Note: Descriptions are shown in the official language in which they were submitted.
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Growth Factor Polypeptides and Nucleic Acids Encoding Same
FIELD OF THE INVENTION
The invention relates to nucleic acids and polypeptides. In particular, this
invention
discloses novel nucleic acids and polypeptides with growth factor activity in
mammals.
Additionally antibodies specific for the polypeptides are disclosed.
BACKGROUND OF THE INVENTION
Polypeptide growth factors exerting effects in a variety of tissues have been
described.
Among these growth factors are bone morphogenetic protein-1 (BMP-1), vascular
endothelial
growth factor (VEGF), and platelet-derived growth factor (PDGF).
Multiple effects have been attributed to BMP-1. For example, BMP-1 is capable
of
inducing formation of cartilage in vivo. BMP1 is also identical to purified
procollagen C
proteinase (PCP), a secreted calcium-dependent metalloprotease that has been
reported to be
required for cartilage and bone formation. BMP-1 cleaves the C-terminal
propeptides of
procollagen I, II, and III and its activity is increased by the procollagen C-
endopeptidase
enhancer protein.
Vascular endothelial growth factor (VEGF) polypeptides have been reported to
act as
mitogens primarily for vascular endothelial cells. The specificity for
vascular endothelial cells
contrasts VEGF polypeptides from other polypeptide mitogens, such as basic
fibroblast
growth factor and platelet-derived growth factors, which are active on a wider
range of cell
types.
VEGF has also been reported to affect tumor angiogenesis. For example, VEGF
has
been shown to stimulate the elongation, network formation, and branching of
nonproliferating
endothelial cells in culture that are deprived of oxygen and nutrients.
The platelet derived growth factor (PDGF) family currently consists of at
least 3
distinct genes, PDGF A, PDGF B, and PDGF C whose gene products selectively
signal
through two PDGFRs to regulate diverse cellular functions. PDGF A, PDGF B, and
PDGF C
dimerize in solution to form homodimers, as well as the heterodimer.
Expression of RNA encoding the PDGF A and PDGF B subunits has been reported in
vascular tissues involved in atherosclerosis. PDGF A and PDGF B mRNA have been
reported
to be present in mesenchymal-appearing intimal cells and endothelial cells,
respectively, of
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atherosclerotic plaques. In addition, PDGF receptor mRNA has also been
localized
predominantly in plaque intimal cells.
The PDGF B is related to the transforming gene (v-sis) of simian sarcoma
virus. The
PDGF B has also been reported to be mitogen for cells of mesenchymal origin.
The PDGF B
has in addition been implicated in autocrine growth stimulation in the
pathologic proliferation
of endothelial cells characteristically found in glioblastomas. PDGF has also
been reported to
promote cellular proliferation and inhibits apoptosis.
SUMMARY OF THE INVENTION
The invention is based in part on the discovery of nucleic acids encoding
polypeptides
related to bone-morphogenetic protein-1 (BMP-1), vascular endothelial growth
factor (VEGF-
E) and platelet derived growth factor (PDGF). The nucleic acids,
polynucleotides, proteins
and polypeptides, or fragments thereof described herein are collectively
referred to as FCTRX
nucleic acids and polypeptides
In one aspect, the invention provides an isolated FCTRX polypeptide or
fragment of a
FCTRX polypeptide. The FCTRX polypeptide can include, e.g., an amino acid
sequence
selected from the group consisting of SEQ ID N0:2, 4, 6, 8, 10 and 12. Also
within the
invention is a FCTRX polypeptide that includes the amino acid sequence of a
variant of a SEQ
ID N0:2, 4, 6, 8, 10 or 12 amino acid sequences. In some embodiments, one or
more of the
amino acids in the variant sequence is changed to a different amino acid. In
some
embodiments, no more than 15% of the amino acid residues in the amino acid
sequence of said
variant are changed. A FCTRX polypeptide of the invention also includes a
mature form of a
SEQ ID N0:2, 4, 6, 8, 10 or 12 polypeptide, e.g., a polypeptide having the
amino acid
sequence of amino acids 24-370 of SEQ ID N0:2, or the corresponding fragments
in SEQ ID
N0:4. In other embodiments, the invention includes a variant of a mature form
of a
polypeptide including amino acid sequence of SEQ ID N0:2, 4, 6, 8, 10 and 12.
In the variant
form, one or more of the amino acids specified in the chosen sequence is
changed to a
different amino acid. In some embodiments, no more than 15% of the amino acid
residues in
the amino acid sequence of the variant of said mature form differ from the
sequence of a SEQ
ID N0:2, 4, 6, 8, 10 or 12 polypeptide.
Also provided by the invention is a fragment of a FCTRX polypeptide, a
fragment of a
variant form of a FCTRX polypeptide, a fragment of a mature form of a FCTRX
polypeptide,
or the fragment of a variant of a mature form of a FCTRX polypeptide.
Fragments of a
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FCTRX polypeptide include, e.g., amino acids 247-370 of SEQ ID N0:2, amino
acids 247-
338 of SEQ ID N0:2, and amino acids 339-370 of SEQ ID N0:2, as well as the
corresponding
homologous fragments in SEQ ID N0:4.
The invention also provides FCTRX nucleic acid molecules, including nucleic
acid
molecules, such as SEQ ID NOS:1, 3, 5, 7, 9 and 11, encoding FCTRX
polypeptides, nucleic
acids encoding variants of FCTRX polypeptides, nucleic acids encoding mature
forms of
FCTRX polypeptides, or nucleic acids encoding variants of mature forms of
FCTRX
polypeptides.
The invention also features an antibody that immunoselectively-binds to FCTRX
polypeptides. The antibody can be, e.g., a monoclonal antibody, a humanized
antibody, or a
human antibody.
In another aspect, the invention includes pharmaceutical compositions
that.include
therapeutically- or prophylactically-effective amounts of a therapeutic and a
pharmaceutically-
acceptable Garner. The therapeutic can be, e.g., a FCTRX nucleic acid, a FCTRX
polypeptide,
or an antibody specific for a FCTRX polypeptide. In a further aspect, the
invention includes,
in one or more containers, a therapeutically- or prophylactically-effective
amount of this
pharmaceutical composition.
In a further aspect, the invention includes a method of producing a
polypeptide by
culturing a cell that includes a FCTRX nucleic acid under conditions allowing
for expression
of the FCTRX polypeptide encoded by the FCTRX nucleic acid. If desired, the
FCTRX
polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence
of a
FCTRX polypeptide in a sample. In the method, a sample is contacted with a
compound that
selectively binds to the polypeptide under conditions allowing for formation
of a complex
between the polypeptide and the compound. The complex is detected, if present,
thereby
identifying the FCTRX polypeptide within the sample. The compound can be,
e.g., an anti-
FCTRX antibody, or another polypeptide that binds to a FCTRX polypeptide.
Also included in the invention is a method of detecting the presence of a
FCTRX
nucleic acid molecule in a sample by contacting the sample with a FCTRX
nucleic acid probe
or primer, and detecting whether the nucleic acid probe or primer bound to a
FCTRX nucleic
acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the
activity of a
FCTRX polypeptide. The method includes contacting a cell sample that includes
the FCTRX
polypeptide with a compound that binds to the FCTRX polypeptide in an amount
sufficient to
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modulate the activity of said polypeptide. The compound can be, e.g., a small
molecule, such
as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid
or other organic
(carbon containing) or inorganic molecule, as further described herein.
The invention further includes a method for screening for a modulator of
disorders or
syndromes including, e.g., cancer. The method includes contacting a test
compound with a
FCTRX polypeptide and determining if the test compound binds to said FCTRX
polypeptide.
Binding of the test compound to the FCTRX polypeptide indicates the test
compound is a
modulator of activity, or of latency or predisposition to the disorder or
syndrome. In one
embodiment, the candidate test compound has a molecular weight not more than
about 1500
Da.
Also within the scope of the invention is a method for screening for a
modulator of
activity, or of latency or predisposition to any FCTRX associated disorders or
syndromes
including, by administering a test compound to a test animal at increased risk
for the
aforementioned disorders or syndromes. The test animal expresses a recombinant
polypeptide
encoded by a FCTRX nucleic acid. Expression or activity of FCTRX polypeptide
is then
measured in the test animal, as is expression or activity of the protein in a
control animal
which recombinantly-expresses FCTRX polypeptide and is not at increased risk
for the
disorder or syndrome. Next, the expression of FCTRX polypeptide in both the
test animal and
the control animal is compared. A change in the activity of FCTRX polypeptide
in the test
animal relative to the control animal indicates the test compound is a
modulator of latency of
the disorder or syndrome.
In yet another aspect, the invention includes a method for determining the
presence of
or predisposition to a disease associated with altered levels of a FCTRX
polypeptide, a
FCTRX nucleic acid, or both, in a subject (e.g., a human subject). The method
includes
measuring the amount of the FCTRX polypeptide in a test sample from the
subject and
comparing the amount of the polypeptide in the test sample to the amount of
the FCTRX
polypeptide present in a control sample. An alteration in the level of the
FCTRX polypeptide
in the test sample as compared to the control sample indicates the presence of
or predisposition
to a disease in the subject.
In a further aspect, the invention includes a method of treating or preventing
a
pathological condition associated with a disorder in a mammal by administering
to the subject
a FCTRX polypeptide, a FCTRX nucleic acid, or a FCTRX-specific antibody to a
subject
(e.g., a human subject), in an amount sufficient to alleviate or prevent the
pathological
condition.
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FCTRX nucleic acids according to the invention can be used to identify various
cell
types, including cancerous cells. For example, Example 7 illustrates that
clone 30664188Ø99
(SEQ ID NO:1) is strongly expressed specifically in CNS cancer, lung cancer
and ovarian
cancer. It is also shown in the Examples that SEQ ID NO:1 produces a gene
product which
either persists intact in conditioned medium arising from transfecting HEK 293
cells, or is
proteolytically cleaved. Evidence presented in Example 13 suggests that the
form of the
30664188Ø99 protein (SEQ ID N0:2) that is active in the various experiments,
which are
reported in the Examples, is a proteolysis product of the 30664188Ø99
protein. As shown in
the Examples, the activities ascribed to either one or both of these
substances include the
ability to stimulate net DNA synthesis as monitored by incorporation of BrdU
into DNA,
proliferation of cell number, the ability to transform cells in culture, and
the ability to induce
tumor formation in vivo. These various activities occur in a variety of cell
types.
FCTRX nucleic acids, and their encoded polypeptides, can also be used to
modulate
cell growth. For example, it is likely that the polypeptide having the amino
acid sequence of
SEQ ID N0:2, 4, 6, 8, 10 and 12, or all, has specific functions in a variety
of cells. In addition
to stimulating growth and proliferation of certain cells, it is endogenously
expressed in certain
specific classes of tumor cell lines. Thus, a FCTRX polypeptide, e.g., a
polypeptide having
the amino acid sequence of SEQ ID N0:2, 4, 6, 8, 10 or 12, can be used where
net cell growth
and proliferation is desired and in different circumstances where cell growth
is to be inhibited
or abrogated.
A FCTRX nucleic acid or gene product, e.g., a nucleic acid encoding SEQ ID
N0:2, 4,
6, 8, 10 or 12, is useful as a therapeutic agent in promoting wound healing,
neovascularization
and tissue growth, and similar tissue regeneration needs. More specifically, a
FCTRX nucleic
acid or polypeptide may be useful in treatment of anemia and leukopenia,
intestinal tract
sensitivity and baldness. Treatment of such conditions may be indicated in,
e.g., patients
having undergone radiation or chemotherapy. It is intended in such cases that
administration
of a FCTRX nucleic acid or polypeptide, e.g., a polypeptide including the
amino acid
sequence of SEQ ID N0:2, 4, 6, 8, 10 or 12, or a nucleic acid sequence
encoding these
polypeptides (e.g., SEQ ID NO:1, 3, 5, 7, 9, or 11) will be controlled in dose
such that any
hyperproliferative side effects are minimized.
Alternatively, in cases of tumors, such as CNS cancer and ovarian cancer, in
which
FCTRX nucleic acids is expressed at high levels, (e.g., a tumor in SEQ ID NO:1
is expressed
in high levels), it is desired to inhibit or eliminate the effects of
production of a FCTRX
nucleic acid or gene product (e.g., SEQ ID N0:2 or SEQ ID N0:4, or a nucleic
acid encoding
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one of these polypeptides). For example, this may be accomplished by
administration of an
antibody directed against a polypeptide having the amino acid sequence of SEQ
ID N0:2 or
SEQ ID N0:4,or fragment thereof. In particular, the antibody can be directed
against the
active fragment p35 (see the Examples) identified herein. An alternative
example involves
identifying the putative protease implicated in the formation of p35 from p85
(see the
Examples). Administration of a substance that specifically inhibits the
activity of this
protease, but not the activity of other proteases, will be effective to
prevent formation of the
active p35 form of a FCTRX polypeptide, e.g., a clone 30664188Ø99
polypeptide.
Based on the roles of molecules related to FCTRX polypeptides and nucleic
acids,
(e.g., BMP-1, VEGF-like polypeptides such as fallotein, and PDGF) in malignant
disease
progression and the gene expression profile described herein, it is foreseen
that, for a subset of
human gliomas and ovarian epithelial carcinomas, targeting of a FCTRX
polypeptide using an
antibody has an inhibitory effect on tumor growth, matrix invasion, chemo-
resistance, radio-
resistance, and metastatic dissemination. In various embodiments, the FCTRX
polypeptide is
linked to a monoclonal antibody, a humanized antibody or a fully human
antibody.
A FCTRX polypeptide can potentially block or limit the extent of tumor
neovascularization. In addition to classical modes of administration of
potential antibody
therapeutics newly developed modalities of administration may be useful. For
example, local
administration of'3~I-labeled monoclonal antibody for treatment of primary
brain tumors after
surgical resection has been reported. Additionally, direct stereotactic
intracerebral injection of
monoclonal antibodies and their fragments is also being studied clinically and
pre-clinically.
Intracarotid hyperosmolar perfusion is an experimental strategy to target
primary brain
malignancy with drug conjugated human monoclonal antibodies.
Additionally, the nucleic acids of the invention, and fragments and variants
thereof,
may be used, by way of nonlimiting example, (a) to direct the biosynthesis of
the
corresponding encoded proteins, polypeptides, fragments and variants as
recombinant or
heterologous gene products, (b) as probes for detection and quantification of
the nucleic acids
disclosed herein, (c) as sequence templates for preparing antisense molecules,
and the like.
Such uses are described more fully in the following disclosure.
Furthermore, the proteins and polypeptides of the invention, and fragments and
variants thereof, may be used, in ways that include (a) serving as an
immunogen to stimulate
the production of an anti-FCTRX antibody, (b) a capture antigen in an
immunogenic assay for
such an antibody, and (c) as a target for screening for substances that bind
to a FCTRX
polypeptide of the invention. These utilities and other utilities for FCTRX
nucleic acids,
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polypeptides, antibodies, agonists, antagonists, and other related compounds
are disclosed
more fully below.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the present invention, suitable methods
and materials are
described below. All publications, patent applications, patents, and other
references
mentioned herein are incorporated by reference in their entirety. In the case
of conflict, the
present specification, including definitions, will control. In addition, the
materials, methods,
and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the
following
detailed description and claims.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a representation of an alignment of the amino acid sequence of clone
30664188Ø99 with the amino acid sequence of a human secretory growth factor-
like protein
VEGF-E amino acid sequence (SEQ ID N0:24).
FIG. 2 is a representation of a Western blot of a 30664188.m99 protein
expressed in E.
coli cells.
FIG. 3 is a representation of a Western blot of a 30664188.m99 protein
secreted by
human 293 cells.
FIG. 4A is a schematic representation of a scheme for the recombinant
production,
purification and apparent molecular weight of a mature form of the protein of
clone
30664188Ø99.
FIG. 4B includes representations of two Western blot analyses showing
expression of a
30664188Øm99 polypeptide.
FIG. S is a graph showing incorporation of BrdU into NIH 3T3 cells and CCD-
1070
cells in response to various treatments.
FIG. 6 is a graph showing proliferation of NIH 3T3 5-24 cells in response to
various
treatments.
FIG. 7 is a graph showing cell number in NIH 3T3 cells exposed to a mock
treatment
or 30664188.
FIG. 8 is a depiction of a photomicrograph showing cell density and cell
morphology
of NIH 3T3 cells in response to treatment with pCEP4sec CM or 30664188
protein.
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FIG. 9 is a depiction of a photomicrograph showing changes in cell number in
NHost
osteoblast cells in response to various treatments.
FIG. 10A is a representation of a western blot of 30664188.m99 expressed by
HEK
293 cells cultured in the absence of serum.
FIG. lOB is a representation of SDS-PAGE 30664188.m99 protein expressed by HEK
293 cells cultured in the presence of serum.
FIG. 11 is a representation of dose titration of BrdU incorporation into NIH
3T3 cells
stimulated by p85 and by p35 fragments of 30664188.m99 protein.
FIG. 12 is a diagram depicting a comparison of core PDGF domains among PDGF
family members. Human and mouse PDGF D core PDGF domains were aligned with
human
PDGF C, human PDGF B and human PDGF A core PDGF domains (GenBank accession
numbers: AAF80597, P01127 and P04085, respectively). Invariant cysteine
residues are
shaded. The asterisk indicates a conserved cysteine residue that is missing in
PDGF D.
FIG. 13 is a representation of the nucleotide and deduced amino acid sequence
of the
human PDGF D gene. Also shown is the human PDGF D genomic structure. The
initiation
and stop codons are boxed, and intron/exon boundaries are depicted with
arrows.
FIG. 14 is a representation of a Western blot and SDS PAGE analysis of PDGF D.
In
Panel A, samples from the conditioned medium of HEK 293 cells transiently
transfected with
pCEP4/Sec (lane 1) or pCEP4/Sec-PDGF D (lanes 2 & 3) and cultured in the
presence (lane 3)
or absence (lanes 1 & 2) of FBS were examined by SDS-PAGE under reducing
conditions,
followed by immunoblot analysis using anti-V5 antibody. In Panel B, purified
PDGF-D from
pCEP4/Sec-PDGF D transfected HEK 293 cells cultured in the presence (lanes 3 &
4) or
absence (lanes 1 & 2) of FBS was resolved by SDS-PAGE and stained with
Coomassie Blue.
Samples were treated with (+) and without (-) DTT. Molecular weight markers
are indicated
on the left.
FIG. 15 is a representation of fragments obtained from p35 and identified by N-
terminal sequencing. In each panel, the upper sequence in black is the
predicted sequence
from the clone, and the lower sequence in gray is the sequence provided by N-
terminal
sequencing. The diagonal shadings represent two fragments of p35. Horizontal
shading
represents the V5 epitope and vertical shading represents the 6His tag, both
of which originate
from vector pCEP4/Sec-30664188 (Example 4). In Panel A, two sequences were
identified,
one beginning with GlyArg (shown with these, two residues underlined), and the
second
beginning with the third residue, Ser.
