001-02-20
WO OO/1I169 PCT/US99/19072
Examples of such disorders include a neoplastic disorder, such as
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; a neurological disorder, such as
epilepsy, ischemic
cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease,
Pick's disease, Huntington's
disease, dementia, Parkinson's disease and other extrapyr~amidal disorders,
amyotrophic lateral
sclerosis and other motor neuron disorders, progressive neural muscular
atrophy, retinitis pigmentosa,
hereditary ataxias, multiple sclerosis and other demyelina~ting diseases,
bacterial and viral meningitis,
brain abscess, subdural empyema, epidural abscess, suppurative intracranial
thrombophlebitis, myelitis
and radiculitis, viral central nervous system disease, prior diseases
including kuru, Creutzfeldt-Jakob
disease, and Gerstmann-Straussler-Scheinker syndrome, f=atal familial
insomnia, nutritional and
metabolic diseases of the nervous system, neurofibromatosis, tuberous
sclerosis, cerebelloretinal
hemangioblastomatosis, encephaIotrigeminal syndrome, mental retardation and
other developmental
disorders of the central nervous system, cerebral palsy, ne;uroskeletal
disorders, autonomic nervous
system disorders, cranial nerve disorders, spinal cord diseases, muscular
dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and poiymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, akathesia,
amnesia, catatonia, diabetic
neuropathy; tardive dyskinesia, dystonias, paranoid psychoses, postherpetic
neuralgia, and Tourette's
disorder; a vesicle trafficking disorder, such as cystic fibrosis, glucose-
galactose malabsorption
syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-
and hypoglycemia,
Grave's disease, goiter, Cushing's disease, and Addison's disease,
gastrointestinal disorders including
ulcerative colitis, gastric and duodenal ulcers, other conditions associated
with abnormal vesicle
trafficking, including acquired immunodeficiency syndrome (AIDS), allergies
including hay fever,
asthma, and urticaria (hives), autoimmune hemolytic anemia, proliferative
glomerulonephritis,
inflammatory bowel disease, multiple sclerosis, myastheniia gravis, rheumatoid
and asteoarthritis,
scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus
erythematosus, toxic shock
syndrome, traumatic tissue damage, and viral, bacterial, fungal, helminthic,
and protozoal infections;
and a smooth muscle disorder. A smooth muscle disorder is defined as any
impairment or alteration in
the normal action of smooth muscle and may include, but i.s not limited to,
angina, anaphylactic shock,
arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension,
hypoglycemia,
myocardial infarction, migraine, and pheochromocytorna, and myopathies
including cardiomyopathy,
encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic acidosis, myoclonic
disorder, and
CA 02339860 2001-02-20
WO 00111169 PCT/I3S99/19072
ophthalmoplegia. Smooth muscle includes, but is not linnited to, that of the
blood vessels,
gastrointestinal tract, heart, and uterus.
In another embodiment, a vector capable of expressing CSIG or a fragment or
derivative
thereof may be administered to a subject to treat.or prevent a disorder
associated with decreased
expression or activity of CSIG including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
CSIG in conjunction with a suitable pharmaceutical carrier may be administered
to a subject to treat or
prevent a disorder associated with decreased expression or activity of CSIG
including, but not limited
to, those provided above.
IO In still another embodiment, an agonist which modulates the activity of
CSIG may be
administered to a subject to treat or prevent a disorder as:>ociated with
decreased expression or activity
of CSIG including, but not limited to, those listed above.
1n a further embodiment, an antagonist of CSIG may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of CSIG.
Such a disorder may
include, but is not limited to, a neoplastic disorder, such as adenocarcinoma,
leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen,
testis, thymus, thyroid, and uterus; an immunological disorder, such as
acquired immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress syndrome,
allergies, ankylosing
spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune
hemolytic anemia, autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic Iymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's
syndrome, rheumatoid
arthritis, scleroderma, Sjogren's syndrome; systemic anaphylaxis, systemic
lupus erythematosus,
systemic sclerosis, thrombocytopenic purpura, ulcerative .colitis, uveitis,
Werner syndrome,
complications of cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal,
parasitic, protozoal, and heiminthic infections, and trauma; a neurological
disorder, such as such as
epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal disorders,
amyotrophic lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy,
26
CA 02339860 2001-02-20
WO 00111169 PCTIUS99/19072
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other
demyelinating diseases, bacterial
and viral meningitis, brain abscess, subdural empyema, c;pidural abscess,
suppurative intracranial
thrombophlebitis, myeIitis and radiculitis, viral central nervous system
disease, priors diseases
including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome, fatal
familial insomnia, nutritional and metabolic diseases of l:he nervous system,
neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomato;>is,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system,
cerebral palsy,
neuroskeletal disorders, autonomic nervous system disorders, cranial nerve
disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders, peripheral
nervous system disorders,
dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic
myopathies, myasthenia
gravis, periodic paralysis, mental disorders including mood, anxiety, and
schizophrenic disorders,
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, and Tourette's disorder; and a vesicle trafficking
disorder, such as cystic
fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia,
diabetes mellitus, diabetes
insipidus, hyper- and hypoglycemia, Grave's disease, goiter, Cushing's
disease, and Addison's
disease, gastrointestinal disorders including ulcerative colitis, gastric and
duodenal ulcers, other
conditions associated with abnormal vesicle trafficking, including acquired
immunodeficiency
syndrome (AIDS), allergies including hay fever, asthma, and urticaria (hives),
autoimmune hemolytic
anemia, proliferative glomeruionephritis, inflammatory bowel disease, multiple
sclerosis, myasthenia
gravis, rheumatoid and osteoarthritis, scleroderma, Chediak-Higashi and
Sjogren's syndromes,
systemic lupus erythematosus, toxic shock syndrome, traumatic tissue damage,
and viral, bacterial,
fungal, helminthic, and protozoa! infections. In one aspect, an antibody which
specifically binds CSIG
may be used directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a
pharmaceutical agent to cells or tissue which express CSIG.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding CSIG may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of CSIG including, but not limited to, those
described above.
1n other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents fon~ use in
combination therapy may be made by
one of ordinary skill in the art, according to conventional ;pharmaceutical
principles. The combination
of therapeutic agents may act synergistically to effect the 'treatment or
prevention of the various
disorders described above. Using this approach, one may be able to achieve
therapeutic efficacy with
lower dosages of each agent, thus reducing the potential for adverse side
effects.
27
CA 02339860 2001-02-20
WO 00/11169 PCT!(JS99119072
An antagonist of CSIG may be produced using methods which are generally known
in the art.
In particular, purified CSIG may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind CSIG. Antibodies to CSIG may
also be generated
using methods that are well known in the art. Such antibodies may include, but
are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments,
and fragments produced
by a Fab expression library. Neutralizing antibodies (i.e., those which
inhibit dimer formation) are
especially preferred fortherapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with CSIG or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending an the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Cor~nebacterium parvum .are especially
preferable.