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FIG. 16 is a depiction of the SDS-PAGE of the 30664188 gene product in the
presence
of fetal bovine serum (Panel B) and Calf Serum (Panel A). Lanes 1 and 2 in
each panel show
authentic 30664188 p35 alone or in the presence of serum, respectively. Lane 3
in each panel
shows p85 in the absence of serum, and lanes 4-6 show p85 in the presence of
increasing
concentrations of the respective serum.
FIG. 17 includes diagrams demonstrating the biological activity and PDGF
receptor
activation of recombinant PDGF DD, including its effects on DNA synthesis and
cell growth.
Panels A & B depict a BrdU incorporation assay. CCD1070 human (A) or NIH 3T3
murine
(B) fibroblasts were serum-starved, incubated with PDGF DD p35 (closed
circles), PDGF DD
p85 (closed diamonds) PDGF BB (open triangles) or PDGF AA (closed squares) for
18 hrs,
and analyzed by BrdU incorporation assay. Panel C depicts, growth assay. NIH
3T3 cells
were incubated with serum-free media supplemented with the indicated factor
(symbols
indicated above) or 5% calf serum (open circles) and counted at the indicated
time intervals.
Panel D shows PDGFR activation in fibroblasts. NIH 3T3 fibroblasts were serum
starved 18
1 S hrs and incubated in the absence or presence of PDGF DD, PDGF AA or PDGF
BB (200
ng/ml) for 10 min. Whole cell lysates were then immunoprecipitated (designated
IP) with
antibody directed against the a or (3 PDGF receptor (PDGFR) and subjected to
Western blot
analysis with anti-phosphotyrosine mAb (anti-PY), anti-a PDGFR antibody or
anti-(3 PDGFR
antibody. The position of the PDGFR is indicated.
FIG. 18 is diagram showing the competition of 30664188 p85 with other growth
factors that induce growth of NIH/3T3 Cells, and the effect of adding a 100-
fold range of
30664188 p85 in the presence of either 30664188 p35 or PDGF BB on the cell
growth of
NIH/3T3 cells.
FIG. 19 is a representation of the differential gene expression analysis after
PDGF DD,
PDGF BB, and PDGF AA treatment. In panel A, primary human foreskin fibroblasts
were
treated with PDGF DD, PDGF BB, PDGF AA or control buffer for 3 hr. Total RNA
was
harvested and subjected to GeneCalling (U. S. Patent No. 5,871,697 andR.
Shimkets et al.,
Nat. Biotechnol. 18, 798-803 (1999)). The number of shared gene fragments
induced (gray
shaded boxes) or suppressed (gray hatched boxes) by each treatment are listed
to right. In
panel B, representative genes induced by PDGF DD and PDGF BB treatment are
shown. The
fold induction (gray shaded box) or suppression (gray hatched box) is
indicated in each box.
FIG. 20 is a diagram showing the results of the competition of growth of CCD
1070
cells in response to growth factors in the absence or presence of receptor
antibodies. CCD
1070 cells were incubated in the presence of the p35 form of 30664188, PDGF
AA, or PDGF
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BB. In each case, the growth factor was incubated by itself, with a
nonspecific antibody
(Rab), with an antibody specific for the alpha PDGF receptor (alpha Rab) or
the beta PDGF
receptor (beta Rab), or in the presence of both specific antibodies.
FIG. 21 is a depiction of the stimulation of the growth of pulmonary artery
smooth
muscle cells by growth factors. Smooth muscle cells were treated with purified
p35 PDGF
DD, PDGF AA or PDGF BB at the concentrations indicated, and the amount of BrdU
incorporated into DNA was determined.
FIG. 22 is a diagram showing the proliferation of pulmonary artery smooth
muscle
cells in response to various treatments.
FIG. 23 is a diagram showing the proliferation of saphenous vein cells in
response to
various treatments.
FIG. 24 is a diagram showing the neutralization of the growth of NIH 3T3 mouse
cells
induced by 30664188 by treatment with a specific antibody.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides nucleic acids that encoded polypeptides related to bone-
morphogen protein-1 (BMP-1) vascular endothelial growth factor (VEGF-E) and
platelet
derived growth factor (PDGF).
Included in the invention are novel nucleic acid sequences and their encoded
polypeptides. The sequences are collectively referred to as "FCTRX nucleic
acids" or
"FCTRX polynucleotides" and the corresponding encoded polypeptide is referred
to as a
"FCTRX polypeptide" or "FCTRX protein". Unless indicated otherwise, "FCTRX" is
meant
to refer to any of the novel sequences disclosed herein. In addition, the
polypeptides and
nucleic acids of the invention are alternately referred to herein collectively
as "PDGFD".
Furthermore, when reference is made to "PDGFXX" wherein "X" is either A, B, C
or D, this
is meant to referr to homodimers of the particular PDGF. Alternately, when
reference is made
to "PDGFXY" wherein X and Y are either the A, B, C or D, and "X" is different
from "Y" this
is meant to refer to PDGF heterodimers.
It is shown herein that the PDGFD has a high molecular weight latent form,
designated
p85, and a low molecular weight active form, designated p35.
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FCTRl Nucleic Acids and Polypeptides
A FCTR1 polynucleotide of the invention includes the nucleic acid present in
clone
30664188Ø99. Clone 30664188Ø99 is 1828 nucleotides in length. The
nucleotide sequence
of FCTR1 (also referred to as 30664188Ø99 or PDGFD) is reported in Table 1
(SEQ ID
NO:1 ). The clone was originally obtained from RNA from pituitary gland
tissues and is also
present in RNA from human uterine microvascular endothelial cells (Clonetics,
San Diego,
CA), human erythroleukemia cells (ATCC, Manassas, VA), thyroid, small
intestine,
lymphocytes, adrenal gland and salivary gland.
TABLE 1. NUCLEOTIDE (SEQ ID NO:1) AND PROTEIN (SEQ ID N0:2)
SEQUENCE OF FCTRl (also referred to as 30664188-0-99
Translated Protein - Frame: 2 - Nucleotide 182 to 1292
1 CTAAAAAATATGTTCTCTACAACACCAAGGCTCATTAAAATATTT
46 TAAATATTAATATACATTTCTTCTGTCAGAAATACATAAAACTTT
91 ATTATATCAGCGCAGGGCGGCGCGGCGTCGGTCCCGGGAGCAGAA
136 CCCGGCTTTTTCTTGGAGCGACGCTGTCTCTAGTCGCTGATCCCA
181 AATGCACCGGCTCATCTTTGTCTACACTCTAATCTGCGCAAACTT
MetHisArgLeuIlePheValTyrThrLeuIleCysAlaAsnPh
226 TTGCAGCTGTCGGGACACTTCTGCAACCCCGCAGAGCGCATCCAT
eCysSerCysArgAspThrSerAlaThrProGlnSerAlaSerI1
271 CAAAGCTTTGCGCAACGCCAACCTCAGGCGAGATGAGAGCAATCA
eLysAlaLeuArgAsnAlaAsnLeuArgArgAspGluSerAsnHi
316 CCTCACAGACTTGTACCGAAGAGATGAGACCATCCAGGTGAAAGG
sLeuThrAspLeuTyrArgArgAspGluThrIleGlnValLysG1
361 AAACGGCTACGTGCAGAGTCCTAGATTCCCGAACAGCTACCCCAG
yAsnGlyTyrValGlnSerProArgPheProAsnSerTyrProAr
406 GAACCTGCTCCTGACATGGCGGCTTCACTCTCAGGAGAATACACG
gAsnLeuLeuLeuThrTrpArgLeuHisSerGlnGluAsnThrAr
451 GATACAGCTAGTGTTTGACAATCAGTTTGGATTAGAGGAAGCAGA
gIleGlnLeuValPheAspAsnGlnPheGlyLeuGluGluAlaG1
496 AAATGATATCTGTAGGTATGATTTTGTGGAAGTTGAAGATATATC
uAsnAspIleCysArgTyrAspPheValGluValGluAspIleSe
541 CGAAACCAGTACCATTATTAGAGGACGATGGTGTGGACACAAGGA
rGluThrSerThrIleIleArgGlyArgTrpCysGlyHisLysGl
586 AGTTCCTCCAAGGATAAAATCAAGAACGAACCAAATTAAAATCAC
uValProProArgIleLysSerArgThrAsnGlnIleLysIleTh
631 ATTCAAGTCCGATGACTACTTTGTGGCTAAACCTGGATTCAAGAT
rPheLysSerAspAspTyrPheValAlaLysProGlyPheLysI1
11
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676 TTATTATTCTTTGCTGGAAGATTTCCAACCCGCAGCAGCTTCAGA
eTyrTyrSerLeuLeuGluAspPheGlnProAlaAlaAlaSerG1
721 GACCAACTGGGAATCTGTCACAAGCTCTATTTCAGGGGTATCCTA
uThrAsnTrpGluSerValThrSerSerIleSerGlyValSerTy
766 TAACTCTCCATCAGTAACGGATCCCACTCTGATTGCGGATGCTCT
rAsnSerProSerValThrAspProThrLeuIleAlaAspAlaLe
811 GGACAAAAAAATTGCAGAATTTGATACAGTGGAAGATCTGCTCAA
uAspLysLysIleAlaGluPheAspThrValGluAspLeuLeuLy
856 GTACTTCAATCCAGAGTCATGGCAAGAAGATCTTGAGAATATGTA
sTyrPheAsnProGluSerTrpGlnGluAspLeuGluAsnMetTy
901 TCTGGACACCCCTCGGTATCGAGGCAGGTCATACCATGACCGGAA
rLeuAspThrProArgTyrArgGlyArgSerTyrHisAspArgLy
2O 946 GTCAAAAGTTGACCTGGATAGGCTCAATGATGATGCCAAGCGTTA
sSerLysValAspLeuAspArgLeuAsnAspAspAlaLysArgTy
991 CAGTTGCACTCCCAGGAATTACTCGGTCAATATAAGAGAAGAGCT
rSerCysThrProArgAsnTyrSerValAsnIleArgGluGluLe
1036 GAAGTTGGCCAATGTGGTCTTCTTTCCACGTTGCCTCCTCGTGCA
uLysLeuAlaAsnValValPhePheProArgCysLeuLeuValG1
1081 GCGCTGTGGAGGAAATTGTGGCTGTGGAACTGTCAACTGGAGGTC
nArgCysGlyGlyAsnCysGlyCysGlyThrValAsnTrpArgSe
1126 CTGCACATGCAATTCAGGGAAAACCGTGAAAAAGTATCATGAGGT
rCysThrCysAsnSerGlyLysThrValLysLysTyrHisGluVa
1171 ATTACAGTTTGAGCCTGGCCACATCAAGAGGAGGGGTAGAGCTAA
lLeuGlnPheGluProGlyHisIleLysArgArgGlyArgAlaLy
1216 GACCATGGCTCTAGTTGACATCCAGTTGGATCACCATGAACGATG
sThrMetAlaLeuValAspIleGlnLeuAspHisHisGluArgCy
1261 TGATTGTATCTGCAGCTCAAGACCACCTCGATAAGAGAATGTGCA
SAspCysIleCysSerSerArgProProArg
1306 CATCCTTACATTAAGCCTGAAAGAACCTTTAGTTTAAGGAGGGTG
1351 AGATAAGAGACCCTTTTCCTACCAGCAACCAAACTTACTACTAGC
1396 CTGCAATGCAATGAACACAAGTGGTTGCTGAGTCTCAGCCTTGCT
1441 TTGTTAATGCCATGGCAAGTAGAAAGGTATATCATCAACTTCTAT
1486 ACCTAAGAATATAGGATTGCATTTAATAATAGTGTTTGAGGTTAT
1531 ATATGCACAAACACACACAGAAATATATTCATGTCTATGTGTATA
1576 TAGATCAAATGTTTTTTTTGGTATATATAACCAGGTACACCAGAG
1621 CTTACATATGTTTGAGTTAGACTCTTAAAATCCTTTGCCAAAATA
1666 AGGGATGGTCAAATATATGAAACATGTCTTTAGAAAATTTAGGAG
1711 ATAAATTTATTTTTAAATTTTGAAACACAAAACAATTTTGAATCT
1756 TGCTCTCTTAAAGAAAGCATCTTGTATATTAAAAATCAAAAGATG
SS 1801 AGGCTTTCTTACATATACATCTTAGTTG
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Nucleotides 182 to 1292 of SEQ ID NO:1 encode a 370 amino acid protein (SEQ ID
N0:2) that includes sequences characteristic of secreted proteins. The
sequence of the
encoded protein, which is also referred to herein as "30664188Ø99 protein",
S "30664188Ø99", "PDGFD", or "human PDGFD" is presented in Table 1. The
predicted
molecular weight of the 30664188Ø99 protein is 42847.8 daltons with a pI of
7.88.
BLASTN and BLASTP analyses indicate the 30664188Ø99 polypeptide has a
likeness to human vascular endothelial growth factor E (VEGF-E), as well as to
VEGF-E from
other vertebrate species. For example, there is a 44% identity to human
secretory growth
factor-like protein (VEGF-E, or fallotein; Acc. No.: AAF00049 which references
GenBank-
ID: AF091434 for the nucleotide sequence). An alignment of the amino acid
sequence of the
30664188Ø99 polypeptide with that of VEGF-E is shown in FIG. 1. BLASTP
analyses also
indicate that FCTR1 is related to human PDGF C, PDGF B, and PDGF A (42%, 27%,
and
25% overall amino acid identity, respectively)
1 S PFAM and PROSITE analyses indicte that 30664188Ø99 polypeptide amino
acid
sequence conatains a PDGF domain (aa 272-362) and a N-linked glycosylation
site (residue
276).
The 30664188Ø99 polypeptide amino acid sequence shows similarity to the
sequence
of human procollagen C-endopeptidase (bone morphogenetic protein-1; BMP-1; PIR-
ID:A58788), which is a polypeptide of 823 residues. Residues 54 to 169 of the
30664188Ø99
polypeptide show 30-41% identity over three segments of the BMP-1 polypeptide.
The
30664188Ø99 polypeptide also shows a similar degree of identity is to BMP-1
from Xenopus
laevis (ACC NO:P98070), which is a 707 residue protein. The latter protein may
act as a zinc
protease in promoting cartilage and bone formation (Wozney et al., Science
242: 1528-34,
1988).
The 30664188Ø99 polypeptide is also related to other growth factors. For
example, it
shows 42% identity and 59% similarity to chicken spinal cord-derived growth
factor
(TREMBLNEW-ACC:BAB03265), 42% identity and 59% identity to human secretory
growth
factor-like protein fallotein (SPTREMBL-ACC:Q9UL22), 42% identity and 39%
similarity to
human platelet-derived growth factor C (TREMBLNEW-ACC:AAF80597), and 39%
identity
and 59% similarity to mouse fallotein (SPTREMBL-ACC:Q9QY71).
The homologies discussed above identify the 30664188Ø99 polypeptide as a
member
of the BMP-1/VEGF-E/PDGF protein family. BMP-1 proteins include an EGF-like
domain,
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three CUB domains, and PDGF/VEGF domains.' BMP-1 proteins are also members of
the
astacin subfamily.
SignalP and PSORT analyses predict that the amino acid sequence for
30664188Ø99
includes a cleavable amino terminal signal peptide with a cleavage site
between positions 23
and 24 (TSA-TP). The protein is most likely secreted and localized outside of
the cell. The
InterPro software program predicts the presence of a CUB domain in
30664188Ø99 from
residue 53 to residue 167, a PDGF domain spanning residues 272-306 and 350-
362, and a
metallothionein domain from residue 302 to residue 365. A FCTR1 polypeptide of
the
invention includes a polypeptide having one, two, three, or four of these
domains, or a
combination thereof.
A FCTR1 polypeptide of the invention includes a mature form of a FCTR1
polypeptide
that includes amino acids 24-370 of SEQ ID N0:2. These sequences are also
encoded in a
construct encoded by clone 30664188Øm99, which is described in more detail
below. Also
within the invention are nucleic acids encoding FCTRX polypeptide fragments
that include
amino acid sequences 247-370, 247-338, or 339-370, or their variant forms. In
some
embodiments, the fragments stimulate proliferation of cells. Also within the
invention are the
FCTRX polypeptide fragments, or their variants, encoded by these nucleic
acids.
FCTR2 Nucleic Acids and Polypeptides
A FCTR2 polynucleotide of the invention includes the nucleic acid sequence
present in
clone 30664188Ø331. Clone 30664188Ø331 is 1587 nucleotides in length and
was
originally isolated from RNA from pituitary gland tissues. The nucleotide
sequence of FCTR2
(also referred to as 30664188Ø331) is shown in Table 2 (SEQ ID N0:3).
TABLE 2. NUCLEOTIDE (SEQ ID N0:3) AND PROTEIN (SEQ ID N0:4) SEQUENCE
OF FCTR2 (30664188-0-331)
Translated Protein - Frame: 3 - Nucleotide 540 to 936
1 AGAGGCTCTCAAATTAGATCAAGAAATGCCTTTAACAGAAGTGAA
46 GAGTGAACCTGCTCCTGACATGGCGGCTTCACTCTCAGGAGAATA
91 CACGGATACAGCTAGTGTTTGACAATCAGTTTGGATTAGAGGAAG
136 CAGAAAATGATATCTGTAGGTATGATTTTGTGGAAGTTGAAGATA
181 TATCCGAAACCAGTACCATTATTAGAGGACGATGGTGTGGACACA
226 AGGAAGTTCCTCCAAGGATAAAATCAAGAACGAACCAAATTAAAA
271 TCACATTCAAGTCCGATGACTACTTTGTGGCTAAACCTGGATTCA
316 AGATTTATTATTCTTTGCTGGAAGATTTCCAACCCGCAGCAGCTT
361 CAGAGACCAACTGGGAATCTGTCACAAGCTCTATTTCAGGGGTAT
406 CCTATAACTCTCCATCAGTAACGGATCCCACTCTGATTGCGGATG
451 CTCTGGACAAAAAAATTGCAGAATTTGATACAGTGGAAGATCTGC
496 TCAAGTACTTCAATCCAGAGTCATGGCAAGAAGATCTTGAGAATA
M
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541 TGTATCTGGACACCCCTCGGTATCGAGGCAGGTCATACCATGACC
etTyrLeuAspThrProArgTyrArgGlyArgSerTyrHisAspA
586 GGAAGTCAAAAGTTGACCTGGATAGGCTCAATGATGATGCCAAGC
S rgLysSerLysValAspLeuAspArgLeuAsnAspAspAlaLysA
631 GTTACAGTTGCACTCCCAGGAATTACTCGGTCAATATAAGAGAAG
rgTyrSerCysThrProArgAsnTyrSerValAsnIleArgGluG
676 AGCTGAAGTTGGCCAATGTGGTCTTCTTTCCACGTTGCCTCCTCG
luLeuLysLeuAlaAsnValValPhePheProArgCysLeuLeuV
721 TGCAGCGCTGTGGAGGAAATTGTGGCTGTGGAACTGTCAACTGGA
alGlnArgCysGlyGlyAsnCysGlyCysGlyThrValAsnTrpA
766 GGTCCTGCACATGCAATTCAGGGAAAACCGTGAAAAAGTATCATG
rgSerCysThrCysAsnSerGlyLysThrValLysLysTyrHisG
811 AGGTATTACAGTTTGAGCCTGGCCACATCAAGAGGAGGGGTAGAG
luValLeuGlnPheGluProGlyHisIleLysArgArgGlyArgA
856 CTAAGACCATGGCTCTAGTTGACATCCAGTTGGATCACCATGAAC
laLysThrMetAlaLeuValAspIleGlnLeuAspHisHisGluA
901 GATGTGATTGTATCTGCAGCTCAAGACCACCTCGATAAGAGAATG
RgCysAspCysIleCysSerSerArgProProArg
946 TGCACATCCTTACATTAAGCCTGAAAGAACCTTTAGTTTAAGGAG
991 GGTGAGATAAGAGACCCTTTTCCTACCAGCAACCAAACTTACTAC
1036 TAGCCTGCAATGCAATGAACACAAGTGGTTGCTGAGTCTCAGCCT
1081 TGCTTTGTTAATGCCATGGCAAGTAGAAAGGTATATCATCAACTT
1126 CTATACCTAAGAATATAGGATTGCATTTAATAATAGTGTTTGAGG
1171 TTATATATGCACAAACACACACAGAAATATATTCATGTCTATGTG
1216 TATATAGATCAAATGTTTTTTTTGGTATATATAACCAGGTACACC
1261 AGAGCTTACATATGTTTGAGTTAGACTCTTAAAATCCTTTGCCAA
1306 AATAAGGGATGGTCAAATATATGAAACATGTCTTTAGAAAATTTA
1351 GGAGATAAATTTATTTTTAAATTTTGAAACACAAAACAATTTTGA
1396 ATCTTGCTCTCTTAAAGAAAGCATCTTGTATATTAAAAATCAAAA
1441 GATGAGGCTTTCTTACATATACATCTTAGTTGATTATTAAAAAAG
1486 GAAAAATATGGTTTCCAGAGAAAAGGCCAATACCTAAGCATTTTT
1531 TCCATGAGAAGCACTGCATACTTACCTATGTGGACTATAATAACC
1576 TGTCTCCAAAAC
Clone 30664188Ø331 includes an open reading frame from nucleotides 540 to
936.
The open reading frame encodes a polypeptide of 132 amino acids (SEQ ID N0:4).
The
encoded polypeptide is referred to herein,as the "30664188Ø331 protein" or
the
"30664188Ø331 polypeptide". The predicted amino acid sequence of the
30664188Ø331
nucleic acid sequence is shown in Table 2 (SEQ ID N0:4).
Nucleotides 50 to 1472 of clone 30664188Ø331 are 100% identical to
nucleotides
406-1828 of clone 30664188Ø99. The 132 amino acids of the clone
30664188Ø331 protein
are 100% identical to the carboxy-terminal region of the protein sequence of
30664188Ø99.
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Thus, the nucleic acids of clones 30664188Ø99 and 30664188Ø331 are
therefore related as
splice variants of a common gene.
The 30664188Ø331 protein shows similarity to human growth factor FIGF (c-fos-
induced growth factor; ptnr:SPTREMBL-ACC:043915), a member of the platelet-
derived
S growth factor/vascular endothelial growth factor (PDGFNEGF) family, and to
rat vascular
endothelial growth factor D (ptnr:SPTREMBL-ACC:03S2S1).
FCTR3 Nucleic Acids and Polypeptides
A FCTR3 (also refered to within the specification as PDGFD or murine PDGFD or
mPDGFD) nucleic acid and polypeptide according to the invention includes the
nucleic acid
and encoded polypeptide sequence shown in Table3 (SEQ ID NO: S and 6). The
FCTR3
nucleic acid sequence was identified from a murine brain library. The
predicted open reading
frame codes for a 370 amino acid long secreted protein. The FCTR3 has a
predicted
molecular weight of 42, 808 daltons and a pI of 7.53.
Protein structure analysis using PFAM and PROSITE identified the core PDGF
I S domain within the FCTR3 polypeptide sequence. Alignment of the domain is
shown in FIG.
12
TABLE 3. NUCLEOTIDE (SEQ ID N0:5) AND PROTEIN (SEQ ID N0:6) SEQUENCE
OF FCTR3
1
ATGCAACGGCTCGTTTTAGTCTCCATTCTCCTGTGCGCGAACTTTAGCTGCTATCCGGACACTTTTGCGACTCCGCAGA
G
M Q R L V L V S I L L C A N F S C Y P D T E A T P Q R
2S 81
AGCATCCATCAAAGCTTTGCGCAATGCCAACCTCAGGAGAGATGAGAGCAATCACCTCACAGACTTGTACCAGAGAGAG
G
A S I. K A L R N A N L R R D E S N H L T D L Y Q R E E
161
AGAACATTCAGGTGACAAGCAATGGCCATGTGCAGAGTCCTCGCTTCCCGAACAGCTACCCAAGGAACCTGCTTCTGAC
A
N I Q V T S N G H V Q S P R F P N S Y P R N L L L T
291
TGGTGGCTCCGTTCCCAGGAGAAAACACGGATACAACTGTCCTTTGACCATCAATTCGGACTAGAGGAAGCAGAAAATG
A
W W L R S Q E K T R I Q L S F D H Q F G L E E A E N D
321
CATTTGTAGGTATGACTTTGTGGAAGTTGAAGAAGTCTCAGAGAGCAGCACTGTTGTCAGAGGAAGATGGTGTGGCCAC
A
3 S I C R Y D F V E V E E V S E S S T V V R G R W C G H K
401
AGGAGATCCCTCCAAGGATAACGTCAAGAACAAACCAGATTAAAATCACATTTAAGTCTGATGACTACTTTGTGGCAAA
A
E I P P R I T S R T N Q I K I T F K S D D Y F V A K
4O 481
CCTGGATTCAAGATTTATTATTCATTTGTGGAAGATTTCCAACCGGAAGCAGCCTCAGAGACCAACTGGGAATCAGTCA
C
P G F K I Y Y S F V E D F Q P E A A S E T N W E S V T
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561
AAGCTCTTTCTCTGGGGTGTCCTATCACTCTCCATCAATAACGGACCCCACTCTCACTGCTGATGCCCTGGACAAAACT
G
S S F S G V S Y H S P S I T D P T L T A D A L D K T V
641
TCGCAGAATTCGATACCGTGGAAGATCTACTTAAGCACTTCAATCCAGTGTCTTGGCAAGATGATCTGGAGAATTTGTA
T
S A E F D T V E D L L K H F N P V S W Q D D L E N L Y
721
CTGGACACCCCTCATTATAGAGGCAGGTCATACCATGATCGGAAGTCCAAAGTGGACCTGGACAGGCTCAATGATGATG
T
L D T P H Y R G R S Y H D R K S K V D L D R L N D D V
1O 801
CAAGCGTTACAGTTGCACTCCCAGGAATCACTCTGTGAACCTCAGGGAGGAGCTGAAGCTGACCAATGCAGTCTTCTTC
C
K R Y S C T P R N H S V N L R E E L K L T N A V F F P
1S
881
CACGATGCCTCCTCGTGCAGCGCTGTGGTGGCAACTGTGGTTGCGGAACTGTCAACTGGAAGTCCTGCACATGCAGCTC
A
R C L L V Q R C G G N C G C G T V N W K S C T C S S
961
GGGAAGACAGTGAAGAAGTATCATGAGGTATTGAAGTTTGAGCCTGGACATTTCAAGAGAAGGGGCAAAGCTAAGAATA
T
G K T V K K Y H E V L K F E P G H F K R R G K A K N M
1041 GGCTCTTGTTGATATCCAGCTGGATCATCATGAGCGATGTGACTGTATCTGCAGCTCAAGACCACCTCGATAA
(SEQ
20 ID No: s)
N0:6).
A L V D I Q L D H H E R C D C I C S S R P P R (SEQ ID
FCTR4 Nucleic Acids and Polypeptides
A FCTR4 (also refered to within the specification as PDGFD or marine PDGFD or
2S mPDGFD) nucleic acid and polypeptide according to the invention includes
the nucleic acid
and encoded polypeptide sequence shown in Table 4 (SEQ ID NO: 7 and 8). The
FCTR4
nucleic acid sequence was identified from a marine brain library and is a
splice variant of
FCTR3 . Unlike FCTR3, however, FCTR4 lacks a significant portion of the PDGF-
like
domain.
TABLE 4. NUCLEOTIDE (SEQ ID N0:7) AND PROTEIN (SEQ ID N0:8) SEQUENCE
OF FCTR4
3S 1
ATGCAACGGCTCGTTTTAGTCTCCATTCTCCTGTGCGCGAACTTTAGCTGCTATCCGGACACTTTTGCGACTCCGCAGA
G
M Q R L V L V S I L L C A N F S C Y P D T F A T .P Q R
B1
AGCATCCATCAAAGCTTTGCGCAATGCCAACCTCAGGAGAGATGAGAGCAATCACCTCACAGACTTGTACCAGAGAGAG
G
A S I K A L R N A N L R R D E S N H L T D L Y Q R E E
161
AGAACATTCAGGTGACAAGCAATGGCCATGTGCAGAGTCCTCGCTTCCCGAACAGCTACCCAAGGAACCTGCTTCTGAC
A
N I Q V T S N G H V Q S P R F P N S Y P R N L L L T
241
TGGTGGCTCCGTTCCCAGGAGAAAACACGGATACAACTGTCCTTTGACCATCAATTCGGACTAGAGGAAGCAGAAAATG
A
4S W W L R S Q E K T R I Q L S F D H Q F G L E E A E N D
321
CATTTGTAGGTATGACTTTGTGGAAGTTGAAGAAGTCTCAGAGAGCAGCACTGTTGTCAGAGGAAGATGGTGTGGCCAC
A
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I C R Y D F V E V E E V S E S S T V V R G R W C G H K
901
AGGAGATCCCTCCAAGGATAACGTCAAGAACAAACCAGATTAAAATCACATTTAAGTCTGATGACTACTTTGTGGCAAA
A
E I P P R I T S R T N Q I K I T F K S D D Y F V A K
981
CCTGGATTCAAGATTTATTATTCATTTGTGGAAGATTTCCAACCGGAAGCAGCCTCAGAGACCAACTGGGAATCAGTCA
C
P G F K I Y Y S F V E D F Q P E A A S E T N W E S V T
561
AAGCTCTTTCTCTGGGGTGTCCTATCACTCTCCATCAATAACGGACCCCACTCTCACTGCTGATGCCCTGGACAAAACT
G
1 O S S F S G V S Y H S P S I T D P T L T A D A L D K T V
691
TCGCAGAATTCGATACCGTGGAAGATCTACTTAAGCACTTCAATCCAGTGTCTTGGCAAGATGATCTGGAGAATTTGTA
T
A E F D T V E D L L K H F N P V S W Q D D L E N L Y
IS 721
CTGGACACCCCTCATTATAGAGGCAGGTCATACCATGATCGGAAGTCCAAAGGTATTGAAGTTTGAGCCTGGACATTTC
A
L D T P H Y R G R S Y H D R K S K G I E V (SEQ ID NO: 10)
801
AGAGAAGGGGCAAAGCTAAGAATATGGCTCTTGTTGATATCCAGCTGGATCATCATGAGCGATGTGACTGTATCTGCAG
C
881 TCAAGACCACCTCGATAA (SEQ ID N0:9).
FCTRS Nucleic Acids and Polypeptides
A FCTRS (also refered to within the specification as PDGFD or human PDGFD or
hPDGFD) nucleic acid and polypeptide according to the invention includes the
nucleic acid
and encoded polypeptide sequence of clone pCR2.1-5852 2B and is shown in Table
5 ( SEQ
ID NO: 9 and 10). The FCTRS nucleic acid sequence was identified as a splice
variant of
FCTR1.
Similar to FCTR1, protein structure analysis programs PSORT , PFAM and PROSITE
predicted that FCTRS contains a characteristic signal peptide (aa 1-23), PDGF
domain (aa
272-362) and a N-linked glycosylation site (residue 276). BLASTP analysis
revealed that the
human FGTRS is most closely related to human PDGF C, PDGF B, and PDGF A (42%,
27%,
and 25% overall amino acid identity, respectively). Alignment of the core PDGF
domains of
PDGF C, PDGF B, and PDGF A with human PDGFD is presented in Fig. 12. From this
alignment it is apparent that PDGF D retains seven of eight invariant
cysteines involved in
intrachain and interchain disulphide bond with a substitution of a glycine
residue for the fifth
cysteine conserved in other sequences (Fig. 12, asterisk).
TABLE 5. NUCLEOTIDE (SEQ ID N0:9) AND PROTEIN (SEQ ID NO:10)
SEQUENCE OF FCTR5 (clone pCR2.1-S852 2B)
4O ATGCACCGGCTCATCTTGTTCTACACTCTAATCTGCGCAAACTTTTGCAGCTGTCGGGACACTTCTGCAA
CCCCGCAGAGCGCATCCATCAAAGCTTTGCGCAACGCCAACCTCAGGCGAGATGTTGACCTGGATAGGCTCAATGA
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TGATGCCAAGCGTTACAGTTGCACTCCCAGGAATTACTCGGTCAATATAAGAGAAGAGCTGAAGTTGGCCAATGTG
GTCTTCTTTCCACGTTGCCTCCTCGTGCAGCGCTGTGGAGGAAATTGTGGCTGTGGAACTGTCAACTGGAGGTCCT
GCACATGCAATTCAGGGAAAACCGTGAAAAAGTATCATGAGGTATTACAGTTTGAGCCTGGCCACATCAAGAGGAG
GGGTAGAGCTAAGACCATGGCTCTAGTTGACATCCAGTTGGATCACCATGAACGATGCGATTGTATCTGCAGCTCA
S AGACCACCTCGA (SEQ ID N0: 9).
MHRLILFYTLICANFCSCRDTSATPQSASIKALRNANLRRDVDLDRLNDDAKRYSCTPRNYSVNIREELK
LANVVFFPRCLLVQRCGGNCGCGTVNWRSCTCNSGKTVKKYHEVLQFEPGHIKRRGRAKTMALVDIQLDHHERCDC
1O ICSSRPPR SEQ ID N0: 10).
FCTR6 Nucleic Acids and Polypeptides
A FCTR6 (also refered to within the specification as PDGFD or human PDGFD or
hPDGFD) nucleic acid and polypeptide according to the invention includes the
nucleic acid
15 and encoded polypeptide sequence of clone pCR2.1-5869 4B and is shown in
Table 6 (SEQ
ID NO: 11 and 12). The FCTR6 nucleic acid sequence was identified as a splice
variant of
FCTR1.
FCTR6 contains much of the 5' end of the full length gene (FCTR1 ), but it is
spliced to
a cryptic, non-consensus splice site at the extreme 3' end of the coding
sequence. This
20 splicing introduces a STOP codon immediately downstream to the splice site.
This splice
variant contains the intact CUB domain of 30664188Ø99, but deletes the PDGF
domains,
indicating a possible regulatory function of the molecule.
Similar to FCTR1, however, protein structure analysis programs PSORT , PFAM
and
PROSITE predicted that FCTR6 contains a characteristic signal peptide (aa 1-
23), ), CUB
25 domain (aa 53-167) and a N-linked glycosylation site (residue 276). BLASTP
analysis
revealed that the human FGTRS is most closely related to human PDGF C, PDGF B,
and
PDGF A (42%, 27%, and 25% overall amino acid identity, respectively).
Alignment of the
core PDGF domains of PDGF C, PDGF B, and PDGF A with human PDGFD is presented
in
Fig. 12. From this alignment it is apparent that PDGF D retains seven of eight
invariant
30 cysteines involved in intrachain and interchain disulphide bond with a
substitution of a glycine
residue for the fifth cysteine conserved in other sequences (Fig. 12,
asterisk).
TABLE 6. NUCLEOTIDE (SEQ ID NO:11) AND PROTEIN (SEQ ID N0:12)
SEQUENCE OF FCTR6 (clone pCR2. 1- s969 4s)
3S
ATGCACCGGCTCATCTTTGTCTACACTCTAATCTGCGCAAACTTTTGCAGCTGTCGGGACACTTCTGCAACCCCGCA
GAGCGCATCCATCAAAGCTTTGCGCAACGCCAACCTCAGGCGAGATGAGAGCAATCACCTCACAGACTTGTACCGAAGA
GATGA
GACCATCCAGGTGAAAGGAAACGGCTACGTGCAGAGTCCTAGATTCCCGAACAGCTACCCCAGGAACCTGCTCCTGACA
TGGCG
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GCTTCACTCTCAGGAGAATACACGGATACAGCTAGTGTTTGACAATCAGTTTGGATTAGAGGAAGCAGAAAATGATATC
TGTAG
GTAGAGCTAAGACCATGGCTCTAGTTGACATCCAGTTGGATCACCATGAACGATGCGATTGTATCTGCAGCTCAAGACC
ACCTC
GA (SEQ ID N0: 11).
MHRLIFVYTLICANFCSCRDTSATPQSASIKALRNANLRRDESNHLTDLYRRDETIQVKGNGYVQSPRFPNSYPRNL
LLTWRLHSQENTRIQLVFDNQFGLEEAENDICR (SEQ ID N0: 12).
The similarities of the disclosed FCTRX polypeptides to previously described
BMP-1
VEG-E and PDGF polypeptides indicate a similarity of functions by the FCTRX
nucleic acids
and polypeptides of the invention. These utilities are described in more
detail below.
FCTRX nucleic acids and polypeptides may be use to induce formation of
cartilage, as
BMP-1 is also capable of inducing formation of cartilage in vivo (Wozney et
al., Science 242:
1528-1534 (1988)).
1 S An additional use for the FCTRX nucleic acids and polypeptides is in the
modulation
of collagen formation. Recombinantly expressed BMP 1 and purified procollagen
C proteinase
(PCP), a secreted metalloprotease requiring calcium and needed for cartilage
and bone
formation, are, in fact, identical. See, Kessler et al., Science 271:360-62
(1996). BMP-1
cleaves .the C-terminal propeptides of procollagen I, II, and III and its
activity is increased by
the procollagen C-endopeptidase enhancer protein. FCTRX nucleic acids and
polypeptides
may play similar roles in collagen modulation pathways.
FCTRX nucleic acids and polypeptides can also be used to stage various
cancers. For
example, bone metastases can almost universally be correlated to the morbidity
and mortality
of certain prostate cancers. For example, bone morphogenetic proteins are
implicated as
having important roles in various cancers. Overexpression of bone
morphogenetic protein
(BMP)-4 and BMP-2 mRNA has been reported in gastric cancer cell lines of
poorly
differentiated type. See, Katoh et al., J. Gastroenterol 31(1):137-9 (1996).