It is preferred that the oligopeptides, peptides, or' fragments used to induce
antibodies to CSIG
have an amino acid sequence consisting of at least about 5 amino acids, and,
more preferably, of at
least about 10 amino acids. It is also preferable that these oligopeptides,
peptides, or fragments are
identical to a portion of the amino acid sequence of the natural protein and
contain the entire amino
acid sequence of a small, naturally occurring molecule. ;Short stretches of
CSIG amino acids may be
fused with those of another protein, such as KLH, and antibodies to the
chimeric molecule may be
produced.
Monoclonal antibodies to CSIG may be preparedl using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. ( 1975) Nature 256:495-497; Kozbor,
D. et al. ( 1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci.
80:2026-2030; and Cole,
S.P. et al. (1984) Mol. CeII Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. ( 1984) Proc.
Natl. Acad. Sci. 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-
608; and Takeda, S. et
al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for the
production of single chain
antibodies may be adapted, using methods known in the au~t, to produce CSIG-
specific single chaiw
antibodies. Antibodies with related specificity, but of distinct idiotypic
composition, may be generated
28
CA 02339860 2001-02-20
WO 00/11169 PCT/US99/I9072
by chain shuffling from random combinatorial immunol;labutin libraries. (See,
e.g., Burton D.R.
(1991 ) Proc. Natl. Acad. Sci. 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See; e.g., Orlandi, R, et al. {1989) Proc. Natl. Acad. Sci.
86: 3833-3837; Winter, G. et
al. ( I 991 ) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for CSIG may also be
generated. For
example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D. et
al. (I989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding, or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established spe;cificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
CSIG and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering CSIG epitopes is preferred, but a competiitive binding assay
may also be employed
(Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for CS1G.. Affinity is
expressed as an association
constant, Ke, which is defined as the molar concentration of CSIG-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka
determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple CSIG epitopes, represents the average affinity, o~r avidity, of the
antibodies for CSIG. The Ka
determined for a preparation of monoclonal antibodies, which are monospecific
for a particular CSIG
epitope, represents a true measure of affinity. High-affiniity antibody
preparations with Ke ranging
from about 109 to 10'2 L/mole are preferred for use in immunoassays in which
the CSIG-antibody
complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka ranging
from about 106 to 10' L/mole are preferred for use in immunopurification and
similar procedures
which ultimately require dissociation of CSIG, preferably in active form, from
the antibody (Catty, D.
( I988) Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC;
Liddell, J. E. and
Cryer, A. (I991) A Practical Guide to Monoclonal Antibodies, John Wiley &
Sons, New York NY).
The titre and avidity of polyclonal antibody preparations may be further
evaluated to
29
CA 02339860 2001-02-20
WO 00!11169 PCTIIJS99/i9072
determine the quality and suitability of such preparation s for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably
5-10 mg specific antibody/ml, is preferred for use in procedures requiring
precipitation of CSIG-
antibody complexes. Procedures for evaluating antibody specificity, titer, and
avidity; and guidelines
for antibody quality and usage in various applications, are generally
available. (See, e.g., Catty, su ra,
and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding CSIG, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
the complement of the
polynucleotide encoding CS1G may be used in situations in which it would be
desirable to block the
transcription of the mRNA. In particular, cells may be transformed with
sequences complementary to
polynucleotides encoding CSIG. Thus, complementary nnolecules or fragments may
be used to
modulate CSIG activity, or to achieve regulation of gene function. Such
technology is now well
known in the art, and sense or antisense oligonucleotides or larger fragments
can be designed from
various locations along the coding or control regions of sequences encoding
CSIG.
Expression vectors derived from retroviruses, ad<~noviruses, or herpes or
vaccinia viruses, or
from various bacterial plasmids, may be used for delivery of nucleotide
sequences to the targeted
organ, tissue, or cell population. Methods which are well known to those
skilled in the art can be used
to construct vectors to express nucleic acid sequences cornplementary to the
polynucleotides encoding
CSIG. (See, e.g.; Sambrook, supra; Ausubel, 1995, supra.)
Genes encoding CSIG can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding CSIG. Such
constructs may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in
the absence of integration into the DNA, such vectors ma;y continue to
transcribe RNA molecules until
they are disabled by endogenous nucleases. Transient expression may last for a
month or more with a
non-replicating vector, and may last even longer if appropriate replication
elements are part of the
vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, :RNA, or FNA) to the
control, 5', or
regulatory regions of the gene encoding CSIG. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 from the start
site, are preferred. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex
DNA have been described in the literature. (See, e.g., Gee:, J.E. et al.
(1994) in Huber, B.E. and B.L.
CA 02339860 2001-02-20
WO 00/11 I69 PCT/US99119072
Carr, Molecular and Immunolo~Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-I77.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding CSIG.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites" including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as soli<i phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding CSIG. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SPti. Alternatively,
these cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition ~of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-mf;thyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated far autolog~ous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
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WO 00/I 1 i69 PCT/US99119072
using methods which are well known in the art. {See, e.g;., Goldman, C.K. et
aI. (1997) Nature
Biotechnology 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as dogs, cats, cows, horses,
rabbits, monkeys, and
most preferably, humans.
An additional embodiment of the invention relates to the administration of a
pharmaceutical or
sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any of the
therapeutic effects discussed above. Such pharmaceutical compositions may
consist of CSIG,
antibodies to CSIG, and mimetics, agonists, antagonists, or inhibitors of
CSIG. The compositions may
be administered alone or in combination with at least one other agent, such as
a stabilizing compound,
which may be administered in any sterile, biocompatible pharmaceutical carrier
including, but not
limited to, saline, buffered saline, dextrose, and water. The compositions may
be administered to a
patient alone, or in combination with other agents; drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal; subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate processing
of the active compounds into preparations which can be used pharmaceutically.
Further details on
techniques for formulation and administration may be foumd in the latest
edition of Remin tg on's
Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral administration.
Such carriers enable the pharmaceutical compositions to be formulated as
tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, and tlhe like, for
ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose; sucrose, mannitol,
and sorbitol; starch from corn, wheat, rice, potato, or other plants;
cellutose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethyllcellulose; gums,
including arabic and
tragacanth; and proteins, such as gelatin and collagen. Ifdesired,
disintegrating or solubilizing agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and
alginic acid or a salt thereof,
32
CA 02339860 2001-02-20
WO OOll.lI69 PCT/US99/19072
such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated sugar
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone,
carbopol gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
product identification or to
characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable liquids, such
as fatty oils, liquid, or
liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parente;ral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution, Ringer's
solution, or physiologically buffered saline. Aqueous injection suspensions
may contain substances
which increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or
dextran. Additionally, suspensions of the active compounds may be prepared as
appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or
synthetic fatty acid esters, such as ethyl oleate, triglycerides, or
Liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the suspension may
also contain suitable
stabilizers or agents to increase the solubility of the compounds and allow
for the preparation of highly
concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants a.re generally known in
the art.