This observation
may have implications regarding the poor prognosis of patients with diffuse
osteoplastic bone
metastasis of gastric cancer. Additionally, osteosarcomas producing bone
morphogenetic
protein (BMP) differed in clinical features from those not producing BMP. See,
Yoshikawa et
al Cancer 56: 1682-7 (1985) They were characterized radiologically by
perpendicular
spicules, histologically by osteoblastic type cells, and clinically by an
increased serum alkaline
phosphatase level, relative resistance to preoperative chemotherapy with
Adriamycin
(doxorubicin) plus high-dose methotrexate, and a tendency to metastasize to
other bones and
the lungs.
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The relatedness of FCTRX polypeptides to VEGF- reveals uses for FCTRX nucleic
acids and polypeptides in modulating angiogenesis. Angiogenesis is a process
which
contributes to the development of new blood vessels. During angiogenesis, new
capillaries
sprout from existing vessels. See, Risau FASEB J. 9(10): 926-33 (1995); Risau
et al.,
Ann.Rev. Cell Dev Biol. 11:73-91 (1995). In adult mammals, new blood vessels
are produced
through angiogenesis. Pathological states in which angiogenesis contributes to
the appearance
and maintenance of the pathology include tumor development and growth.
Vascular
endothelial growth factor F has been reported to be involved in angiogenesis.
Vascular endothelial growth factor (VEGF) is a multifunctional cytokine
expressed
and secreted at high levels by many tumor cells in both nonhumans and humans.
See review
in Ferrara, Curr Top Microbiol Immunol 237: 1-30 (1999). VEGF exerts its
effects on the
vascular endothelium through at least two receptors that are expressed on the
cell surface. The
first is kinase insert domain-containing receptor (KDR)/fetal liver kinase 1
(Flk-1), and the
second is FLT-1 (Warren et al., J Clin Invest 95(4): 1789-97 (1995)). These
two receptors
have different affinities for VEGF and appear to have different cellular
responses. See,
Athanassiades et al., Placenta 19(7): 465-73 (1998); Li et al. Cell Res 9(1):
11-25 (1999).
FLT-1 null mice die in the embryonic stage, at about day 8.5, whereas KDR null
mice survive
through birth and retain endothelial and hematopoietic cell development.
Activation of KDR
leads to mitogenesis and to up-regulation of e-nitric oxide synthase (eNOS)
and inducible
NOS, enzymes in the nitric oxide pathway that contribute to regulation of
vasodilation and that
play a role in vascular tumor development.
It has been also been reported that VEGF acts as a survival factor for newly
formed
blood vessels. In the developing retina, for example, vascular regression in
response to
hyperoxia has been correlated with inhibition of VEGF release by glial cells.
See, Alon et al,
Nat Med 1(10): 1024-8(1995). Furthermore, administration of anti-VEGF
monoclonal
antibodies results in regression of already established tumor-associated
vasculature in
xenograft models. See, Yuan, et al., Proc Natl Acad Sci U S A 93(25): 14765-
70(1996).
Therefore, antibodies to FCTRX polypeptides may also be used to induce or
promote
regression of newly formed blood vessels.
Tumor cells additionally respond to hypoxia by secreting VEGF. This response
promotes neovascularization and consequently permits tumor growth.
Furthermore, it has
been found that several tumor cells, including hematopoietic cells (Bellamy et
al., Cancer Res
59(3): 728-33 (1999)), breast cancer cells (Speirs et al., Br J Cancer 80(5-
6): 898-903(1999)),
and Kaposi's sarcoma (Masood et al., Proc Natl Acad Sci U S A 94(3): 979-84
(1997)),
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express the KDR receptor. Such results suggest that in these tumors VEGF is
acting not only
in a paracrine fashion to stimulate angiogenesis, but also via an autocrine
mechanism as well
to stimulate proliferation and/or survival of endothelial cells, and/or
promoting survival of
tumor cells. Accordingly, modulation of angiogenesis by FCTRX antibodies, or
other
antagonists of FCTRX nucleic acid or polypeptide function, can be used in
anoxia-associated
conditions to inhibit endothelial cell proliferation, and/or tumor cells such
as hematopoietic
cells, breast cancer cells, and Kaposi's sarcoma cells.
The similarity between FCTRX polypeptides and VEGF polypeptides suggests that
FCTRX nucleic acids and their encoded polypeptides can be used to modulate
cell survival. It
has been reported that VEGF signaling is important for cell survival. Binding
of VEGF to its
receptor, VEGF receptor-2 (VEGFR-2/Flkl/KDR), is reported to induce the
formation of a
complex of VE-cadherin,13-catenin, phosphoinositide-3-OH kinase (PI3-K), and
KDR. PI3-K
in this complex activates the serine/threonine protein kinase Akt (protein
kinase B) by
phosphorylation. See, Carmeliet et al., 1999 Cell 98(2): 147-57. Activated Akt
is then thought
to be necessary and sufficient to mediate the VEGF-dependent survival signal.
See, Gerber et
al. 1998 J. Biol. Chem. 273(46): 30336-43. These findings indicate that there
is a relationship
between VEGF signaling and cell survival.
The similarity between FCTRX polypeptides and PDGF polypeptides suggests that
FCTRX nucleic acids and their encoded polypeptides can be used in various
therapeutic and
diagnostic applications. For example, FCTRX nucleic acids and their encoded
polypeptides
can be used to treat cancer, cardiovascular and fibrotic diseases and diabetic
ulcers. In
addition, FCTRX nucleic acids and their encoded polypeptides will be
therapeutically useful
for the prevention of aneurysms and the the acceleration of wound closure
through gene
therapy. Furthermore, FCTRX nucleic acids and their encoded polypeptides can
be utilized
to stimulate cellular growth.
FCTRX nucleic acids according to the invention can be used to identify various
cell
types, including cancerous cells. For example, Example 7 illustrates that
clone 30664188Ø99
(SEQ ID NO:1 ) is strongly expressed specifically in CNS cancer, lung cancer
and ovarian
cancer. It is also shown in the Examples that SEQ ID NO:1 produces a gene
product which
either persists intact in conditioned medium arising from transfecting HEK 293
cells, or is
proteolytically cleaved. Evidence presented in Example 13 suggests that the
form of the
30664188Ø99 protein (SEQ ID N0:2) that is active in the experiments reported
in the
Examples is a proteolysis product of the 30664188Ø99 protein. The activities
ascribed to
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either one or both of these substances include the ability to stimulate net
DNA synthesis as
monitored by incorporation of BrdU into DNA, proliferation of cell number, the
ability to
transform cells in culture, and the ability to induce tumor formation in vivo.
These various
activities occur in a variety of cell types.
A FCTRX nucleic acid or gene product, e.g., a nucleic acid encoding SEQ ID
N0:2 or
SEQ ID N0:4, is useful as a therapeutic agent in promoting wound healing,
neovascularization
and tissue growth, and similar tissue regeneration needs. More specifically, a
FCTRX nucleic
acid or polypeptide may be useful in treatment of anemia and leukopenia,
intestinal tract
sensitivity and baldness. Treatment of such conditions may be indicated in,
e.g., patients
having undergone radiation or chemotherapy. It is intended in such cases that
administration
of a FCTX nucleic acid or polypeptide, e.g., a polypeptide including the amino
acid sequence
of SEQ ID N0:2 or SEQ ID N0:4, or a nucleic acid sequence encoding these
polypeptides
(e.g., SEQ ID NO:1 or SEQ ID N0:3) will be controlled in dose such that any
hyperproliferative side effects are minimized.
Alternatively, in cases of tumors, such as CNS cancer and ovarian cancer, in
which
FCTRX nucleic acids is expressed at high levels, (e.g., a tumor in SEQ ID NO:1
is expressed
in high levels), it is desired to inhibit or eliminate the effects of
production of a FCTRX
nucleic acid or gene product (e.g., SEQ ID N0:2 or SEQ ID N0:4, or a nucleic
acid encoding
one of these polypeptides). For example, this may be accomplished by
administration of an
antibody directed against a polypeptide having the amino acid sequence of SEQ
ID N0:2 or
SEQ ID N0:4,or fragment thereof. In particular, the antibody can be directed
against the
active fragment p35 (see the Examples) identified herein. An alternative
example involves
identifying the putative protease implicated in the formation of p35 from p85
(see the
Examples). Administration of a substance that specifically inhibits the
activity of this
protease, but not the activity of other proteases, will be effective to
prevent formation of the
active p35 form of a FCTRX polypeptide, e.g., a clone 30664188Ø99
polypeptide.
Based on the roles of molecules related to FCTRX polypeptides and nucleic
acids,
(e.g., BMP-1 and VEGF-like polypeptides such as fallotein) in malignant
disease progression
and the gene expression profile described herein, it is foreseen that, for a
subset of human
gliomas and ovarian epithelial carcinomas, targeting of a FCTRX polypeptide
using an
antibody has an inhibitory effect on tumor growth, matrix invasion, chemo-
resistance, radio-
resistance, and metastatic dissemination. In various embodiments, the FCTRX
polypeptide is
linked to a monoclonal antibody, a humanized antibody or a fully human
antibody.
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Furthermore, based on chomosomal location analysis (See EXAMPLE 15) the PDGFD
nucleic acids localize to chromosome 11 q23-24. This chromosomal locus to D
maps is a
region of genomic instability (H. Kurahashi et al., Hum. Mol. Genet. 9, 1665-
1670 (2000))
altered in various neoplasias (A. Ferti-Passantonopoulou, A. Panani, S.
Raptis, Cancer Genet.
S Cytogenet. 51, 183-188 (1991); M. Tarkkanen et al., Genes Chromosomes Cancer
25, 323-331
(1999)) and Jacobsen's syndrome (E. Pivnick et al., J. Med. Genet. 33, 772-778
(1996)) that
might be explained in part through abnormal growth factor expression.
Jacobsen's syndrome is
marked by craniofacila abnormalities, heart defects, glandular abnormalities
and lack of brain
development (E. Pivnick et al. (1996)). Accordingly, the FCTRX nucleic acids
and
polypeptides according to the invention may be used in various diagnostic and
therpeutic
applications of these disease state.
Additionally, rearrangements resulting in amplification or deletions about the
11 q23-
241ocus have been reported in breast cancer (A. Ferti-Passantonopoulou, A.
Panani, S. Raptis,
Cancer Genet. Cytogenet. 51, 183-188 (1991); K. Shen et al., J. Surg. Oncol.
74, 100-107
(2000)), primary sarcomas, their pulmonary metastasis (M. Tarkkanen et al.
(1999)), and
myeloid leukemias (L. Michaux et al., Genes Chromosomes Cancer 29, 40-47
(2000); P.
Crossen, L. Savage, D. Heaton, M. Morrison, Cancer Genet. Cytogenet. 112, 144-
148 (1999)).
Thus, FCTRX nucleic acids polypeptides and antobodies according to the
invention may also
have diagnostic and therapeutic applications in the detection and treatment
these cancers.
A~FCTRX polypeptide can potentially block or limit the extent of tumor
neovascularization. In addition to classical modes of administration of
potential antibody
therapeutics newly developed modalities of administration may be useful. For
example, local
administration of 1311-labeled monoclonal antibody for treatment of primary
brain tumors after
surgical resection has been reported. Additionally, direct stereotactic
intracerebral injection of
monoclonal antibodies and their fragments is also being studied clinically and
pre-clinically.
Intracarotid hyperosmolar perfusion is an experimental strategy to target
primary brain
malignancy with drug conjugated human monoclonal antibodies.
Additionally, the nucleic acids of the invention, and fragments and variants
thereof,
may be used, by way of nonlimiting example, (a) to direct the biosynthesis of
the
corresponding encoded proteins, polypeptides, fragments and variants as
recombinant or
heterologous gene products, (b) as probes for detection and quantification of
the nucleic acids
disclosed herein, (c) as sequence templates for preparing antisense molecules,
and the like.
Such uses are described more fully in the following disclosure.
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Furthermore, the proteins and polypeptides of the invention, and fragments and
variants thereof, may be used, in ways that include (a) serving as an
immunogen to stimulate
the production of an anti-FCTRX antibody, (b) a capture antigen in an
immunogenic assay for
such an antibody, and (c) as a target for screening for substances that bind
to a FCTR.X
polypeptide of the,invention. These utilities and other utilities for FCTRX
nucleic acids,
polypeptides, antibodies, agonists, antagonists, and other related compounds
uses are disclosed
more fully below. In view of its strong effects in modulating cell growth, an
increase of
FCTRX polypeptide expression or activity can be used to promote cell survival.
Conversely, a
decrease in FCTRX polypeptide expression can be used to induce cell death.
FCTRX Nucleic Acids
The novel nucleic acids of the invention include those that encode a FCTRX
polypeptide or biologically active portions thereof. The nucleic acids include
nucleic acids
encoding FCTRX polypeptides that include the amino acid sequence of one or
more of SEQ
ID NOS:2, 4, 6, 8, 10 and 12. In some embodiments, a nucleic acid encoding a
polypeptide
having the amino acid sequence of one or more of SEQ ID NOS:2, 4, 6, 8, 10 and
12 includes
the nucleic acid sequence of any of SEQ ID NOS:1, 3, 5, 7, 9 and 11, or a
fragment thereof.
Additionally, a FCTRX nucleic acid of the invention includes mutant or variant
nucleic
acids of any of SEQ ID NOS:1, 3, 5, 7, 9 and 11, or a fragment thereof, any of
whose bases
may be changed from the disclosed sequence while still encoding a protein that
maintains its
FCTRX -like activities and physiological functions. The invention further
includes the
complement of the nucleic acid sequence of any of SEQ ID NOS:1, 3, 5, 7, 9 and
11,
including fragments, derivatives, analogs and homolog thereof. The invention
additionally
includes nucleic acids or nucleic acid fragments, or complements thereto,
whose structures
include chemical modifications.
A FCTRX nucleic acid of the invention can encode a mature form of a FCTRX
polypeptide. As used herein, a "mature" form of a polypeptide or protein is
the product of a
naturally occurnng polypeptide or precursor form or proprotein. The naturally
occurnng
polypeptide, precursor or proprotein includes, by way of nonlimiting example,
the full length
gene product, encoded by the corresponding gene. Alternatively, it may be
defined as the
polypeptide, precursor or proprotein encoded by an open reading frame
described herein. The
product "mature" form arises, again by way of nonlimiting example, as a result
of one or more
naturally occurnng processing steps as they may take place within the cell, or
host cell, in
CA 02386383 2002-04-04
WO 01/25437 PCT/US00/27671
which the gene product arises. Examples of such processing steps leading to a
"mature" form
of a polypeptide or protein include the cleavage of the N-terminal methionine
residue encoded
by the initiation codon of an open reading frame, or the proteolytic cleavage
of a signal
peptide or leader sequence. Thus a mature form arising from a precursor
polypeptide or
protein that has residues 1 to N, where residue 1 is the N-terminal
methionine, would have
residues 2 through N remaining after removal of the N-terminal methionine.
Alternatively, a
mature form arising from a precursor polypeptide or protein having residues 1
to N, in which
an N-terminal signal sequence from residue 1 to residue M is cleaved, would
have the residues
from residue M+1 to residue N remaining. Additionally, a "mature" protein or
fragment may
arise from a cleavage event other than removal of an initiating methionine or
removal of a
signal peptide. Further as used herein, a "mature" form of a polypeptide or
protein may arise
from a step of post-translational modification other than a proteolytic
cleavage event. Such
additional processes include, by way of non-limiting example, glycosylation,
myristylation or
phosphorylation. In general, a mature polypeptide or protein may result from
the operation of
only one of these processes, or a combination of any of them.
Also included are nucleic acid fragments sufficient for use as hybridization
probes to
identify nucleic acids encoding FCTRX polypeptides (e.g., a FCTRX mRNA
encoding SEQ
ID N0:2 or SEQ ID N0:4) and fragments for use as polymerase chain reaction
(PCR) primers
for the amplification or mutation of FCTRX nucleic acid molecules. As used
herein, the term
"nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic
DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using
nucleotide analogs, and derivatives, fragments and homologs thereof. The
nucleic acid
molecule can be single-stranded or double-stranded, but preferably is double-
stranded DNA.
"Probes" refer to nucleic acid sequences of variable length, preferably
between at least
about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt,
depending on use.
Probes are used in the detection of identical, similar, or complementary
nucleic acid
sequences. Longer length probes are usually obtained from a natural or
recombinant source
(although they may be prepared by chemical synthesis as well), are highly
specific and much
slower to hybridize than oligomers. Probes may be single- or double-stranded
and designed
to have specificity in PCR, membrane-based hybridization technologies, or
ELISA-like
technologies.
An "isolated" nucleic acid molecule is one that is separated from other
nucleic acid
molecules that are present in the natural source of the nucleic acid. Examples
of isolated
nucleic acid molecules include, but are not limited to, recombinant DNA
molecules contained
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in a vector, recombinant DNA molecules maintained in a heterologous host cell,
partially or
substantially purified nucleic acid molecules, and synthetic DNA or RNA
molecules.
Preferably, an "isolated" nucleic acid is free of sequences which naturally
flank the nucleic
acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in
the genomic DNA of
the organism from which the nucleic acid is derived. For example, in various
embodiments,
the isolated FCTRX nucleic acid molecule can contain less than about 50 kb, 25
kb, 5 kb, 4
kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally
flank the nucleic
acid molecule in genomic DNA of the cell from which the nucleic acid is
derived. Moreover,
an "isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of
other cellular material or culture medium when produced by recombinant
techniques, or of
chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid
molecule having
the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9 and 11, or a complement of
any of this
nucleotide sequence, can be isolated using standard molecular biology
techniques and the
sequence information provided herein. Using all or a portion of the nucleic
acid sequence of
any of SEQ ID NOS:1, 3, S, 7, 9 and 11 as a hybridization probe, FCTRX nucleic
acid
sequences can be isolated using standard hybridization and cloning techniques
(e.g., as
described in Sambrook et al., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2"d
Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and
Ausubel, et al.,
eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY,
1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers according
to standard
PCR amplification techniques. The nucleic acid so amplified can be cloned into
an
appropriate vector and characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to FCTRX nucleotide sequences can be prepared
by standard
synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked
nucleotide
residues, which oligonucleotide has a sufficient number of nucleotide bases to
be used in a
PCR reaction. A short oligonucleotide sequence may be based on, or designed
from, a
genomic or cDNA sequence and is used to amplify, confirm, or reveal the
presence of an
identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10
nt, 50 nt, or
100 nt in length, preferably about 15 nt to 30 nt in length. In one
embodiment, an
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oligonucleotide comprising a nucleic acid molecule less than 100 nt in length
would further
comprise at lease 6 contiguous nucleotides of any of SEQ ID NOS:1, 3, 5, 7, 9
and 11, or a
complement thereof. Oligonucleotides may be chemically synthesized and may be
used as
probes.
In another embodiment, an isolated nucleic acid molecule of the invention
comprises a
nucleic acid molecule that is a complement of the nucleotide sequence shown in
any of SEQ
ID NOS:1, 3, 5, 7, 9 and 11. In another embodiment, an isolated nucleic acid
molecule of the
invention comprises a nucleic acid molecule that is a complement of the
nucleotide sequence
shown in any of SEQ ID NOS:1, 3, 5, 7, 9 and 11, or a portion of this
nucleotide sequence. A
nucleic acid molecule that is complementary to the nucleotide sequence shown
in is one that
is sufficiently complementary to the nucleotide sequence shown in of any of
SEQ ID NOS:1,
3, S, 7, 9 and 11 that it can hydrogen bond with little or no mismatches to
the nucleotide
sequence shown in of any of SEQ ID NOS:1, 3, 5, 7, 9 and 11, thereby forming a
stable
duplex.
1 S As used herein, the term "complementary" refers to Watson-Crick or
Hoogsteen base
pairing between nucleotides units of a nucleic acid molecule, and the term
"binding" means
the physical or chemical interaction between two polypeptides or compounds or
associated
polypeptides or compounds or combinations thereof. Binding includes ionic, non-
ionic, van
der Waals, hydrophobic interactions, etc. A physical interaction can be either
direct or
indirect. Indirect interactions may be through or due to the effects of
another polypeptide or
compound. Direct binding refers to interactions that do not take place
through, or due to, the
effect of another polypeptide or compound, but instead are without other
substantial chemical
intermediates.
Moreover, the nucleic acid molecule of the invention can comprise only a
portion of
the nucleic acid sequence of any of SEQ ID NOS:1, 3, 5, 7, 9 and 11, e.g., a
fragment that can
be used as a probe or primer, or a fragment encoding a biologically active
portion of a FCTRX
polypeptide. Fragments provided herein are defined as sequences of at least 6
(contiguous)
nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to
allow for specific
hybridization in the case of nucleic acids or for specific recognition of an
epitope in the case of
amino acids, respectively, and are at most some portion less than a full
length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or
amino acid
sequence of choice. Derivatives are nucleic acid sequences or amino acid
sequences formed
from the native compounds either directly or by modification or partial
substitution. Analogs
are nucleic acid sequences or amino acid sequences that have a structure
similar to, but not
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identical to, the native compound but differs from it in respect to certain
components or side
chains. Analogs may be synthetic or from a different evolutionary origin and
may have a
similar or opposite metabolic activity compared to wild type.
Derivatives and analogs may be full length or other than full length, if the
derivative or
analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or
analogs of the nucleic acids or proteins of the invention include, but are not
limited to,
molecules comprising regions that are substantially homologous to the nucleic
acids or
proteins of the invention, in various embodiments, by at least about 70%, 80%,
85%, 90%,
95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a
nucleic acid or
amino acid sequence of identical size or when compared to an aligned sequence
in which the
alignment is done by a computer homology program known in the art, or whose
encoding
nucleic acid is capable of hybridizing to the complement of a sequence
encoding the
aforementioned proteins under stringent, moderately stringent, or low
stringent conditions.
See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons,
1 S New York, NY, 1993, and below. An exemplary program is the Gap program
(Wisconsin
Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group,
University
Research Park, Madison, WI) using the default settings, which uses the
algorithm of Smith and
Waterman (Adv. Appl. Math., 1981, 2: 482-489, which is incorporated herein by
reference in
its entirety).
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the
nucleotide level or
amino acid level as discussed above. Homologous nucleotide sequences encode
those
sequences coding for isoforms of a FCTRX polypeptide. Isoforms can be
expressed in
different tissues of the same organism as a result of, for example,
alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the present
invention,
homologous nucleotide sequences include nucleotide sequences encoding for a
FCTRX
polypeptide of species other than humans, including, but not limited to,
mammals, and thus
can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other
organisms. Homologous
nucleotide sequences also include, but are not limited to, naturally occurnng
allelic variations
and mutations of the nucleotide sequences set forth herein. A homologous
nucleotide
sequence does not, however, include the nucleotide sequence encoding human
FCTRX
protein. Homologous nucleic acid sequences include those nucleic acid
sequences that encode
conservative amino acid substitutions (see below) in any of SEQ ID NOS:2, 4,
6, 8, 10 and 12
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as well as a polypeptide having FCTRX activity. Biological activities of the
FCTRX proteins
are described herein.
As used herein, "identical" residues correspond to those residues in a
comparison
between two sequences where the equivalent nucleotide base or amino acid
residue in an
alignment of two sequences is the same residue. Residues are alternatively
described as
"similar" or "positive" when the comparisons between two sequences in an
alignment show
that residues in an equivalent position in a comparison are either the same
amino acid or a
conserved amino acid as defined below.
The nucleotide sequence determined from the cloning of the human FCTRX gene
allows for the generation of probes and primers designed for use in
identifying the cell types
disclosed and/or cloning FCTRX protein homologues in other cell types, e.g.,
from other
tissues, as well as FCTRX homologues from other mammals. The probe/primer
typically
comprises a substantially purified oligonucleotide. The oligonucleotide
typically comprises a
region of nucleotide sequence that hybridizes under stringent conditions to at
least about 12,
25, 50, 100, 150, 200, 250, 300, 350 or 400 or more consecutive sense strand
nucleotide
sequence of SEQ ID NOS:1, 3, 5, 7, 9 and 11; or an anti-sense strand
nucleotide sequence of
SEQ ID NOS:1, 3, 5, 7, 9 and 11; or of a naturally occurring mutant of SEQ ID
NOS:1, 3, 5, 7,
9 and 11.
Probes based on a human FCTRX nucleotide sequence can be used to detect
transcripts
or genomic sequences encoding the same or homologous proteins. In various
embodiments,
the probe further comprises a label group attached thereto, e.g., the label
group can be a
radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such
probes can
be used as a part of a diagnostic test kit for identifying cells or tissue
which misexpress a
FCTRX protein, such as by measuring a level of a FCTRX protein-encoding
nucleic acid in a
sample of cells from a subject e.g., detecting mRNA levels or determining
whether a genomic
FCTRX gene has been mutated or deleted.
"A polypeptide having a biologically active portion of a FCTRX" refers to
polypeptides exhibiting activity similar, but not necessarily identical to, an
activity of a
polypeptide of the present invention, including mature forms, as measured in a
particular
biological assay, with or without dose dependency. A nucleic acid fragment
encoding a
"biologically active portion of a FCTRX polypeptide" can be prepared by
isolating a portion of
SEQ ID NOS:1 or 3 that encodes a polypeptide having a FCTRX polypeptide
biological
activity such as those disclosed herein, expressing the encoded portion of
FCTRX protein
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(e.g., by recombinant expression in vitro) and assessing the activity of the
encoded portion of
the FCTIRX polypeptide.
FCTRX Variants
The invention further encompasses nucleic acid molecules that differ from the
disclosed FCT1RX nucleotide sequences due to degeneracy of the genetic code.
These nucleic
acids thus encode the same FCT1RX protein as that encoded by the nucleotide
sequence shown
in SEQ ID NOS:1, 3, 5, 7, 9 and 11. In another embodiment, an isolated nucleic
acid
molecule of the invention has a nucleotide sequence encoding a protein having
an amino acid
sequence shown in any of SEQ ID NOS:2, 4, 6, 8, 10 and 12 .
In addition to the human FCTItX nucleotide sequence shown in any of SEQ ID
NOS:1,
3, 5, 7, 9 and 11, it will be appreciated by those skilled in the art that DNA
sequence
polymorphisms that lead to changes in the amino acid sequences of a FCTRX may
exist within
a population (e.g., the human population). Such genetic polymorphism in the
FCT1ZX gene
may exist among individuals within a population due to natural allelic
variation. As used
herein, the terms "gene" and "recombinant gene" refer to nucleic acid
molecules comprising an
open reading frame encoding a FCT1RX protein, preferably a mammalian FCTItX
protein.
Such natural allelic variations can typically result in 1-5% variance in the
nucleotide sequence
of the FCT1RX gene. Any and all such nucleotide variations and resulting amino
acid
polymorphisms in the FCTRX gene that are the result of natural allelic
variation and that do
not alter the functional activity of the FCT12X polypeptide are intended to be
within the scope
of the invention.
Moreover, nucleic acid molecules encoding FCTRX proteins from other species,
and
thus that have a nucleotide sequence that differs from the human sequence of
any of SEQ ID
NOS:1, 3, 5, 7, 9 and 11, are intended to be within the scope of the
invention. Nucleic acid
molecules corresponding to natural allelic variants and homologues of the
FCTRX cDNAs of
the invention can be isolated based on their homology to the human FCT1RX
nucleic acids
disclosed herein using the human cDNAs, or a portion thereof, as a
hybridization probe
according to standard hybridization techniques under stringent hybridization
conditions.
In another embodiment, an isolated nucleic acid molecule of the invention is
at least 6
nucleotides in length and hybridizes under stringent conditions to the nucleic
acid molecule
comprising the nucleotide sequence of any of SEQ ID NOS:1, 3, 5, 7, 9 and 11.
In another
embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750
nucleotides iri length.
In another embodiment, an isolated nucleic acid molecule of the invention
hybridizes to the
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coding region. As used herein, the term "hybridizes under stringent
conditions" is intended to
describe conditions for hybridization and washing under which nucleotide
sequences that
exceed a minimum degree of similarity to each other typically remain
hybridized to each
other. For example, depending on the degree of stringency imposed, nucleotide
sequences at
least about 60% similar to each other may hybridize.
As used herein, the phrase "stringent hybridization conditions" refers to
conditions
under which a probe, primer or oligonucleotide will hybridize to a target
sequence; optimally
the probe will hybridize to no other sequences, and more generally will not
hybridize to
sequences below a specified degree of similarity to the probe. Stringent
conditions are
sequence-dependent and will be different in different circumstances. Longer
sequences
hybridize specifically at higher temperatures than shorter sequences.
Generally, stringent
conditions are selected to be about 5°C lower than the thermal melting
point (Tm) for the
specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined
ionic strength, pH and nucleic acid concentration) at which SO% of the probes
complementary
to the target sequence hybridize to the target sequence at equilibrium. Since
the target
sequences are generally present at excess, at Tm, 50% of the probes are
occupied at
equilibrium. Typically, stringent conditions will be those in which the salt
concentration is
less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or
other salts) at
pH 7.0 to 8.3 and the temperature is at least about 30°C for short
probes, primers or
oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for
longer probes, primers and
oligonucleotides. Stringent conditions may also be achieved with the addition
of destabilizing
agents, such as formamide.
Stringent conditions such as described above are known to those skilled in the
art and
can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,
N.Y.
(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at
least about 65%,
70%, 75%, 85%, 90%, 95%, 98%, or 99% identical to each other typically remain
hybridized
to each other. A non-limiting example of stringent hybridization conditions is
hybridization in
a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA,
0.02% PVP,
0.02% Ficoll, 0.02% BSA, and S00 mg/ml denatured salmon sperm DNA at
65°C. This
hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at
50°C. An
isolated nucleic acid molecule of the invention that hybridizes under
stringent conditions to the
sequence of any of SEQ ID NOS:1, 3, 5, 7, 9 and 11 corresponds to a naturally
occurnng
nucleic acid molecule. As used herein, a "naturally-occurnng" nucleic acid
molecule refers to
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an RNA or DNA molecule having a nucleotide sequence that occurs in nature
(e.g., encodes a
natural protein).
Homologs (i.e., nucleic acids encoding FCTRX proteins derived from species
other
than human) or other related sequences (e.g., paralogs) can be obtained by
low, moderate or
high stringency hybridization with all or a portion of the particular human
sequence as a probe
using methods well known in the art for nucleic acid hybridization and
cloning.
In a second embodiment, a nucleic acid sequence that is hybridizable to the
nucleic
acid molecule comprising the nucleotide sequence of any of SEQ ID NOS:l, 3, S,
7, 9 and 11,
or fragments, analogs or derivatives thereof, under conditions of moderate
stringency is
provided. A non-limiting example of moderate stringency hybridization
conditions are
hybridization in 6X SSC, SX Denhardt's solution, 0.5% SDS and 100 mg/ml
denatured salmon
sperm DNA at 55°C, followed by one or more washes in 1X SSC, 0.1% SDS
at 37°C. Other
conditions of moderate stringency that may be used are well known in the art.
See, e.g.,
Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John
Wiley &
Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL,
Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule
comprising the nucleotide sequence of any of SEQ ID NOS:1,.3, 5, 7, 9 and 11,
or fragments,
analogs or derivatives thereof, under conditions of low stringency, is
provided. A non-limiting
example of low stringency hybridization conditions are hybridization in 35%
formamide, SX
SSC, 50 mM Tris-HCl (pH 7.5), S mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA,
100
mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C,
followed by one
or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at
50°C.
Other conditions of low stringency that may be used are well known in the art
(e.g., as
employed for cross-species hybridizations). See, e.g., Ausubel et al. (eds.),
1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990,
GENE
TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and
Weinberg, 1981, Proc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of a FCTRX nucleotide
sequence,
e.g., a gene sequence, that may exist in the population, the skilled artisan
will further
appreciate that changes can be introduced by mutation into the nucleotide
sequence of any of
SEQ ID NOS:1, 3, 5, 7, 9 and 11, thereby leading to changes in the amino acid
sequence of the
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encoded FCTRX protein, without altering the functional ability of the FCTRX
protein. For
example, nucleotide substitutions leading to amino acid substitutions at "non-
essential" amino
acid residues can be made in the sequence of any of SEQ ID NOS:1, 3, 5, 7, 9
and 11. A
"non-essential" amino acid residue is a residue at a position in the sequence
that can be altered
from the wild-type sequence of the FCTRX polypeptide without altering the
biological
activity, whereas an "essential" amino acid residue is a residue at a position
that is required for
biological activity. For example, amino acid residues that are conserved among
members of a
family of FCTRX proteins, of which the FCTRX proteins of the present invention
are
members, are predicted to be particularly unamenable to alteration.
For example, a FCTRX protein according to the present invention can contain at
least
one domain that is a typically conserved region in a FCTRX protein family
member. As such,
these conserved domains are not likely to be amenable to mutation. Other amino
acid
residues, however, (e.g., those that are poorly conserved among members of the
FCTRX
protein family) may not be as essential for activity and thus are more likely
to be amenable to
alteration.
Another aspect of the invention pertains to nucleic acid molecules encoding
FCTRX
proteins that contain changes in amino acid residues relative to the amino
acid sequence of
SEQ IDN0:2 or SEQ ID N0:4 that are not essential for activity. In one
embodiment, the
isolated nucleic acid molecule comprises a nucleotide sequence encoding a
protein, wherein
the protein comprises an amino acid sequence at least about 75% similar to the
amino acid
sequence of any of SEQ ID NOS:2, 4, 6, 8, 10 and 12. Preferably, the protein
encoded by the
nucleic acid is at least about 80% identical to any of SEQ ID NOS:2, 4, 6, 8,
10 and 12, more
preferably at least about 90%, 95%, 98%, and most preferably at least about
99% identical to
SEQ ID N0:2.
An isolated nucleic acid molecule encoding a protein homologous to the protein
of any
of SEQ ID NOS:2, 4, 6, 8, 10 and 12 can be created by introducing one or more
nucleotide
substitutions, additions or deletions into the corresponding nucleotide
sequence, such that one
or more amino acid substitutions, additions or deletions are introduced into
the encoded
protein.
Mutations can be introduced into SEQ ID NOS:1, 3, 5, 7, 9 and 11 by standard
techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
Preferably,
conservative amino acid substitutions are made at one or more predicted non-
essential amino
acid residues. A "conservative amino acid substitution" is one in which the
amino acid residue
is replaced with an amino acid residue having a similar side chain. Families
of amino acid
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residues having similar side chains have been defined in the art. Certain
amino acids have side
chains with more than one classifiable characteristic. These families include
amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine,
threonine, tyrosine, tryptophan, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), beta-
branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino
acid residue in a
FCTRX polypeptide is replaced with another amino acid residue from the same
side chain
family. Alternatively, in another embodiment, mutations can be introduced
randomly along all
or part of a FCTRX coding sequence, such as by saturation mutagenesis, and the
resultant
mutants can be screened for FCTRX polypeptide biological activity to identify
mutants that
retain activity. Following mutagenesis of SEQ ID NOS:1, 3, 5, 7, 9 and 11 the
encoded
protein can be expressed by any recombinant technology known in the art and
the activity of
the protein can be determined.
The relatedness of amino acid families may also be determined based on side
chain
interactions. Substituted amino acids may be fully conserved "strong" residues
or fully
conserved "weak" residues. The "strong" group of conserved amino acid residues
may be any
one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW,
wherein the single letter amino acid codes are grouped by those amino acids
that may be
substituted for each other. Likewise, the "weak" group of conserved residues
may be any one
of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK,
VLIM, HFY.
In one embodiment, a mutant FCTRX polypeptide can be assayed for (1) the
ability to
form protein:protein interactions-with other FCTRX proteins, other cell-
surface proteins, or
biologically active portions thereof, (2) complex formation between a mutant
FCTRX protein
and a FCTRX receptor; (3) the ability of a mutant FCTRX protein to bind to an
intracellular
target protein or biologically active portion thereof; (e.g., avidin
proteins); (4) the ability to
bind BR.A protein; or (5) the ability to specifically bind an antibody to a
FCTRX polypeptide.
In other embodiments, a mutant FCTRX protein can be assayed for its ability to
induce
tumor formation, or to transform cells, such as NIH 3T3 cells, as described in
the Examples
below.
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Antisense FCTRX Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules
that are hybridizable to or complementary to a FCT1RX nucleic acid, e.g., the
antisense nucleic
acid can be complementary to a nucleic acid molecule comprising the nucleotide
sequence of
SEQ ID NOS:1, 3, S, 7, 9 and 11, or fragments, analogs or derivatives thereof.
An "antisense"
nucleic acid includes a nucleotide sequence that is complementary to a "sense"
nucleic acid
encoding a protein, e.g., complementary to the coding strand of a double-
stranded cDNA
molecule or complementary to an mRNA sequence. In specific aspects, antisense
nucleic acid
molecules are provided that comprise a sequence complementary to at least
about 10, 25, S0,
100, 250 or 500 nucleotides or an entire FCTRX coding strand, or to only a
portion thereof.
Nucleic acid molecules encoding fragments, homologs, derivatives and analogs
of a FCTRX
protein of any of SEQ ID NOS:2, 4, 6, 8, 10 and 12 or antisense nucleic acids
complementary
to a FCTRX nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7, 9 and 11 are
additionally
provided.
1 S In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding
region" of the coding strand of a nucleotide sequence encoding a FCTRX
polypeptide. The
term "coding region" refers to the region of the nucleotide sequence
comprising codons which
are translated into amino acid residues (e.g., the protein coding region of a
FCTRX
polypeptide that corresponds to any of SEQ ID NOS:2, 4, 6, 8, 10 and 12). In
another
embodiment, the antisense nucleic acid molecule is antisense to a "noncoding
region" of the
coding strand of a nucleotide sequence encoding a FCT1RX polypeptide. The term
"noncoding
region" refers to 5' and 3' sequences which flank the coding region that are
not translated into
amino acids (i.e., also referred to as 5' and 3' untranslated regions).