2S The pharmaceutical compositions of the present invention may be
manufactured in a manner
that is known in the art, e.g., by means of conventional mixing, dissolving,
granulating,
dragee-making, ievigating, emulsifying, encapsulating. entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and succinic
acid. Salts tend to be more soluble in aqueous or other protonic solvents than
are the corresponding
free base forms. In other cases, the preferred preparation may be a
lyophilized powder which may
contain any or all of the following: 1 mM to 50 mM histid!.ine, 0.1 % to 2%
sucrose, and 2% to 7%
mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to
use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate
33
CA 02339860 2001-02-20
WO 00/11169 PCT/US99119072
container and labeled for treatment of an indicated condlition. For
administration of CSIG, such
labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in. the invention include
compositions wherein
the active ingredients are.contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
For any compound, the therapeutically effective. dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells or in animal models such as mice,
rats, rabbits, dogs, or pigs.
An animal model may also be used to determine the appropriate concentration
range and route of
administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example CSIG
or fragments thereof, antibodies of CSIG, and agonists, antagonists or
inhibitors of CSIG, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
IS calculating the EDS° (the dose therapeutically effective in 50% of
the population) or LDS° (the dose
lethal to SO% of the population) statistics. The dose ratio of therapeutic to
toxic effects is the
therapeutic index, and it can be expressed as the EDS°/LDS°
ratio. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained from cell
culture assays and animal
studies are used to formulate a range of dosage for human use. The dosage
contained in such
compositions is preferably within a range of circulating concentrations that
includes the EDS° with
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors vvhich may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting pharmaceutical compositions m<~y be administered every
3 to 4 days, every
week, or biweekly depending on the half life and clearance rate of the
particular formulation.
Normal dosage amounts may vary from about 0. if p.g to i 00,000 pg, up to a
total dose of about
1 gram, depending upon the route of administration. Guidance as to particular
dosages and methods of
delivery is provided in the literature and generally available to
practitioners in the art. Those skilled in
the art will employ different formulations for nucleotides than for proteins
or their inhibitors.
Similarly, delivery of polynucleotides or polypeptides will be specific to
particular cells, conditions,
34
CA 02339860 2001-02-20
WO 00/11169 PCTIUS99/19072
locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind CS1G may be used for
the
diagnosis of neopIastic, neurological, immunological, vesicle trafficking, and
smooth muscle disorders
characterized by expression of CSIG, or in assays to monitor patients being
treated with CSIG or
agonists, antagonists, or inhibitors of CSIG. Antibodies useful for diagnostic
purposes may be
prepared in the same manner as described above for therapeutics. Diagnostic
assays for CSIG include
methods which utilize the antibody and a label to detect CSIG in human body
fluids or in extracts of
cells or tissues. The antibodies may be used with or without modification, and
may be labeled by
covalent or non-covalent attachment of a reporter molecule. A wide variety of
reporter molecules,
several of which are described above, are known in the art and may be used.
A variety of protocols for measuring CSIG, including ELISAs, RIAs, and FACS,
are known in
the art and provide a basis for diagnosing altered ar abnormal levels of CSIG
expression. Normal or
standard values for CSIG expression are established by combining body fluids
or cell extracts taken
IS from normal mammalian subjects, preferably human, wil:h antibody to CSIG
under conditions suitable
for complex formation. The amount of standard complex formation may be
quantitated by various
methods, preferably by photometric means. Quantities of CS1G expressed in
subject, control, and
disease samples from biopsied tissues are compared with the standard values.
Deviation between
standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding CSIG may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect and
quantitate gene expression in biopsied tissues in which e~,:pression of CSIG
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
CSIG, and to monitor regulation of CS1G levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes whiich are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding CSIG or closely related
molecules may be used to
identify nucleic acid sequences which encode CSIG. The specificity ofthe
probe, whether it is made
from a highly specific region, e.g., the 5' regulatory region, or from a less
specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplifcation
(maximal, high, intermediate,
or low), will determine whether the probe identifies only naturally occurring
sequences encoding
CSIG, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should
preferably have at
least 50% sequence identity to any of the CSIG encoding sequences. The
hybridization probes of the
CA 02339860 2001-02-20
WO 00/11169 PCT/US99/19072
subject invention may be DNA or RNA and may be derived from the sequence of
SEQ ID N0:2 or
from genomic sequences including promoters, enhancers, and introns of the CSIG
gene.
Means for producing specific hybridization probes for DNAs encoding CSIG
include the
cloning of polynucleotide sequences encoding CSIG or CSIG derivatives into
vectors for the
production of mRNA probes.Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate Labeled nucleotides. H;y~bridization probes
may be labeled by a
variety of reporter groups, for example, by radionucIides such as'ZP or 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidinlbiotin coupling
systems, and the like.
Polynucleotide sequences encoding CSIG may b~e used for the diagnosis of
neoplastic,
neurological, immunological, vesicle trafficking, and smooth muscle disorders
associated with
expression of CSIG. Examples of such disorders include, but are not limited
to, a neoplastic disorder,
such as adenocarcinoma, leukemia, lymphoma, melanorria, myeloma, sarcoma,
teratocarcinoma, and,
in particular, cancers of the adrenal gland, bladder, bone; bone marrow,
brain, breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis; prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus; a.neurological
disorder, such as such as epilepsy, ischemic cerebrovascular disease, stroke,
cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous
system disease, priors diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases ofthe
nervous system, neurofbromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and otlher developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis,
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, akathesia, amnesia, catatonia,
diabetic neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic ne;uratgia, and
Tourette's disorder; an
immunological disorder, such as acquired immunodeficiency syndrome (AIDS),
Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma,
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WO 00/11169 PCT/US99/19072
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
bronchitis, cholecystitis,
contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,
diabetes mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nadosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, I;out, Graves' disease,
Hashimoto's tttyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatifis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, and trauma; a vesicle
trafficking disorder, such
as cystic fibrosis, glucose-galactose malabsorption syndrome,
hypercholesterolemia, diabetes mellitus,
diabetes insipidus, hyper- and hypoglycemia, Grave's di<.>ease, goiter,
Cushing's disease, and
Addison's disease, gastrointestinal disorders including ulcerative colitis,
gastric and duodenal ulcers,
other conditions associated with abnormal vesicle trafficking, including
acquired immunodeficiency
syndrome (AIDS), allergies including hay fever, asthma, and urticaria (hives),
autoimmune hemolytic
anemia, proliferative glomerulonephritis, inflammatory bowel disease, multiple
sclerosis, myasthenia
gravis, rheumatoid and osteoarthritis, scleroderma, Chediak-Higashi and
Sjogren's syndromes,
systemic lupus erythematosus, toxic shock syndrome, traumatic tissue damage,
and viral, bacterial,
fungal, helminthic, and protozoal infections; and a smooth muscle disorder. A
smooth muscle disorder
is defined as any impairment or alteration in the normal action of smooth
muscle and may include, but
is not limited to, angina, anaphylactic shock, arrhythmias, asthma,
cardiovascular shock, Cushing's
syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, and
pheochromocytoma, and
myopathies including cardiornyopathy, encephalopathy, epilepsy, Kearns-Sayre
syndrome, lactic
acidosis, myoclonic disorder, and ophthalmoplegia. Smooth muscle includes, but
is not limited to, that
of the blood vessels, gastrointestinal tract, heart, and uterus. The
polynucleotide sequences encoding
CSIG may be used in Southern or northern analysis, dot blot, or other membrane-
based technologies;
in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and
in microarrays utilizing
fluids or tissues from patients to detect altered CSIG expression. Such
qualitative or quantitative
methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding CSIG may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding CSIG may be labeled by standard mel:hods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation ~of hybridization
complexes. After a suitable
incubation period, the sample is washed and the signal is q~uantitated and
compared with a standard
37
CA 02339860 2001-02-20
WO 00!11169 PCT/US99/19072
value. If the amount of signal in the patient sample is significantly altered
in comparison to a control
sample then the presence of altered levels of nucleotide sequences encoding
CSIG in the sample
indicates the presence of the associated disorder. Such .assays may also be
used to evaluate the
efficacy of a particular therapeutic treatment regimen in animal studies, in
clinical trials, or to monitor
the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of CSIG,
a normal or standard profile for expression is established. This may be
accomplished by combining
body fluids or cell extracts taken from normal subjects, either animal or
human, with a sequence, or a
fragment thereof, encoding CSIG, under conditions suitable for hybridization
or amplification.
Standard hybridization may be quantified by comparing the values obtained from
normal subjects with
values from an experiment in which a known amount of a substantially purified
polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained
from samples from
patients who are symptomatic for a disorder. Deviation :from standard values
is used to establish the
presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization.assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in tlhe normal subject.
The results obtained from
successive assays may be used to show the efficacy of tre>atment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnornnal amount of transcript
(either under- or
over-expressed) in biopsied tissue from an individual ma;y indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A mare definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding CSIG
may involve the use of PCR. These oiigorners may be chemically synthesized,
generated
enzyrnatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding CSIG, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
CSIG, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantitation of closely related DNA or RNA sequences.
Methods which may also be used to quantitate the expression of CSIG include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
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WO 00/11169 PCT/US99/19072
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C. et
al. (1993) Anal. Biochem. 229-236.) The speed of quantitation of multiple
samples may be
accelerated by running the assay in an ELISA format where the oligomer of
interest is presented in
various dilutions and a spectcophotometric or colorimetric response gives
rapid quantitation.
S 1n further embodiments, oligonucleotides or longer fragments derived from
any of the
polynucleotide sequences described herein may be used as targets in a
microarray. The microarray
can be used to monitor the expression level of large numbers of genes
simultaneously and to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder; to diagnose a
disorder, and to develop and
monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc: Natl. Acad. Sci.
93:106I4-10619; Baldeschweiler et al. (1995) PCT applfication W09S/2S i 1 I6;
Shalon, D. et al. (1995)
PCT application W09S/35SOS; Heller, R.A. et al. (1997) Proc. Natl. Acad: Sci.
94:21 SO-21 SS; and
IS Heller, M.J. et al. (1997) U:S. Patent No. 5,605,662.)
In another embodiment of the invention; nucleic: acid sequences encoding CSIG
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. The
sequences may be mapped to a particular chromosome, to a specific region of a
chromosome, or to
artificial chromosome constructions, e.g., human artificial chromosomes
(HACs), yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial Pl
constructions, or single
chromosome cDNA libraries. (See, e.g.,.Harrington, J.J. et at. (1997) Nat
Genet. 1 S:34S-355; Price,
C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-
154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome
mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al.
(1995} in Meyers, supra, pp.
2S 965-968.) Examples of genetic map data can be found ire various scientific
journals or at the Online
Mendelian Inheritance in Man (OMIM) site. Correlation between the location of
the gene encoding
CSIG on a physical chromosomal map and a specific disorder, or a
predisposition to a specific
disorder, may help define the region of DNA associated with that disorder. The
nucleotide sequences
of the invention may be used to detect differences in gen<: sequences among
normal, carrier, and
affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the number or arm of a particular human
chromosome is not known.
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WO 00/11169 PCT/US99/I9072
New sequences can be assigned to chromosomal arms by physical mapping. This
provides valuable
information to investigators searching for disease genes using positional
cloning or other gene
discovery techniques. Once the disease or syndrome ha.s been crudely localized
by genetic linkage to
a particular genomic region, e.g., ataxia-telangiectasia h~ l 1q22-23, any
sequences mapping to that
area may represent associated or regulatory genes for further investigation.
{See, e.g., Gatti, R.A. et
al. ( 1988) Nature 336:577-580.) The nucleotide sequence of the subject
invention may also be used to
detect differences in the chromosomal location due to translocation,
inversion, etc., among nornnal,
carrier, or affected individuals.
In another embodiment of the invention, CSIG, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between CSIG and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et ai. ( 1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are; reacted with CSIG,
or fragments thereof, and
washed. Bound CSIG is then detected by methods well 1';nown in the art.
Purified CSIG can also be
coated directly onto plates for use in the aforementioned drug screening
techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and immobilize
it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding CSIG specifically compete with a test compound
for binding CS1G. In
this manner, antibodies can be used to detect the presence; of any peptide
which shares one or more
antigenic determinants with CSIG.
In additional embodiments, the nucleotide sequences which encode CSIG may be
used in any
molecular biology techniques that have yet to be develop<;d, provided the new
techniques rely on
properties of nucleotide sequences that are currently knov~n, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one; skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. [Atty Docket No. PF-0572 P), f led August 21, 1998,
and U.S. Ser. No. [Atty
CA 02339860 2001-02-20
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Docket Na. PF-0586 PJ, fled August 21; 1998, are hereby expressly incorporated
by reference.
EXAMPL>E;S
I. Construction of cDNA Libraries
KIDNTUTOl
The KIDNTUTOI cDNA library was constructed from nephroblastoma. The donor was
an
8-month-old female who was diagnosed with Wilms tumior involving 90% of the
renal parenchyma.
No metastases of the tumor into the lymph nodes were detected. Prior to
surgery, the patient was
undergoing heparin anticoagulant therapy.
The frozen kidney tissue was homogenized and Iysed using a Brinkmann
Homogenizer
Polytron PT-3000 (Brinkmann Instruments, Westbury, NJ) in guanidinium
isothiocyanate solution.
The lysate was centrifuged over a 5.7 M CsCI cushion using a Beckman SW28
rotor in a Beckman
L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm at
ambient temperature.
The RNA was extracted with phenol chloroform pH 4.0, precipitated using 0.3 M
sodium acetate and
2.5 volumes of ethanol, resuspended in DEPC-treated water and treated with
DNase at 37°C. RNA
was extracted and precipitated as before. The mRNA was isolated using the
OL1GOTEX kit
(QIAGEN Inc.) and used to construct the cDNA library.
The mRNA was handled according to the recommended protocols in the SUPERSCRIPT
plasmid system (Life Technologies). The cDNAs were fractionated on a SEPHAROSE
CL4B column
(Amersham Pharmacia Biotech), and those cDNAs exceeding 400 by were ligated
into pSPORT I
(Life Technologies}. The plasmid pSPORT 1 was subsequently transformed into
DHSaTM competent
cells (Life Technologies).