The FCTRX coding strand sequences disclosed herein (e.g., SEQ ID NOS:1, 3, 5,
7, 9
and 11 ) allow for antisense nucleic acids to be designed according to the
rules of Watson and
Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be
complementary
to the entire coding region of a FCT1RX mRNA. Alternatively, the antisense
nucleic acid
molecule can be an oligonucleotide that is antisense to only a portion of the
coding or
noncoding region of a FCTRX mRNA. For example, the antisense oligonucleotide
can be
complementary to the region surrounding the translation start site of the
FCT1RX mRNA. An
antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30,
35, 40, 45 or 50
nucleotides in length.
An antisense nucleic acid of the invention can be constructed using chemical
synthesis
or enzymatic ligation reactions using procedures known in the art. For
example, an antisense
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nucleic acid (e.g., an antisense oligonucleotide) can be chemically
synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to increase
the biological
stability of the molecules or to increase the physical stability of the duplex
formed between the
antisense and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted
nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense
nucleic
acid include: 5-fluorouracil, S-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-
2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-
dimethylguanine, 2-
methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine, 7-
methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-
mannosylqueosine, S'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-
N6-
isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine, 2-
thiocytosine, S-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil, uracil-5-
oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil, 3-(3-amino-3-
N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the
antisense
nucleic acid can be produced biologically using an expression vector into
which a nucleic acid
has been subcloned in an antisense orientation (i.e., RNA transcribed from the
inserted nucleic
acid will be of an antisense orientation to a target nucleic acid of interest,
described further in
the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated in situ such that they hybridize with or bind to cellular
mRNA and/or
genomic DNA encoding a FCTRX protein to thereby inhibit expression of the
protein, e.g., by
inhibiting transcription and/or translation. The hybridization can be by
conventional
nucleotide complementarity to form a stable duplex, or, for example, in the
case of an
antisense nucleic acid molecule that binds to DNA duplexes, through specific
interactions in
the major groove of the double helix. An example of a route of administration
of antisense
nucleic acid molecules of the invention includes direct injection at a tissue
site. Alternatively,
antisense nucleic acid molecules can be modified to target selected cells and
then administered
systemically. For example, for systemic administration, antisense molecules
can be modified
such that they specifically bind to receptors or antigens expressed on a
selected cell surface,
e.g., by linking the antisense nucleic acid molecules to peptides or
antibodies that bind to cell
surface receptors or antigens. The antisense nucleic acid molecules can also
be delivered to
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cells using the vectors described herein. To achieve sufficient intracellular
concentrations of
antisense molecules, vector constructs in which the antisense nucleic acid
molecule is placed
under the control of a strong pol II or pol III promoter are generally
preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is an
S a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms
specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
(3-units, the
strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res
15: 6625-6641).
The antisense nucleic acid molecule can also comprise a 2'-O-
methylribonucleotide (moue et
al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue
(moue et al.
(1987) FEBSLett 215: 327-330).
Such modifications include, by way of nonlimiting example, modified bases, and
nucleic acids whose sugar phosphate backbones are modified or derivatized.
These
modifications are carned out at least in part to enhance the chemical
stability of the modified
nucleic acid, such that they may be used, for example, as antisense binding
nucleic acids in
therapeutic applications in a subject.
Also within the invention is a FCTRX ribozymes. Ribozymes are catalytic RNA
molecules with ribonuclease activity that are capable of cleaving a single-
stranded nucleic
acid, such as a FCTRX mRNA, to which they have a complementary region. Thus,
ribozymes
(e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature
334:585-
591 )) can be used to catalytically cleave the FCTRX mRNA transcripts to
thereby inhibit
translation of the FCTRX mRNA. A ribozyme having specificity for a FCTRX-
encoding
nucleic acid can be designed based upon the nucleotide sequence of a FCTRX
nucleic acid
disclosed herein (i.e., SEQ ID NOS:1, 3, 5, 7, 9 and 11). For example, a
derivative of a
Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence
of the
active site is complementary to the nucleotide sequence to be cleaved in a
FCTRX-encoding
mRNA. See, e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech et al. U.S.
Pat. No.
5,116,742. Alternatively, a FCTRX mRNA can be used to select a catalytic RNA
having a
specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel
et al., (1993)
Science 261:1411-1418.
Alternatively, FCTRX gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of a FCTRX gene (e.g., the
FCTRX gene
promoter and/or enhancers) to form triple helical structures that prevent
transcription of the
FCTRX gene in target cells. See generally, Helene. (1991) Anticancer Drug Des.
6: 569-84;
Helene. et al. (1992) Ann. N Y. Acad. Sci. 660:27-36; and Maher (1992)
Bioassays 14: 807-15.
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In various embodiments, the FCTRX nucleic acids can be modified at the base
moiety,
sugar moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility
of the molecule. For example, the deoxyribosephosphate backbone of the nucleic
acids can be
modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med
Chem 4: 5-
23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to
nucleic acid mimics,
e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a
pseudopeptide backbone and only the four natural nucleobases are retained. The
neutral
backbone of PNAs has been shown to allow for specific hybridization to DNA and
RNA under
conditions of low ionic strength. The synthesis of PNA oligomers can be
performed using
standard solid phase peptide synthesis protocols as described in Hyrup et al.
(1996) above;
Perry-O'Keefe et al. (1996) Proc. Nat. Acad. Sci. (USA) 93: 14670-675.
PNAs based on FCTRX nucleic acids can be used in therapeutic and diagnostic
applications. For example, PNAs can be used as antisense or antigene agents
for sequence
specific modulation of gene expression by, e.g., inducing transcription or
translation arrest or
inhibiting replication. PNA based on FCTRX nucleic acids can also be used,
e.g., in the
analysis of single base pair mutations in a gene by, e.g., PNA directed PCR
clamping; as
artificial restriction enzymes when used in combination with other enzymes,
e.g., S 1 nucleases
(Hyrup B. (1996) above); or as probes or primers for DNA sequence and
hybridization (Hyrup
et al. (1996), above; Perry-O'Keefe (1996), above).
In a further embodiment, PNAs of FCTRX nucleic acids can be modified, e.g., to
enhance their stability or cellular uptake, by attaching lipophilic or other
helper groups to
PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other
techniques
of drug delivery known in the art. For example, PNA-DNA chimeras of the
nucleic acids can
be generated that may combine the advantageous properties of PNA and DNA. Such
chimeras
allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact
with the
DNA portion while the PNA portion would provide high binding affinity and
specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths selected
in terms of
base stacking, number of bonds between the nucleobases, and orientation (Hyrup
(1996)
above). The synthesis of PNA-DNA chimeras can be performed as described in
Hyrup (1996)
above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA
chain can be
synthesized on a solid support using standard phosphoramidite coupling
chemistry, and
modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-
thymidine
phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al.
(1989)
Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner
to
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produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn
et al. (1996)
above). Alternatively, chimeric molecules can be synthesized with a 5' DNA
segment and a 3'
PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
In other embodiments, a FCTRX nucleic acid or antisense nucleic acid may
include
other appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or
agents facilitating transport across the cell membrane (see, e.g., Letsinger
et al., 1989, Proc.
Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad.
Sci. 84:648-
652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g.,
PCT Publication
No. W089/10134). In addition, oligonucleotides can be modified with
hybridization triggered
cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or
intercalating agents.
(See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, the
oligonucleotide may be
conjugated to another molecule, e.g., a peptide, a hybridization triggered
cross-linking agent, a
transport agent, a hybridization-triggered cleavage agent, etc.
FCTRX Polypeptides
A FCTRX polypeptide of the invention includes a protein whose sequence is
provided
in SEQ ID N0:2 or 4 . The invention also includes a mature form of a FCT1ZX
polypeptide, as
well as a mutant or variant form of a FCTRX polypeptide. In some embodiments,
a mutant or
variant FCT1RX includes a protein in which any residues may be changed from
the
corresponding residue shown in FIG. 1, while still encoding a protein that
maintains its
FCTRX-like activities and physiological functions, or a functional fragment
thereof. The
invention includes the polypeptides encoded by the variant FCTRX nucleic acids
described
above. In the mutant or variant protein, up to 20% or more of the residues may
be so changed.
In general, a FCTRX polypeptide variant that preserves FCTItX function
includes any
FCT1ZX polypeptide variant in which residues at a particular position in the
sequence have
been substituted by other amino acids. A FCT1RX variant polypeptide also
includes a FCT1RX
polypeptide in which an additional residue or residues has been inserted
between two residues
of the parent protein as well as a protein in which one or more residues have
been deleted from
a reference FCTRX polypeptide sequence (e.g., SEQ ID N0:2 or SEQ ID N0:4, or a
mature
form of SEQ ID N0:2 or SEQ ID N0:4). Thus, any amino acid substitution,
insertion, or
deletion with respect to a reference FCT1RX polypeptide sequence (e.g., SEQ ID
N0:2 or SEQ
ID N0:4, or a mature form of SEQ ID N0:2 or SEQ ID N0:4) is encompassed by the
invention. In some embodiments, a mutant or variant proteins may include one
or more
substitutions, insertions, or deletions with respect to a reference FCT12X
sequence.
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The invention also includes isolated FCTRX proteins, and biologically active
portions
thereof, or derivatives, fragments, analogs or homologs thereof. Also provided
are
polypeptide fragments suitable for use as immunogens to raise anti-FCTRX
antibodies. In one
embodiment, native FCTRX proteins can be isolated from cells or tissue sources
by an
appropriate purification scheme using standard protein purification
techniques. In another
embodiment, FCTRX proteins are produced by recombinant DNA techniques.
Alternative to
recombinant expression, a FCTRX protein or polypeptide can be synthesized
chemically using
standard peptide synthesis techniques.
An "isolated" or "purified" protein or biologically active portion thereof is
substantially
free of cellular material or other contaminating proteins from the cell or
tissue source from
which the FCTRX protein is derived, or substantially free from chemical
precursors or other
chemicals when chemically synthesized. The language "substantially free of
cellular material"
includes preparations of a FCTRX protein in which the protein is separated
from cellular
components of the cells from which it is isolated or recombinantly produced.
In one
1 S embodiment, the language "substantially free of cellular material"
includes preparations of a
FCTRX protein having less than about 30% (by dry weight) of non-FCTRX protein
(also
referred to herein as a "contaminating protein"), more preferably less than
about 20% of non-
FCTRX protein, still more preferably less than about 10% of non-FCTRX protein,
and most
preferably less than about 5% non-FCTRX protein. When the FCTRX protein or
biologically
active portion thereof is recombinantly produced, it is also preferably
substantially free of
culture medium, i.e., culture medium represents less than about 20%, more
preferably less
than about 10%, and most preferably less than about 5% of the volume of the
protein
preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of a FCTRX protein in which the protein is separated from
chemical precursors
or other chemicals that are involved in the synthesis of the protein. In one
embodiment, the
language "substantially free of chemical precursors or other chemicals"
includes preparations
of a FCTRX protein having less than about 30% (by dry weight) of chemical
precursors or non
FCTRX polypeptides, more preferably less than about 20% chemical precursors or
non-
FCTRX polypeptides, still more preferably less than about 10% chemical
precursors or non-
FCTRX polypeptides, and most preferably less than about S% chemical precursors
or non-
FCTRX polypeptides.
Biologically active portions of a FCTRX protein include peptides comprising
amino
acid sequences sufficiently homologous to or derived from the amino acid
sequence of the
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FCTRX protein, e.g., the amino acid sequence shown in SEQ ID N0:2 that include
fewer
amino acids than the full length FCT1RX proteins, and exhibit at least one
activity of a FCT1RX
protein. Typically, biologically active portions comprise a domain or motif
with at least one
activity of the FCTRX protein. A biologically active portion of a FCTRX
protein can be a
S polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in
length.
A biologically active portion of a FCTRX of the present invention may contain
at least
one of the above-identified domains conserved among the FCT1RX family of
proteins.
Moreover, other biologically active portions, in which other regions of the
protein are deleted,
can be prepared by recombinant techniques and evaluated for one or more of the
functional
activities of a native FCTIRX protein.
In some embodiments, the FCTRX protein is substantially homologous to any of
SEQ
ID NOS:2, 4, 6, 8, 10 and 12 and retains the functional activity of the
protein of any of SEQ
ID NOS:2, 4, 6, 8, 10 and 12, yet differs in amino acid sequence due to
natural allelic variation
or mutagenesis, as described in detail below. Accordingly, in another
embodiment, the
FCTRX protein is a protein that comprises an amino acid sequence at least
about 45%
homologous, and more preferably about 55, 65, 70, 75, 80, 85, 90, 95, 98 or
even 99%
homologous to the amino acid sequence of any of SEQ ID NOS:2, 4, 6, 8, 10 and
12 and
retains the functional activity of the FCT1RX proteins of the corresponding
polypeptide having
the sequence of SEQ ID NOS:2, 4, 6, 8, 10 and 12.
Determining Homology Between Two Or More Sequences
To determine the percent homology of two amino acid sequences or of two
nucleic
acids, the sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced
in either of the sequences being compared for optimal alignment between the
sequences). The
amino acid residues or nucleotides at corresponding amino acid positions or
nucleotide
positions are then compared. When a position in the first sequence is occupied
by the same
amino acid residue or nucleotide as the corresponding position in the second
sequence, then
the molecules are homologous at that position (i.e., as used herein amino acid
or nucleic acid
"homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity
between two sequences. The homology may be determined using computer programs
known
in the art, such as GAP software provided in the GCG program package. See,
Needleman and
Wunsch 1970 JMoI Biol 48: 443-453. Using GCG GAP software with the following
settings
for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP
extension
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penalty of 0.3, the coding region of the analogous nucleic acid sequences
referred to above
exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%,
95%, 98%, or
99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS:1,
3, 5, 7, 9
and 11. Equivalent software procedures for determining the extent of sequence
identity are
widely known in the art may be used in the present context.
The term "sequence identity" refers to the degree to which two polynucleotide
or
polypeptide sequences are identical on a residue-by-residue basis over a
particular region of
comparison. The term "percentage of sequence identity" is calculated by
comparing two
optimally aligned sequences over that region of comparison, determining the
number of
positions at which the identical nucleic acid base (e.g., A, T or U, C, G, or
I, in the case of
nucleic acids) occurs in both sequences to yield the number of matched
positions, dividing the
number of matched positions by the total number of positions in the region of
comparison (i.e.,
the window size), and multiplying the result by 100 to yield the percentage of
sequence
identity. The term "substantial identity" as used herein denotes a
characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a sequence that
has at least 80
percent sequence identity, preferably at least 85 percent identity and often
90 to 95 percent
sequence identity, more usually at least 99 percent sequence identity as
compared to a
reference sequence over a comparison region. The term "percentage of positive
residues" is
calculated by comparing two optimally aligned sequences over that region of
comparison,
determining the number of positions at which the identical and conservative
amino acid
substitutions, as defined above, occur in both sequences to yield the number
of matched
positions, dividing the number of matched positions by the total number of
positions in the
region of comparison (i.e., the window size), and multiplying the result by
100 to yield the
percentage of positive residues.
Chimeric And Fusion FCTRX Proteins
The invention also provides FCTRX chimeric or fusion proteins. As used herein,
a
FCTRX "chimeric protein" or "fusion protein" includes a FCTRX polypeptide
operatively
linked to a non-FCTRX polypeptide. A "FCTRX polypeptide" refers to a
polypeptide having
an amino acid sequence corresponding to a FCTRX polypeptide, or a fragment,
variant or
derivative thereof, whereas a "non-FCTRX polypeptide" refers to a polypeptide
having an
amino acid sequence corresponding to a protein that is not substantially
homologous to the
FCTRX protein, e.g., a protein that is different from the FCTRX protein and
that is derived
from the same or a different organism. Thus, within a FCTRX fusion protein,
the FCTRX
polypeptide can correspond to all or a portion of a FCTRX protein. In one
embodiment, a
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FCTRX fusion protein comprises at least one biologically active portion of a
FCTRX protein.
In another embodiment, a FCTRX fusion protein comprises at least two
biologically active
portions of a FCTRX protein. Within the fusion protein, the term "operatively
linked" is
intended to indicate that the FCTRX polypeptide and the non-FCTRX polypeptide
are fused
in-frame to each other. The non-FCTRX polypeptide can be fused to the N-
terminus or C-
terminus of the FCTRX polypeptide.
For example, in one embodiment a FCTRX fusion protein comprises a FCTRX
polypeptide operably linked to the extracellular domain of a second protein.
Such fusion
proteins can be further utilized in screening assays for compounds that
modulate FCTRX
activity (such assays are described in detail below).
In another embodiment, the fusion protein is a GST-FCTRX fusion protein in
which
the FCTRX sequences are fused to the C-terminus of the GST (i.e., glutathione
S-transferase)
sequences. Such fusion proteins can facilitate the purification of recombinant
FCTRX.
In yet another embodiment, the fusion protein is a FCTRX protein containing a
heterologous signal sequence at its N-terminus. For example, the native FCTRX
signal
sequence can be removed and replaced with a signal sequence from another
protein. In certain
host cells (e.g., mammalian host cells), expression and/or secretion of the
FCTRX can be
increased through use of a heterologous signal sequence.
In a further embodiment, the fusion protein is a FCTRX-immunoglobulin fusion
protein in which the FCTRX sequences comprising one or more domains are fused
to
sequences derived from a member of the immunoglobulin protein family. The
FCTRX-
immunoglobulin fusion proteins of the invention can be incorporated into
pharmaceutical
compositions and administered to a subject to inhibit an interaction between a
FCTRX ligand
and a FCTRX protein on the surface of a cell, to thereby suppress FCTRX-
mediated signal
transduction in vivo. In one example, a contemplated FCTRX ligand of the
invention is a
FCTRX receptor. The FCTRX-immunoglobulin fusion proteins can be used to
modulate the
bioavailability of a FCTRX cognate ligand. Inhibition of the FCTRX
ligand/FCTRX
interaction may be useful therapeutically for both the treatment of
proliferative and
differentiative disorders, as well as modulating (e.g., promoting or
inhibiting) cell survival.
Moreover, the FCTRX-immunoglobulin fusion proteins of the invention can be
used as
immunogens to produce anti-FCTRX antibodies in a subject, to purify FCTRX
ligands, and in
screening assays to identify molecules that inhibit the interaction of a FCTRX
with a FCTRX
ligand. A FCTRX chimeric or fusion protein of the invention can be produced by
standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
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polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation, restriction
enzyme digestion to provide for appropriate termini, filling-in of cohesive
ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and enzymatic
ligation. In
another embodiment, the fusion gene can be synthesized by conventional
techniques including
automated DNA synthesizers. Alternatively, PCR amplification of gene fragments
can be
carried out using anchor primers that give rise to complementary overhangs
between two
consecutive gene fragments that can subsequently be annealed and reamplified
to generate a
chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT
PROTOCOLS ~~r
1 O MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression
vectors are
commercially available that already encode a fusion moiety (e.g., a GST
polypeptide). A
FCTRX-encoding nucleic acid can be cloned into such an expression vector such
that the
fusion moiety is linked in-frame to the FCTRX protein.