BRAINOT12
The BRA1NOT12 library was constructed using RNA isolated from brain tissue
removed from
the right frontal lobe of a 5-year-old Caucasian male during a
hemispherectomy. Pathology indicated
extensive poiymicrogyria and mild to moderate gIiosis (predominantly subpial
and subcortical) and
was consistent with chronic seizure disorder.
The frozen tissue was homogenized and lysed in T.RIZOL reagent (I gm tissue/10
ml
TRIZOL; Life Technologies), a monophasic solution of phenol and guanidine
isothiocyanate, using a
Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, NY).
After a brief
incubation on ice, chloroform was added ( 1:5 v/v), and the lysate was
centrifuged. The upper
chloroform layer was removed to a fresh tube, and the RNA extracted with
isopropanol, resuspended
in DEPC-treated water, and treated with DNase for 25 min at 37°C. The
RNA was re-extracted twice
with acid phenol-chloroform pH 4.7 and precipitated using ~0.3M sodium acetate
and 2.5 volumes
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ethanol. The mRNA was isolated with the OLIGOTEX kit (QIAGEN, Inc.,
Chatsworth, CA) and used
to construct the cDNA library.
The mRNA was handled according to the recommended protocols in the SUPERSCRIPT
plasmid system (Life Technologies). The cDNAs were fractionated on a SEPHAROSE
CL4B column
{Amersham Pharmacia Biotech), and those cDNAs exceeding 400 by were ligated
into pINCYI
(Incyte Pharmaceuticals, Palo Alto CA). The plasmid pIINCYI was subsequently
transformed into
DHSa competent cells (Life Technologies).
II. Isolation of cDNA Clones
Plasmid DNA was released from the cells and purified using the REAL Prep 96
plasmid kit
(QIAGEN). The recommended protocol was employed except for the following
changes: 1 ) the
bacteria were cultured in 1 ml of sterile Terrific Broth (L,ife Technologies)
with carbenicillin at 25
mglL and glycerol at 0.4%; 2) after inoculation, the cultures were incubated
for 19 hours and at the
end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3)
following isopropanol
precipitation, the plasmid DNA pellet was resuspended uz 0.1 ml of distilled
water. After the last step
in the protocol, samples were transferred to a 96-well block for storage at
4° C.
III. Sequencing and Analysis
The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-
Elmer) or
the HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton)
systems in
combination with the PTC-200 thermal cyolers (MJ Research). The cDNAs were
sequenced using the
ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI
protocols, base calling
software, and kits. In one alternative, cDNAs were sequenced using the
MEGABACE 1000 DNA
sequencing system (Molecular Dynamics}: In another alternative, the cDNAs were
amplified and
sequenced using the ABI PRISM BIGDYE Terminator cycle sequencing ready
reaction kit (Perkin-
Elmer). In yet another alternative, cDNAs were sequenced using solutions and
dyes from Amersham
Pharmacia Biotech. Reading frames for the ESTs were determined using standard
methods (reviewed
in Ausubel, 1997, suara, unit 7.7). Some of the cDNA sequences were selected
for extension using the
techniques disclosed in Example V.
The polynucleotide sequences derived from cDNA, extension, and shotgun
sequencing were
assembled and analyzed using a combination of software programs which utilize
algorithms well
known to those skilled in the art. Table 1 summarizes the software programs,
descriptions, references,
and threshold parameters used. The first column of Table 1 shows the tools,
programs, and algorithms
used, the second column provides a brief description thereof, the third column
presents the references
which are incorporated by reference herein, and the fourth column presents,
where applicable, the
scores, probability values, and other. parameters used to evaluate the
strength ofa match between two
4z
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WO 00/11169 PCT/US99/19072
sequences (the higher the probability the greater the homology). Sequences
were analyzed using
MACDNASIS PRO software (Hitachi Software Engineering) and LASERGENE software
(DNASTAR).
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programming, and dinucIeotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as GenBank primate, rodent,
mammalian, vertebrate, and
eukaryote databases, and BLOCKS to acquire annotation, using programs based on
BLAST, FASTA,
and BLIMPS. The sequences were assembled into full length poiynucleotide
sequences using
programs based on Phred, Phrap; and Conned, and were screened for open reading
frames using
programs based on GeneMark, BLAST, and FASTA. T'he full length polynncleotide
sequences were
translated to derive the corresponding full length amino acid sequences, and
these full length
sequences were subsequently analyzed by querying agaiinst databases such as
the GenBank databases
(described above), SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.
The programs described above for the assembly and analysis of full length
polynucleotide and
amino acid sequences were used to identify polynucleotide sequence fragments
from SEQ ID N0:3
and SEQ ID N0:4. Fragments from about 20 to about 4000 nucleotides which are
useful in
hybridization and amplification technologies were described in The Invention
section above.
IV. Northern Analysis
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound. (See, e.g.., Sambrook, supra,
ch. 7; AusubeI, 1995,
supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in nucleotide databases such as GenBank or L:IFESEQ database,(Incyte
Pharmaceuticals,
Palo Alto CA). This analysis is much faster than multiple membrane-based
hybridizations. In
addition, the sensitivity of the computer search can be modified to determine
whether any particular
match is categorized as exact or similar. The basis of the search is the
product score, which is defined
as:
% seguence identity x % maxinnum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact within
a 1 % to 2% error, and, with a product score of 70, the match will be exact.
Similar molecules are
43
CA 02339860 2001-02-20
WO OOI11169 PCT/US99/19072
usually identified by selecting those which show product scores between 15 and
40, although lower
scores may identify related molecules.
The results of northern analyses are reported a percentage distribution of
libraries in which the
transcript encoding CSIG occurred. Analysis involved i:he categorization of
cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease categories included cancer,
inflammation/trauma, fetal,
neurological, and pooled. For each category, the number of libraries
expressing the sequence of
interest was counted and divided by the total number of libraries across all
categories. Percentage
1o values of tissue-specific and disease expression are reported in the
description of the invention.
V. Extension of CSIG Encoding Poiynucleotides
Full length nucleic acid sequences of SEQ ID N~0:3 and SEQ ID N0:4 were
produced by
extension of an appropriate fragment of the full length molecule using
oligonucleotide primers
designed from this fragment. One primer was synthesized to initiate 5'
extension of the known
fragment, and the other primer, to initiate 3' extension of"the known
fragment. The initial primers
were designed using OLIGO 4.06 software (National Biosciences), or another
appropriate program, to
be about 22 to 30 nucleotides in length, to have a GC content of about 50% or
more, and to anneal to
the target sequence at temperatures of about 68°C to about 72°C.
Any stretch of nucleotides which
would result in hairpin structures and primer-primer dimerizations was
avoided.
Selected human cDNA libraries were used to exlend the sequence. 1f more than
one extension
was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCh; using methods well known in
the art: PCR
was performed in 96-well plates using the PTC-200 thennai cycler (MJ
Research). The reaction mix
contained DNA template, 200 nmol of each primer, reaction buffer containing
Mg2+, (NH4)~SO4, and
~-mercaptoethanol, Taq DNA polymerase (Amersham Plharmacia Biotech), ELONGASE
enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C;, IS
sec; Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3; and 4 repeated 20 times; Step 6~: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, I min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ul
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1 X TE
and 0.5 pl of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
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CA 02339860 2001-02-20
WO 00/11169 PCT/US99/19072
Acton MA), allowing the DNA to bind to the reagent. T'he plate was scanned in
a Fluoroskan II
{Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,ul to 10 ~cl aliquot of the reaiction mixture was
analyzed by electrophoresis
on a L % agarose mini-gel to determine which reactions were successful in
extending the sequence.
T'he extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecullar Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0:6 to 0.8%)
agarose gels, fragments were excised, and agar digested 'with Agar ACE
(Promega). Extended clones .
were relegated using T4 ligase (New England Biolabs, Bf;verly MA) into pUC I8
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polyrnerase ('Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. 'Cransformed cells
were selected on antibiotic-
containing media, individual colonies were picked and cultured overnight at
37°C in 384-well plates in
LB/2x carb liquid media.
IS The cells were lysed, and DNA was amplified b;y PCR using Taq DNA
polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following parameters:
Step l: 94°C, 3 min; Step 2: 94°C, 35 sec; Step 3: 60°C,
1 min; Step 4: 72°C, 2 min; Step 5: steps 2,
3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at
4°C. DNA was quantified by
PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries
were reamplified using the same conditions as described above. Samples were
diluted with 20%
dimethysuiphoxide (I:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing primers
and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM
BIGDYE
Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID N0:3 and SEQ ID N0:4 are
used to
obtain 5' regulatory sequences using the procedure above, oligonucleotides
designed for such
extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:3 and SEQ ID N0:4 are employed to
screen
cDNAs, genomic DNAs, or mRNAs. Although the labeling of oliganucleotides,
consisting of about
20 base pairs, is specifically described, essentially the same procedure is
used with larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oIigomer, 250 pCi of
['zP]-adenosine triphosphate (Amersham Pharmacia Biotec:h), and T4
polynucleotide kinase (DuPont
NEN, Boston MA). The labeled oligonucleotides are subsl;antially purified
using a SEPHADEX G-25
CA 02339860 2001-02-20
WO 00/11169 PCT/US99/19072
superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An
aliquot containing
10'counts per minute of the labeled probe is used in a typical membrane-based
hybridization analysis
of human genornic DNA digested with one of the following endonucleases: Ase I,
Bgl II, Eco-RI, Pst
I, Xbal, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7°./° agarose
gel and transferred to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at roam temperature
under increasingly stringent conditions up to O.lx saline ,sodium citrate and
0.5% sodium dodecyl
sulfate. After XOMAT-AR film (Eastman Kodak, Rocht;ster NY) is exposed to the
blots,
hybridization patterns are compared visually.
VII. Microarrays
A chemical coupling procedure and an ink jet dE;vice can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldeschweiler, s, upra.)
An array analogous to a dot
or slot blot may also be used to arrange and link elements. to the surface of
a substrate using thermal,
UV, chemical, or mechanical bonding procedures. A typ;ical.array may be
produced by hand or using
available methods and machines and contain any appropriate number of elements.
After hybridization,
nonhybridized probes are removed and a scanner used to determine the levels
and patterns of
fluorescence. The degree of complementarily and the relative abundance of each
probe which
hybridizes to an element on the microarray may be assessed through analysis of
the scanned images.
Full-length cDNAs, Expressed Sequence Tags (SSTs), or fragments thereof may
comprise
the elements of the microarray. Fragments suitable for hybridization can be
selected using software
well known in the art such as LASERGENE software (DTJASTAR). Full-length
cDNAs, ESTs, or
fragments thereof corresponding to one of the nucleotide sequences of the
present invention, or
selected at random from a cDNA library relevant to the present invention, are
arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide
using, e.g., UV cross-linking
followed by thermal and chemical treatments and subsequent drying. (See, e.g.,
Schena, M. et al.
(1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.)
Fluorescent probes
are prepared and used for hybridization to the elements on the substrate. The
substrate is analyzed by
procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the CSIG-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring CSIG. Although
use of oligonucleotides
comprising from about i S to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
46
CA 02339860 2001-02-20
WO OOI11169 PCTIUS99119072
4.06 software (National Biosciences) and the coding sequence of CSIG. To
inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5' sequence and
used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotiile is
designed to prevent ribosomal binding to he CSIG-encoding transcript:
IX. Expression of CSIG
Expression and purification of CSIG is achieved using bacterial or virus-based
expression
systems. For expression of CSIG in bacteria, cDNA is subcloned into an
appropriate vector containing
an antibiotic resistance gene and an inducible promoter that directs high
levels of cDNA transcription.
Examples of such promoters include, but are not limited to, the trp-lac (tac)
hybrid promoter and the
TS or T7 bacteriophage promoter in conjunction with the: lac operator
regulatory element.
Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21
(DE3). Antibiotic
resistant bacteria express CSIG upon induction with isopropyl beta-D-
thiogalactopyranoside (IPTG).
Expression of CSIG in eukaryotic cells is achieved by infecting insect or
mammalian cell lines with
recombinant Auto raphica californica nuclear polyhedrosis virus (AcMNPV),
commonly known as
baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with
cDNA encoding CSIG
by either homologous recombination or bacterial-mediated transposition
involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong polyhedrin
promoter drives high levels of
cDNA transcription. Recombinant baculovirus is used to infect Spodoptera
fru;yrda (Sf9) insect
cells in most cases, or human hepatocytes, in some cases.. Infection of the
latter requires additional
genetic modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA
9 i :3224-3227; Sandig, V. et al. ( 1996) Hum. Gene Ther. 7:1937-1945.)
In most expression systems, CSIG is synthesized as a fusion protein with,
e.g., glutathione S-
transferase {GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma japonicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purifcation, the GST moiety can be proteolytically cleaved
from CSIG at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, supra,
ch 10 and 16). Purified CSIG obtained by these methods can be used directly in
the following activity
assay.
X. Demonstration ofCSIG Activity
47
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WO 00!11169 PCTIUS99119072
CSIG-1 activity is demonstrated by its effect on mitosis in quiescent cells
transfected with
eDNA encoding CSIG-1. CSIG-I is expressed by transfecting a mammalian cell
line such as COS7,
HeLa, or, CHO with an eukaryotic expression vector encoding CSIG-1. Eukaryotic
expression vectors
are commercially available, and the techniques to introduce them into cells
are well known to those
skilled in the art. The cells are incubated for 48-72 hours after transfection
under conditions
appropriate for the cell line to allow expression of CSIG-1. Non-transfected
cells are cultured in a
similar manner as controls. Phase microscopy is used to compare the mitotic
index of transfected
versus control cells. The change in the mitotic index is proportional to the
activity of CSIG-1 in the
transfected cells.
CSIG-2 activity is demonstrated by a prototypical assay for growth factor
activity, which
measures the stimulation of DNA synthesis in Swiss mouse 3T3 cells (McKay, I.
and Leigh, I. (1993)
Growth Factors: A Practical Approach, Oxford Universil:y Press, New York NY.)