FCTRX Agonists And Antagonists
The present invention also pertains to variants of a FCTRX protein that
function as
either FCTRX agonists (mimetics) or as FCTRX antagonists. Variants of a FCTRX
protein
can be generated by mutagenesis, e.g., discrete point mutation or truncation
of the FCTRX
protein. An agonist of the FCTRX protein can retain substantially the same, or
a subset of, the
biological activities of the naturally occurring form of the FCTRX protein. An
antagonist of
the FCTRX protein can inhibit one or more of the activities of the naturally
occurnng form of
the FCTRX protein by, for example, competitively binding to a downstream or
upstream
member of a cellular signaling cascade which includes the FCTRX protein. Thus,
specific
biological effects can be elicited by treatment with a variant of limited
function. In one
embodiment, treatment of a subject with a variant having a subset of the
biological activities
of the naturally occurnng form of the protein has fewer side effects in a
subject relative to
treatment with the naturally occurnng form of the FCTRX protein.
Variants of the FCTRX protein that function as either FCTRX agonists
(mimetics) or
as FCTRX antagonists can be identified by screening combinatorial libraries of
mutants, e.g.,
truncation mutants, of the FCTRX protein for FCTRX protein agonist or
antagonist activity.
In one embodiment, a variegated library of FCTRX variants is generated by
combinatorial
mutagenesis at the nucleic acid level and is encoded by a variegated gene
library. A
variegated library of FCTRX variants can be produced by, for example,
enzymatically ligating
a mixture of synthetic oligonucleotides into gene sequences such that a
degenerate set of
CA 02386383 2002-04-04
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potential FCTRX sequences is expressible as individual polypeptides, or
alternatively, as a set
of larger fusion proteins (e.g., for phage display) containing the set of
FCTRX sequences
therein. There are a variety of methods which can be used to produce libraries
of potential
FCTRX variants from a degenerate oligonucleotide sequence. Chemical synthesis
of a
degenerate gene sequence can be performed in an automatic DNA synthesizer, and
the
synthetic gene then ligated into an appropriate expression vector. Use of a
degenerate set of
genes allows for the provision, in one mixture, of all of the sequences
encoding the desired set
of potential FCTRX variant sequences. Methods for synthesizing degenerate
oligonucleotides
are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et
al. (1984) Annu
Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)
Nucl Acid Res
11:477.
Polypeptide Libraries
In addition, libraries of fragments of the FCTRX protein coding sequence can
be used
to generate a variegated population of growth promoter fragments for screening
and
subsequent selection of variants of a FCTRX protein. In one embodiment, a
library of coding
sequence fragments can be generated by treating a double stranded PCR fragment
of a FCTRX
coding sequence with a nuclease under conditions wherein nicking occurs only
about once per
molecule, denaturing the double stranded DNA, renaturing the DNA to form
double stranded
DNA that can include sense/antisense pairs from different nicked products,
removing single
stranded portions from reformed duplexes by treatment with S 1 nuclease, and
ligating the
resulting fragment library into an expression vector. By this method, an
expression library can
be derived which encodes N-terminal and internal fragments of various sizes of
the FCTRX
protein.
Several techniques are known in the art for screening gene products of
combinatorial
libraries made by point mutations or truncation, and for screening cDNA
libraries for gene
products having a selected property. Such techniques are adaptable for rapid
screening of the
gene libraries generated by the combinatorial mutagenesis of FCTRX proteins.
The most
widely used techniques, which are amenable to high throughput analysis, for
screening large
gene libraries typically include cloning the gene library into replicable
expression vectors,
transforming appropriate cells with the resulting library of vectors, and
expressing the
combinatorial genes under conditions in which detection of a desired activity
facilitates
isolation of the vector encoding the gene whose product was detected.
Recursive ensemble
mutagenesis (REM), a new technique that enhances the frequency of functional
mutants in the
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libraries, can be used in combination with the screening assays to identify
FCTRX variants
(Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering
6:327-331).
Anti-FCTRX Antibodies
The term "antibody" as used herein refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin (Ig) molecules, i.e.,
molecules that
contain an antigen binding site that specifically binds (immunoreacts with) an
antigen. Such
antibodies include, but are not limited to, polyclonal, monoclonal, chimeric,
single chain, Fab,
Fab' and F(ab')2 fragments, and an Fab expression library. In general,
antibody molecules
obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD,
which differ
from one another by the nature of the heavy chain present in the molecule.
Certain classes
have subclasses as well, such as IgGI, IgG2, and others. Furthermore, in
humans, the light
chain may be a kappa chain or a lambda chain. Reference herein to antibodies
includes a
reference to all such classes, subclasses and types of human antibody species.
An isolated protein of the invention intended to serve as an antigen, or a
portion or
fragment thereof, can be used as an immunogen to generate antibodies that
immunospecifically bind the antigen, using standard techniques for polyclonal
and monoclonal
antibody preparation. The full-length protein can be used or, alternatively,
the invention
provides antigenic peptide fragments of the antigen for use as immunogens. An
antigenic
peptide fragment comprises at least 6 amino acid residues of the amino acid
sequence of the
full length protein, such as an amino acid sequence shown in SEQ ID NOS:2, 4,
6, 8, 10 and
12, and encompasses an epitope thereof such that an antibody raised against
the peptide forms
a specific immune complex with the full length protein or with any fragment
that contains the
epitope. Preferably, the antigenic peptide comprises at least 10 amino acid
residues, or at least
15 amino acid residues, or at least 20 amino acid residues, or at least 30
amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of the
protein that are
located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of the FCTRX that is located on the surface of
the protein, e.g., a
hydrophilic region. A hydrophobicity analysis of the human FCTRX protein
sequence will
indicate which regions of a FCTRX polypeptide are particularly hydrophilic
and, therefore, are
likely to encode surface residues useful for targeting antibody production. As
a means for
targeting antibody production, hydropathy plots showing regions of
hydrophilicity and
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hydrophobicity may be generated by any method well known in the art,
including, for
example, the Kyte Doolittle or the Hopp Woods methods, either with or without
Fourier
transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78:
3824-3828;
Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein
by reference in
their entirety. Antibodies that are specific for one or more domains within an
antigenic protein,
or derivatives, fragments, analogs or homologs thereof, are also provided
herein.
A protein of the invention, or a derivative, fragment, analog, homolog or
ortholog
thereof, may be utilized as an immunogen in the generation of antibodies that
immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of
polyclonal
or monoclonal antibodies directed against a protein of the invention, or
against derivatives,
fragments, analogs homologs or orthologs thereof (see, for example,
Antibodies: A Laboratory
Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, NY, incorporated herein by reference). Some of these antibodies are
discussed below.
Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals
(e.g., rabbit,
goat, mouse or other mammal) may be immunized by one or more injections with
the native
protein, a synthetic variant thereof, or a derivative of the foregoing. An
appropriate
immunogenic preparation can contain, for example, the naturally occurring
immunogenic
protein, a chemically synthesized polypeptide representing the immunogenic
protein, or a
recombinantly expressed immunogenic protein. Furthermore, the protein may be
conjugated
to a second protein known to be immunogenic in the mammal being immunized.
Examples of
such immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum
albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation
can further
include an adjuvant. Various adjuvants used to increase the immunological
response include,
but are not limited to, Freund's (complete and incomplete), mineral gels
(e.g., aluminum
hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols,
polyanions,
peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such
as Bacille
Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory
agents.
Additional examples of adjuvants which can be employed include MPL-TDM
adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be
isolated from the mammal (e.g., from the blood) and further purified by well
known
techniques, such as affinity chromatography using protein A or protein G,
which provide
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primarily the IgG fraction of immune serum. Subsequently, or alternatively,
the specific
antigen which is the target of the immunoglobulin sought, or an epitope
thereof, may be
immobilized on a column to purify the immune specific antibody by
immunoaffmity
chromatography. Purification of immunoglobulins is discussed, for example, by
D. Wilkinson
S (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14,
No. 8 (April 17,
2000), pp. 25-28).
Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used herein, refers to a population of antibody molecules that contain only
one molecular
species of antibody molecule consisting of a unique light chain gene product
and a unique
heavy chain gene product. In particular, the complementarity determining
regions (CDRs) of
the monoclonal antibody are identical in all the molecules of the population.
MAbs thus
contain an antigen binding site capable of immunoreacting with a particular
epitope of the
antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those
described in the art. See, e.g., Kohler and Milstein, 1975 Nature, 256:495. In
a hybridoma
method, a mouse, hamster, or other appropriate host animal, is typically
immunized with an
immunizing agent to elicit lymphocytes that produce or are capable of
producing antibodies
that will specifically bind to the immunizing agent. Alternatively, the
lymphocytes can be
immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment
thereof or
a fusion protein thereof. Generally, either peripheral blood lymphocytes are
used if cells of
human origin are desired, or spleen cells or lymph node cells are used if non-
human
mammalian sources are desired. The lymphocytes are then fused with an
immortalized cell
line using a suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell
(Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press,
(1986) pp.
59-103). Immortalized cell lines are usually transformed mammalian cells,
particularly
myeloma cells of rodent, bovine and human origin. Usually, rat or mouse
myeloma cell lines
are employed. The hybridoma cells can be cultured in a suitable culture medium
that
preferably contains one or more substances that inhibit the growth or survival
of the unfused,
immortalized cells. For example, if the parental cells lack the enzyme
hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas
typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which
substances prevent the growth of HGPRT-deficient cells.
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Preferred immortalized cell lines are those that fuse efficiently, support
stable high
level expression of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. More preferred immortalized cell lines are murine
myeloma
lines, which can be obtained, for instance, from the Salk Institute Cell
Distribution Center, San
Diego, California and the American Type Culture Collection, Manassas,
Virginia. Human
myeloma and mouse-human heteromyeloma cell lines also have been described for
the
production of human monoclonal antibodies. See, e.g. Kozbor 1984 J. Immunol.,
133:3001;
Brodeur et al. MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS,
Marcel
Dekker, Inc., New York, (1987) pp. S1-63.
The culture medium in which the hybridoma cells are cultured can then be
assayed for
the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma cells is
determined by
immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay
(RIA) or
enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are
known in
the art. The binding affinity of the monoclonal antibody can, for example, be
determined by
the Scatchard analysis. See, e.g. Munson and Pollard 1980 Anal. Biochem. 107:
220. It is an
objective, especially important in therapeutic applications of monoclonal
antibodies, to
identify antibodies having a high degree of specificity and a high binding
affinity for the target
antigen.
After the desired hybridoma cells are identified, the clones can be subcloned
by
limiting dilution procedures and grown by standard methods (Goding,1986).
Suitable culture
media for this purpose include, for example, Dulbecco's Modified Eagle's
Medium and RPMI-
1640 medium. Alternatively, the hybridoma cells can be grown in vivo as
ascites in a
mammal.
The monoclonal antibodies secreted by the subclones can be isolated or
purified from
the culture medium or ascites fluid by conventional immunoglobulin
purification procedures
such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as
those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies of
the invention can be readily isolated and sequenced using conventional
procedures (e.g., by
using oligonucleotide probes that are capable of binding specifically to genes
encoding the
heavy and light chains of murine antibodies). The hybridoma cells of the
invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed into
expression vectors,
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which are then transfected into host cells such as simian COS cells, Chinese
hamster ovary
(CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin
protein, to
obtain the synthesis of monoclonal antibodies in the recombinant host cells.
The DNA also
can be modified, for example, by substituting the coding sequence for human
heavy and light
chain constant domains in place of the homologous marine sequences (U.5.
Patent No.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to
the
immunoglobulin coding sequence all or part of the coding sequence for a non-
immunoglobulin
polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the
constant
domains of an antibody of the invention, or can be substituted for the
variable domains of one
antigen-combining site of an antibody of the invention to create a chimeric
bivalent antibody.
Humanized Antibodies
The antibodies directed against the protein antigens of the invention can
further
comprise humanized antibodies or human antibodies. These antibodies are
suitable for
administration to humans without engendering an immune response by the human
against the
administered immunoglobulin. Humanized forms of antibodies are chimeric
immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')Z or
other antigen-
binding subsequences of antibodies) that are principally comprised of the
sequence of a human
immunoglobulin, and contain minimal sequence derived from a non-human
immunoglobulin.
Humanization can be performed following the method of Winter and co-workers
(Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al.,
Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the
corresponding sequences of a human antibody. (See also U.S. Patent No.
5,225,539.) In some
instances, Fv framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies can also comprise
residues which
are found neither in the recipient antibody nor in the imported CDR or
framework sequences.
In general, the humanized antibody will comprise substantially all of at least
one, and typically
two, variable domains, in which all or substantially all of the CDR regions
correspond to those
of a non-human immunoglobulin and all or substantially all of the framework
regions are
those of a human immunoglobulin consensus sequence. The humanized antibody
optimally
also will comprise at least a portion of an immunoglobulin constant region
(Fc), typically that
of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and
Presta, Curr. On.
Struct. Biol., 2:593-596 (1992)).
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Human Antibodies
Fully human antibodies essentially relate to antibody molecules in which the
entire
sequence of both the light chain and the heavy chain, including the CDRs,
arise from human
genes. Such antibodies are termed "human antibodies", or "fully human
antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human
B-cell
hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV
hybridoma
technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human
monoclonal
antibodies may be utilized in the practice of the present invention and may be
produced by
using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or
by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et
al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional
techniques,
including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can
be made by
introducing human immunoglobulin loci into transgenic animals, e.g., mice in
which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
challenge, human antibody production is observed, which closely resembles that
seen in
humans in all respects, including gene rearrangement, assembly, and antibody
repertoire. This
approach is described, for example, in U.S. Patent Nos. 5,545;807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-
783 (1992));
Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13
(1994)); Fishwild
et al,( Nature Biotechnolo~y 14, 845-51 (1996)); Neuberger (Nature
Biotechnolo~y 14, 826
(1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman
animals
which are modified so as to produce fully human antibodies rather than the
animal's
endogenous antibodies in response to challenge by an antigen. (See publication
WO
94/02602). The endogenous genes encoding the heavy and light immunoglobulin
chains in the
nonhuman host have been incapacitated, and active loci encoding human heavy
and light chain
immunoglobulins are inserted into the host's genome. The human genes are
incorporated, for
example, using yeast artificial chromosomes containing the requisite human DNA
segments.
An animal which provides all the desired modifications is then obtained as
progeny by
crossbreeding intermediate transgenic animals containing fewer than the full
complement of
the modifications. The preferred embodiment of such a nonhuman animal is a
mouse, and is
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WO 01/25437 PCT/US00/27671
termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO
96/34096.
This animal produces B cells which secrete fully human immunoglobulins. The
antibodies
can be obtained directly from the animal after immunization with an immunogen
of interest,
as, for example, a preparation of a polyclonal antibody, or alternatively from
immortalized B
S cells derived from the animal, such as hybridomas producing monoclonal
antibodies.
Additionally, the genes encoding the immunoglobulins with human variable
regions can be
recovered and expressed to obtain the antibodies directly, or can be further
modified to obtain
analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous immunoglobulin heavy chain is disclosed in
U.S. Patent
No. 5,939,598. It can be obtained by a method including deleting the J segment
genes from at
least one endogenous heavy chain locus in an embryonic stem cell to prevent
rearrangement of
the locus and to prevent formation of a transcript of a rearranged
immunoglobulin heavy chain
locus, the deletion being effected by a targeting vector containing a gene
encoding a selectable
1 S marker; and producing from the embryonic stem cell a transgenic mouse
whose somatic and
germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is
disclosed
in U.S. Patent No. 5,916,771. It includes introducing an expression vector
that contains a
nucleotide sequence encoding a heavy chain into one mammalian host cell in
culture,
introducing an expression vector containing a nucleotide sequence encoding a
light chain into
another mammalian host cell, and fusing the two cells to form a hybrid cell.
The hybrid cell
expresses an antibody containing the heavy chain and the light chain.
' In a further improvement on this procedure, a method for identifying a
clinically
relevant epitope on an immunogen, and a correlative method for selecting an
antibody that
binds immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT
publication WO 99/53049. .
Fab Fragments and Single Chain Antibodies
Techniques can be adapted for the production of single-chain antibodies
specific to an
antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In
addition, methods
can be adapted for the construction of Fab expression libraries (see e.g.,
Huse, et al., 1989
Science 246: 1275-1281) to allow rapid and effective identification of
monoclonal Fab
fragments with the desired specificity for a protein or derivatives,
fragments, analogs or
homologs thereof. Antibody fragments that contain the idiotypes to a protein
antigen may be
produced by techniques known in the art including, but not limited to: (i) an
F~ab')z fragment
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CA 02386383 2002-04-04
WO 01/25437 PCT/US00/27671
produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment
generated by
reducing the disulfide bridges of an F~ab')2 fragment; (iii) an Fab fragment
generated by the
treatment of the antibody molecule with papain and a reducing agent and (iv)
F" fragments.
Bispecifc Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that
have binding specificities for at least two different antigens. In the present
case, one of the
binding specificities is for an antigenic protein of the invention. The second
binding target is
any other antigen, and advantageously is a cell-surface protein or receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispecific antibodies is based on the co-expression
of two
immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have
different
specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of
the random
assortment of immunoglobulin heavy and light chains, these hybridomas
(quadromas) produce
a potential mixture of ten different antibody molecules, of which only one has
the correct
bispecific structure. The purification of the correct molecule is usually
accomplished by
affinity chromatography steps. Similar procedures are disclosed in WO
93/08829, published
13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen
combining sites) can be fused to immunoglobulin constant domain sequences. The
fusion
preferably is with an immunoglobulin heavy-chain constant domain, comprising
at least part
of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-
chain constant
region (CH1) containing the site necessary for light-chain binding present in
at least one of the
fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired,
the
immunoglobulin light chain, are inserted into separate expression vectors, and
are co-
transfected into a suitable host organism. For further details of generating
bispecific
antibodies see, for example, Suresh et al., Methods in Enzymolo~y, 121:210
(1986).
According to another approach described in WO 96/27011, the interface between
a pair
of antibody molecules can be engineered to maximize the percentage of
heterodimers which
are recovered from recombinant cell culture. The preferred interface comprises
at least a part
of the CH3 region of an antibody constant domain. In this method, one or more
small amino
acid side chains from the interface of the first antibody molecule are
replaced with larger side
chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the
large side chains) are created on the interface of the second antibody
molecule by replacing
large amino acid side chains with smaller ones (e.g. alanine or threonine).
This provides a
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WO 01/25437 PCT/US00/27671
mechanism for increasing the yield of the heterodimer over other unwanted end-
products such
as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments
(e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific
antibodies from
antibody fragments have been described in the literature. For example,
bispecific antibodies
can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985)
describe a
procedure wherein intact antibodies are proteolytically cleaved to generate
F(ab')2 fragments.