Initiation of DNA
synthesis indicates the cells' entry into the mitotic cycle and their
commitment to undergo later
division. 3T3 cells are competent to respond to most growth factors, not only
those that are mitogenic,
but also those that are involved in embryonic induction. This competency is
possible because the in
vivo specificity demonstrated by some growth factors is not necessarily
inherent but is determined by
the responding tissue. Therefore, this assay is generally applicable to CSIG-
2. In this assay, varying
amounts of CSIG-2 are added to quiescent 3T3 cultured cells in the presence of
['H]thymidine, a
radioactive DNA precursor. CSIG-2 for this assay can be: obtained by
recombinant means or from
biochemical preparations. Incorporation of [3H]thymidine into acid-
precipitable DNA is measured
over an appropriate time interval, and the amount incorporated is directly
proportional to the amount
of newly synthesized DNA. A linear dose-response curve over at least a hundred-
fold CS1G-2
concentration range is indicative of growth factor activity. One unit of
activity per milliliter is defined
as the concentration of CSIG-2 producing a 50% response level, where 100%
represents maximal
incorporation of ['H]thymidine into acid-precipitable DNA.
XI. Functional Assays
CSIG function is assessed by expressing the sequences encoding CSIG at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-I O ug of recombinant vector are
transiently transfected into
a human cell line, preferably of endothelial or hematopoietic origin, using
either liposome
formulations or electroporation. 1-2 ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
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WO 00/11169 PC'lYUS99/19472
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP, and to evaluate
cellular properties, for example, their apoptotic state. FCM detects and
quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane <;omposition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M. G. ( 1994) Flow Cytometry, Oxford, New York NY.
The influence of CS1G on gene expression can be assessed using highly purified
populations
1 S of cells transfected with sequences encoding CSIG and either CD64 or CD64-
GFP. CD64 and CD64-
GFP are expressed on the surface of transfected cells and bind to conserved
regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success NY).
mRNA can be purified from the cells using methods well known by those of skill
in the art.
Expression of mRNA encoding CSIG and other genes of interest can be analyzed
by northern analysis
or microarray techniques.
XII. Production of CSIG Specific Antibodies
CSIG substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-49S), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the CSIG amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 111.)
Typically, oligopeptides I S residues in length are synthesized using an ABI
431 A Peptide
Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KL,H (Sigma-
Aldrich, St. Louis
MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to
increase
immunogenicity. (See, e.g., Ausubel, 1995; supra.) Rabbets are immunized with
the oligopeptide-
49
CA 02339860 2001-02-20
WO 00!11169 PCT/US99/19072
ICLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide activity by,
for example, binding the peptide to plastic, blocking with 1 % BSA, reacting
with rabbit antisera,
washing, and reacting with radio-iodinated goat anti-rabbiit IgG.
XIII. Purification of Naturally Occurring CSIG Using Specific Antibodies
Naturally occurring or recombinant CSIG is substantially purified by
immunoaffinity
chromatography using antibodies specific for CSIG. An immunoaffinity column is
constructed by
covalently coupling anti-CSIG antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing CSIG are passed~over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absort>ance of CSIG (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/CSIG binding (e.g., a buffer of pH 2 to pH 3, or ;a high
concentration of a chaotrope, such as
urea or thiocyanate ion), and CSIG is collected.
XIV. Identification of Molecules Which Interact wilth CSIG
CSIG, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules
previously arrayed in the
wells of a multi-well plate are incubated with the labeled CSIG, washed, and
any wells with labeled
CSIG complex are assayed. Data obtained using different concentrations of CSIG
are used to
calculate values for the number, affinity, and association of CS1G with the
candidate molecules.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection vvith specific
preferred embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the descrilbed modes for
carrying out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
CA 02339860 2001-02-20
WO 00/11169 PCT/US99/19072
H
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CA 02339860 2001-02-20
WO 00/11169 PCTNS99/19072
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CA 02339860 2001-02-20
WO 00/11169 PCT/US99/19072
SEQUENCE LISTI:N'G
<110> INCYTE PHARMACEUTICALS, INC.
TANG, Y. Tom
CORLEY, Neil C.
PATTERSON, Chandra
GUEGLER, Karl J.
BAUGHN, Mariah R.
<120> CELL SIGNALING PROTEINS
<130> PF-0572 PCT
<140> To Be Assigned
<141> Herewith
<150> 09/137,945; unassigned; 09/137,578; unassigned
<151> 1998-08-21; 1998-08-21; 1998-OS-21; 1998-08-21
<160> 5
<170> PERL Program
<210> 1
<211> 147
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 999661
<400> 1
Met Leu Thr Phe Leu Pro Pro Pro Trp Ala Gly Ile Gly Arg Leu
1 5 10 15
Ile Ala Glu.Cys His Leu Asn Pro Ile Ile Leu Pro Leu Trp His
20 25 30
Val G1y Glu Pro Gly Asp Gly Asp Arg Glu Met Ala Ser Gly Val
35 40 45
Gly Gly Leu Gly Leu Pro Leu Val Pro Gly Cys Pro Ala Pro Pro
50 55 60
His Val Trp Pro Ser Val His Cys Ala Ala Gly Me t Asn Asp Val
65 70 75
Leu Pro Asn Ser Pro Pro Tyr Phe Pro Arg Phe G:Ly Gln Lys Ile
80 85 90
Thr Val Leu Ile Gly Lys Pro Phe Ser Ala Leu Pro Val Leu Glu
95 100 105
Arg Leu Arg Ala Glu Asn Lys Ser Ala Val Glu Mea Arg Lys Ala
110 115 120
Leu Thr Asp Phe Ile Gln Glu Glu Phe Gln His Le:u Lys Thr Gln
I25 130 135
Ala Glu Gln Leu His Asn His Leu Gln Pra Gly Arg
140 145
1/4
CA 02339860 2001-02-20
WO 00/11169 PCTIUS99/19072
<210> 2
<211> 133
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1415354
<400> 2
Met Ala Met VaI Ser Ala Met Ser Trp Val Leu '.Pyr Leu Trp Ile
1 5 10 15
Ser Ala Cys Ala Met Leu Leu Cys His Gly Ser heu Gln His Thr
20 25 30
Phe Gln Gln His His Leu His Arg Pro Glu Gly Gly Thr Cys Glu
35 40 45
Val Ile Ala Ala His Arg Cys Cys Asn Lys Asn Arg Ile Glu Glu
50 55 60
Arg Ser Gln Thr Val Lys Cys Ser Cys Leu Pro Gly Lys Va1 Ala
65 70 75
Gly Thr Thr Arg Asn Arg Pro Ser Cys Val Asp Fvla Ser Ile Val
80 85 90
Ile Gly Lys Trp Trp Cys Glu Met Glu Pro Cys heu Glu Gly Glu
95 100 105
GIu Cys Lys Thr Leu Pro Asp Asn Ser Gly Trp Met Cys Ala Thr
110 115 120
Gly Asn Lys Ile Lys Thr Thr Arg Ile His Pro A.rg Thr
125 I30
<210> 3
<211> 1705
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Cione No: 999661
<400> 3
ggctttccca gcaagcaggg actgaggggg atagtcccca a<:acatgggc cccagggaga 60
agggcctgtt tcattgaggt ttggagagtg ttgagggtaa gcaaacctgt caccccacgc 120
ccccgagaat ggttactgat agggagaggc cttttccttg caggagatgg cgtctaccag 180
aaggggatgg acttcatttt ggagaagctc aaccatgggg ac;tgggtgca tatcttccca 240
gaaggtcagc agggctgact gggtcgagcc cccccagtat gagcgggatg ggctcccaag 300
cctcgcctct gtgctctctc accagggaaa gtgaacatga gtaccgaatt cctgcgtttc 360
aagtggggta agggctgctg gtctctggcc acagccatcc tcccggccca gagatggccc 420
tgtgggcccc tggctcccgc cccctcgggc tggcttgtat ggrgggtagat gggcgtgttt 480
gtagcgccag gaaggggaca ggtgctgaga ctaggcctgc ct.ctcgcagg ggct:tgccca 540
agggagctga attgaactgg aggatgtcgg gggtggcagt gg~ccagaggc tggcacagaa 600
gcttggctca gggcccagct tatgctaaca tttctacctc ccccctgggc aggaatcggg 660
cgcctgattg ctgagtgtca tctcaacccc atcatcctgc ccctgtggca tgtcggtgag 720
cctggggacg gggacagaga gatggcatct ggggtggggg gcctgggact ccctctggtc 780
ccaggctgcc ctgctccacc ccacgtctgg ccttctgtcc actgtgctgc aggaatgaat 840
gacgtccttc ctaacagtcc gccctacttc ccccgctttg gacagaaaat cactgtgctg 900
2I4
CA 02339860 2001-02-20
WO 00/11169 PCT/US99I19072
atcgggaagc ccttcagtgc cctgcctgta ctcgagcggc tccgggcgga gaacaagtcg 960
gctgtggaga tgcggaaagc cctgacggac ttcattcaag aggaattcca gcatctgaag 1020
actcaggcag agcagctcca caaccacctc cagcctggga gataggcctt gcttgctgcc 1080
ttctggattc ttggcccgca cagagctggg gctgagggat ggactgatgc ttttagctca 1140
aacgtggctt ttagacagat ttgttcatag accctctcaa gtgccctctc cgagctggta 1200
ggcattccag ctcctccgtg cttcctcagt tacacaaagg acctcagctg cttctcccac 1260
ttggccaagc agggaggaag aagcttaggc agggctctct ttccttcttg ccttcagatg 1320
ttctctccca ggggctggct tcaggaggga gcatagaagg caggtgagca accagttggc 1380
taggggagca gggggcccac cagagctgtg gagaggggac cctaagactc ctcggcctgg 1440
ctcctaccca ccgcccttgc cgaaccagga gctgctcact acctcctcag gtgggccgca 150 0
ggcgggaaaa gcagcccttg gccagaagtc aagcccagcc acgtggagcc tagagtgagg 1560
gcctgaggtc tggctgcttg cccccatgct ggcgccaaca acttctccat cctttctgcc 1620
tctcaacatc acttgaatcc tagggcctgg gttttcatgt ttttgaaaca gaaccataaa 1680
gcatatgtgt tgggccaaaa ggttt 1705
<210> 4
<211> 812
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1415354
<400> 4
cactggagtg gggatggtcc atcggcaact ataaactgat tctcatcagg aaactgcaca 60
ttatctcccc atcacttcaa aggtctcgtc aggcagaggt gacgccagga gatgatttaa 120
aggtgaaaat gacaaggttt ccacccctca aaccttggct ~ccttttctga caatacagtc 180
tgaatgaacc cgatgtcttt ttttttactg tggaaatagg atcggaagag agtaacattt 240
tttttttaat cctgataaag aagattgttg ggaagctctt tgaaaaaaaa ttttaaattg 300
tggcacagat ggattttaaa aagtgttaga tctttccaat gaacactaat agagtactct 360
gctcttggct ggatttttca gagaatggca atggtctctg cgatgtcctg ggtcctgtat 420
ttgtggataa gtgcttgtgc aatgctactc tgccatggat cccttcagca cactttccag 480
cagcatcacc tgcacagacc agaaggaggg acgtgtgaag tgatagcagc acaccgatgt 540
tgtaacaaga atcgcattga ggagcggtca caaacagtaa agtgttcctg tctacctgga 600
aaagtggctg gaacaacaag aaaccggcct tcttgcgtcg atgcctccat agtgattggg 660
aaatggtggt gtgagatgga gccttgccta gaaggagaag aatgtaagac actccctgac 720
aattctggat ggatgtgcgc aacaggcaac aaaattaaga c:cacgagaat tcacccaaga 780
acctaacaga agcatttgtg gtagtaaagg as 812
<210> 5
<211> 292
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> GenBank ID No: 1263110
<400> 5
Met Pro Leu His Val Lys Trp Pro Phe Pro Ala Val Pro Pro Leu
1 5 10 15
Thr Trp Thr Leu Ala Ser Ser Val Vai Met Gly Leu Val Gly Thr
20 25 30
Tyr Ser Cys Phe Trp Thr Lys Tyr Met Asn His Leu Thr Val His
3I4
CA 02339860 2001-02-20
WO 00111169 PCTIUS99/I9072
35 40 45
Asn Arg Glu Val Leu Tyr G1u Leu Ile Glu Lys ,'Arg Gly Pro Ala
50 55 60
Thr Pro Leu Ile Thr Va1 Ser Asn His Gln Ser Cys Met Asp Asp
65 70 75
Pro His Leu Trp Gly Ile Leu Lys Leu Arg His :Lle Trp Asn Leu
80 85 90
Lys Leu Met Arg Trp Thr Pro Ala Ala Ala Asp :Ele Gys Phe Thr
95 100 105
Lys Glu Leu His Ser His Phe Phe Ser Leu Gly Lys Cys Val Pro
110 115 120
Val Cys Arg Giy Ala Glu Phe Phe Gln Ala Glu Asn Glu Gly Lys
125 130 135
Gly Val Leu Asp Thr Gly Arg His Met Pro Gly Ala Gly Lys Arg
140 145 150
Arg Glu Lys Gly Asp Gly Val Tyr Gln Lys Gly biet Asp Phe Ile
155 160 165
Leu Glu Lys Leu Asn His Gly Asp Trp Val His 3:1e Phe Pro Glu
170 17s iso
G1y Lys Val Asn Met Ser Ser Glu Phe Leu Arg F~he Lys Trp Gly
185 190 195
Ile Gly Arg Leu Ile Ala Glu Cys His Leu Asn Fro Ile Ile Leu
200 205 210
Pro Leu Trp His Val Gly Met Asn Asp Val Leu Pro Asn Ser Pro
215 220 225
Pro Tyr Phe Pro Arg Phe Gly Gln Lys Ile Thr V'al Leu Ile Gly
230 235 240
Lys Pro Phe Ser Ala Leu Pro Val Leu Glu Arg Leu Arg Ala Glu
245 250 255
Asn Lys Ser Ala Val Glu Met Arg Lys Ala Leu Thr Asp Phe Ile
260 265 270
Gln Glu Glu Phe Gln His Leu Lys Thr Gln Ala Glu Gln Leu His
275 280 285
Asn His Leu Gln Pro Gly Arg
290
4/4