These fragments are reduced in the presence of the dithiol complexing agent
sodium arsenite
to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
The Fab'
fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the
Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative
to form the bispecific antibody. The bispecific antibodies produced can be
used as agents for
the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and
chemically
coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-
225 (1992)
describe the production of a fully humanized bispecific antibody F(ab')Z
molecule. Each Fab'
fragment was separately secreted from E. coli and subjected to directed
chemical coupling in
vitro to form the bispecific antibody. The bispecific antibody thus formed was
able to bind to
cells overexpressing the ErbB2 receptor and normal human T cells, as well as
trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments
directly
from recombinant cell culture have also been described. For example,
bispecific antibodies
have been produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553
(1992). The leucine zipper peptides from the Fos and Jun proteins were linked
to the Fab'
portions of two different antibodies by gene fusion. The antibody homodimers
were reduced
at the hinge region to form monomers and then re-oxidized to form the antibody
heterodimers.
This method can also be utilized for the production of antibody homodimers.
The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-
6448 (1993) has
provided an alternative mechanism for making bispecific antibody fragments.
The fragments
comprise a heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL)
by a linker which is too short to allow pairing between the two domains on the
same chain.
Accordingly, the VH and VL domains of one fragment are forced to pair with the
complementary V~ and VH domains of another fragment, thereby forming two
antigen-binding
CA 02386383 2002-04-04
WO 01/25437 PCT/US00/27671
sites. Another strategy for making bispecific antibody fragments by the use of
single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368
(1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific
antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least
one of
which originates in the protein antigen of the invention. Alternatively, an
anti-antigenic arm
of an immunoglobulin molecule can be combined with ax arm which binds to a
triggering
molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3,
CD28, or B7), or
Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII
(CD 16) so as
to focus cellular defense mechanisms to the cell expressing the particular
antigen. Bispecific
antibodies can also be used to direct cytotoxic agents to cells which express
a particular
antigen. These antibodies possess an antigen-binding arm and an arm which
binds a cytotoxic
agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another
bispecific antibody of interest binds the protein antigen described herein and
further binds
1 S tissue factor (TF).
Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such
antibodies have, for example, been proposed to target immune system cells to
unwanted cells
(U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360;
WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared
in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinking agents.
For example, immunotoxins can be constructed using a disulfide exchange
reaction or by
forming a thioether bond. Examples of suitable reagents for this purpose
include iminothiolate
and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Patent No.
4,676,980.
Effector Function Engineering
It can be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. For
example, cysteine residues) can be introduced into the Fc region, thereby
allowing interchain
disulfide bond formation in this region. The homodimeric antibody thus
generated can have
improved internalization capability and/or increased complement-mediated cell
killing and
antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp
Med., 176: 1191-
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WO 01/25437 PCT/US00/27671
1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric
antibodies with
enhanced anti-tumor activity can also be prepared using heterobifunctional
cross-linkers as
described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have enhanced
complement lysis
and ADCC capabilities. See Stevenson et al., Anti-Cancer Dru~Desi~n, 3: 219-
230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated
to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an
enzymatically active
toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or
a radioactive
isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above. Enzymatically active toxins and fragments thereof that
can be used
include diphtheria A chain, nonbinding active fragments of diphtheria toxin,
exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, croon, sapaonaria officinalis
inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of
radionuclides are available for the production of radioconjugated antibodies.
Examples
include z~2Bi, 13~I, ~3~In, 9°Y, and la6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling agents such as N-succinimidyl-3-(2-
pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as
dimethyl
adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes
(such as
glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates
(such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as
1,5-difluoro-
2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as
described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-
isothiocyanatobenzyl-3-
methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating
agent for
conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate is
administered to the patient, followed by removal of unbound conjugate from the
circulation
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using a clearing agent and then administration of a "ligand" (e.g., avidin)
that is in turn
conjugated to a cytotoxic agent.
Immunoliposomes
The antibodies disclosed herein can also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in the art,
such as
described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985);
Hwang et al., Proc.
Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and
4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Patent No.
5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation
method with a lipid composition comprising phosphatidylcholine, cholesterol,
and PEG-
derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of
defined pore size to yield liposomes with the desired diameter. Fab' fragments
of the antibody
of the present invention can be conjugated to the liposomes as described in
Martin et al., J.
Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A
chemotherapeutic
1 S agent (such as Doxorubicin) is optionally contained within the liposome.
See Gabizon et al.,
J. National Cancer Inst., 81(19): 1484 (1989).
Diagnostic Applications of Antibodies Directed Against the Proteins of the
Invention
Antibodies directed against a protein of the invention may be used in methods
known
within the art relating to the localization and/or quantitation of the protein
(e.g., for use in
measuring levels of the protein within appropriate physiological samples, for
use in diagnostic
methods, for use in imaging the protein, and the like). In a given embodiment,
antibodies
against the proteins, or derivatives, fragments, analogs or homologs thereof,
that contain the
antigen binding domain, are utilized as pharmacologically-active compounds
(see below).
An antibody specific for a protein of the invention can be used to isolate the
protein by
standard techniques, such as immunoaffinity chromatography or
immunoprecipitation. Such
an antibody can facilitate the purification of the natural protein antigen
from cells and of
recombinantly produced antigen expressed in host cells. Moreover, such an
antibody can be
used to detect the antigenic protein (e.g., in a cellular lysate or cell
supernatant) in order to
evaluate the abundance and pattern of expression of the antigenic protein.
Antibodies directed
against the protein can be used diagnostically to monitor protein levels in
tissue as part of a
clinical testing procedure, e.g., to, for example, determine the efficacy of a
given treatment
regimen. Detection can be facilitated by coupling (i.e., physically linking)
the antibody to a
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WO 01/25437 PCT/US00/27671
detectable substance. Examples of detectable substances include various
enzymes, prosthetic
groups, fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive
materials. Examples of suitable enzymes include horseradish peroxidase,
alkaline
phosphatase, (3-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent
materials include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes luminol; examples of bioluminescent materials
include
luciferase, luciferin, and aequorin, and examples of suitable radioactive
material include ~zSI,
1 O 131I' 35s or 3H.
Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a protein of the invention, as well as other
molecules
identified by the screening assays disclosed herein, can be administered for
the treatment of
various disorders in the form of pharmaceutical compositions. Principles and
considerations
involved in preparing such compositions, as well as guidance in the choice of
components are
provided, for example, in Remington : THE SCIENCE AND PRACTICE OF PHARMACY
19th ed.
(Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. 1995; DRUG
ABSORPTION
ENHANCEMENT: CONCEPTS, POSSIBILITIES, LIMITATIONS, AND TRENDS, Harwood
Academic
Publishers, Langhorne, Pa., 1994; arid PEPTIDE AND PROTEIN DRUG DELIVERY
(Advances In
Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
If the antigenic protein is intracellular and whole antibodies are used as
inhibitors,
internalizing antibodies are preferred. However, liposomes can also be used to
deliver the
antibody, or an antibody fragment, into cells. Where antibody fragments are
used, the smallest
inhibitory fragment that specifically binds to the binding domain of the
target protein is
preferred. For example, based upon the variable-region sequences of an
antibody, peptide
molecules can be designed that retain the ability to bind the target protein
sequence. Such
peptides can be synthesized chemically and/or produced by recombinant DNA
technology.
See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
The formulation
herein can also contain more than one active compound as necessary for the
particular
indication being treated, preferably those with complementary activities that
do not adversely
affect each other. Alternatively, or in addition, the composition can comprise
an agent that
enhances its function, such as, for example, a cytotoxic agent, cytokine,
chemotherapeutic
agent, or growth-inhibitory agent. Such molecules are suitably present in
combination in
amounts that are effective for the purpose intended.
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The active ingredients can also be entrapped in microcapsules prepared, for
example,
by coacervation techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems (for example,
liposomes,
albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in
macroemulsions.
The formulations to be used for in vivo administration must be sterile. This
is readily
accomplished by filtration through sterile filtration membranes.
Antibody Therapeutics
Antibodies of the invention, including polyclonal, monoclonal, humanized and
fully
human antibodies, may used as therapeutic agents. Such agents will generally
be employed to
treat or prevent a disease or pathology in a subject. An antibody preparation,
preferably one
having high specificity and high affinity for its target antigen, is
administered to the subject
and will generally have an effect due to its binding with the target. Such an
effect may be one
of two kinds, depending on the specific nature of the interaction between the
given antibody
molecule and the target antigen in question. In the first instance,
administration of the
antibody may abrogate or inhibit the binding of the target with an endogenous
ligand to which
it naturally binds. In this case, the antibody binds to the target and masks a
binding site of the
naturally occurring ligand, wherein the ligand serves as an effector molecule.
Thus the
receptor mediates a signal transduction pathway for which ligand is
responsible.
Alternatively, the effect may be one in which the antibody elicits a
physiological result
by virtue of binding to an effector binding site on the target molecule. In
this case the target, a
receptor having an endogenous ligand which may be absent or defective in the
disease or
pathology, binds the antibody as a surrogate effector ligand, initiating a
receptor-based signal
transduction event by the receptor.
A therapeutically effective amount of an antibody of the invention relates
generally to
the amount needed to achieve a therapeutic objective. As noted above, this may
be a binding
interaction between the antibody and its target antigen that, in certain
cases, interferes with the
functioning of the target, and in other cases, promotes a physiological
response. The amount
required to be administered will furthermore depend on the binding affinity of
the antibody for
its specific antigen, and will also depend on the rate at which an
administered antibody is
depleted from the free volume other subject to which it is administered.
Common ranges for
therapeutically effective dosing of an antibody or antibody fragment of the
invention may be,
by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50
mg/kg body
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WO 01/25437 PCT/US00/27671
weight. Common dosing frequencies may range, for example, from twice daily to
once a
week.
FCTRX Recombinant Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding a FCTRX protein, or derivatives, fragments,
analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable
of transporting another nucleic acid to which it has been linked. One type of
vector is a
"plasmid", which refers to a circular double stranded DNA loop into which
additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein
additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of
autonomous
replication in a host cell into which they are introduced (e.g., bacterial
vectors having a
bacterial origin of replication and episomal mammalian vectors). Other vectors
(e.g., non-
episomal mammalian vectors) are integrated into the genome of a host cell upon
introduction
into the host cell, and thereby are replicated along with the host genome.
Moreover, certain
vectors are capable of directing the expression of genes to which they are
operatively linked.
Such vectors are referred to herein as "expression vectors". In general,
expression vectors of
utility in recombinant DNA techniques are often in the form of plasmids. In
the present
specification, "plasmid" and "vector" can be used interchangeably as the
plasmid is the most
commonly used form of vector. However, the invention is intended to include
such other
forms of expression vectors, such as viral vectors (e.g., replication
defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means that
the recombinant expression vectors include one or more regulatory sequences,
selected on the
basis of the host cells to be used for expression, that is operatively linked
to the nucleic acid
sequence to be expressed. Within a recombinant expression vector, "operably
linked" is
intended to mean that the nucleotide sequence of interest is linked to the
regulatory
sequences) in a manner that allows for expression of the nucleotide sequence
(e.g., in an in
vitro transcription/translation system or in a host cell when the vector is
introduced into the
host cell). The term "regulatory sequence" is intended to includes promoters,
enhancers and
other expression control elements (e.g., polyadenylation signals). Such
regulatory sequences
are described, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences
include
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those that direct constitutive expression of a nucleotide sequence in many
types of host cell
and those that direct expression of the nucleotide sequence only in certain
host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by those skilled
in the art that the
design of the expression vector can depend on such factors as the choice of
the host cell to be
transformed, the level of expression of protein desired, etc. The expression
vectors of the
invention can be introduced into host cells to thereby produce proteins or
peptides, including
fusion proteins or peptides, encoded by nucleic acids as described herein
(e.g., FCTRX
proteins, mutant forms of the FCTRX, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of
a FCTRX nucleic acid in prokaryotic or eukaryotic cells. For example, the
FCTRX can be
expressed in bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors)
yeast cells or mammalian cells. Suitable host cells are discussed further in
Goeddel, GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego,
Calif. (1990). Alternatively, the recombinant expression vector can be
transcribed and
translated in vitro, for example, using T7 promoter regulatory sequences and
T7 polymerise.
Expression of proteins in prokaryotes is most often carned out in E. coli with
vectors
containing constitutive or inducible promoters directing the expression of
either fusion or non-
fusion proteins. Fusion vectors add a number of amino acids to a protein
encoded therein,
usually to the amino terminus of the recombinant protein. Such fusion vectors
typically serve
three purposes: (1) to increase expression of recombinant protein; (2) to
increase the solubility
of the recombinant protein; and (3) to aid in the purification of the
recombinant protein by
acting as a ligand in affinity purification. Oftena proteolytic cleavage site
is introduced in
fusion expression vectors at the junction of the fusion moiety and the
recombinant protein to
enable separation of the recombinant protein from the fusion moiety subsequent
to purification
of the fusion protein. Such enzymes, and their cognate recognition sequences,
include Factor
Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia
Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England
Biolabs,
Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione
S-transferase
(GST), maltose E binding protein, or protein A, respectively, to the target
recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc
(Amrann et al., (1988) Gene 69:301-315) and pET l 1d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990) 60-
89).
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One strategy to maximize recombinant protein expression in E. coli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the recombinant
protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY
185,
Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter
the nucleic
acid sequence of the nucleic acid to be inserted into an expression vector so
that the individual
codons for each amino acid are those preferentially utilized in E. coli (Wads
et al., (1992)
Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of
the invention
can be carned out by standard DNA synthesis techniques.
In another embodiment, the FCTRX expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast S. cerivisae include pYepSecl
(Baldari, et al.,
(1987) EMBOJ6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88
(Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San
Diego, Calif.),
and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, the FCTRX nucleic acid can be expressed in insect cells using
baculovirus expression vectors. Baculovirus vectors available for expression
of proteins in
cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al.
(1983) Mol Cell Biol
3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-
39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian
cells using a mammalian expression vector. Examples of mammalian expression
vectors
include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufinan et al. (1987)
EMBO J
6: 187-195). When used in mammalian cells, the expression vector's control
functions are
often provided by viral regulatory elements. For example, commonly used
promoters are
derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other
suitable expression systems for both prokaryotic and eukaryotic cells. See,
e.g., Chapters 16
and 17 of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed.,
Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.,
1989.
In another embodiment, the recombinant mammalian expression vector is capable
of
directing expression of the nucleic acid preferentially in a particular cell
type (e.g., tissue-
specific regulatory elements are used to express the nucleic acid). Tissue-
specific regulatory
elements are known in the art. Non-limiting examples of suitable tissue-
specific promoters
include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev
1:268-277),
lymphoid-specific promoters (Calame and Eaton (1988) Adv Immunol 43:235-275),
in
particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J
8:729-733)
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and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983)
Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter;
Byrne and
Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al.
(1985)
Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey
promoter;
U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the marine hox
promoters
(Kessel and Grass (1990) Science 249:374-379) and the a-fetoprotein promoter
(Campes and
Tilghman (1989) Genes Dev 3:537-546).
The invention further provides a recombinant expression vector comprising a
DNA
molecule of the invention cloned into the expression vector in an antisense
orientation. That
is, the DNA molecule is operatively linked to a regulatory sequence in a
manner that allows
for expression (by transcription of the DNA molecule) of an RNA molecule that
is antisense to
a FCTRX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned
in the
antisense orientation can be chosen that direct the continuous expression of
the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or
enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific or cell type
specific expression
of antisense RNA. The antisense expression vector can be in the form of a
recombinant
plasmid, phagemid or attenuated virus in which antisense nucleic acids are
produced under the
control of a high efficiency regulatory region, the activity of which can be
determined by the
cell type into which the vector is introduced. For a discussion of the
regulation of gene
expression using antisense genes see Weintraub et al., "Antisense RNA as a
molecular tool for
genetic analysis," Reviews--Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such terms refer
not only to the particular subject cell but to the progeny or potential
progeny of such a cell.
Because certain modifications may occur in succeeding generations due to
either mutation or
environmental influences, such progeny may not, in fact, be identical to the
parent cell, but are
still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, the FCTRX
protein
can be expressed in bacterial cells such as E. coli, insect cells, yeast or
mammalian cells (such
as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to
those skilled in the art.
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Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the terms
"transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques
for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate
or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting host cells
can be found in
Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989),
and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may integrate
the foreign DNA into their genome. In order to identify and select these
integrants, a gene that
encodes a selectable marker (e.g., resistance to antibiotics) is generally
introduced into the
host cells along with the gene of interest. Various selectable markers include
those that confer
resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid
encoding a
selectable marker can be introduced into a host cell on the same vector as
that encoding the
growth promoter or can be introduced on a separate vector. Cells stably
transfected with the
introduced nucleic acid can be identified by drug selection (e.g., cells that
have incorporated
the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can
be used to produce (i.e., express) the FCTRX protein. Accordingly, the
invention further
provides methods for producing the FCTRX protein using the host cells of the
invention. In
one embodiment, the method comprises culturing the host cell of invention
(into which a
recombinant expression vector encoding the FCTRX polypeptide has been
introduced) in a
suitable medium such that the FCTItX protein is produced. In another
embodiment, the
method further comprises isolating the FCTRX from the medium or the host cell.
Transgenic Animals
The host cells of the invention can also be used to produce nonhuman
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized oocyte or
an embryonic stem cell into which FCTRX-coding sequences have been introduced.
Such
host cells can then be used to create non-human transgenic animals in which
exogenous
FCTRX sequences have been introduced into their genome or homologous
recombinant
animals in which endogenous FCTRX sequences have been altered. Such animals
are useful
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for studying the function and/or activity of the FCTRX sequences and for
identifying and/or
evaluating modulators of FCTRX activity. As used herein, a "transgenic animal"
is a non-
human animal, preferably a mammal, more preferably a rodent such as a rat or
mouse, in
which one or more of the cells of the animal includes a transgene. Other
examples of
transgenic animals include non-human primates, sheep, dogs, cows, goats,
chickens,
amphibians, etc. A transgene is exogenous DNA that is integrated into the
genome of a cell
from which a transgenic animal develops and that remains in the genome of the
mature
animal, thereby directing the expression of an encoded gene product in one or
more cell types
or tissues of the transgenic animal. As used herein, a "homologous recombinant
animal" is a
non-human animal, preferably a mammal, more preferably a mouse, in which an
endogenous
FCTRX gene has been altered by homologous recombination between the endogenous
gene
and an exogenous DNA molecule introduced into a cell of the animal, e.g., an
embryonic cell
of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing FCTRX-
encoding
nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral
infection, and allowing the oocyte to develop in a pseudopregnant female
foster animal. The
human FCTRX DNA sequence of SEQ ID NOS:1, 3, 5, 7, 9 and 11 can be introduced
as a
transgene into the genome of a non-human animal. Alternatively, a nonhuman
homologue of
the human FCTRX gene, such as a mouse FCTRX gene, can be isolated based on
hybridization to the human FCTRX cDNA (described further above) and used as a
transgene.
Intronic sequences and polyadenylation signals can also be included in the
transgene to
increase the efficiency of expression of the transgene. A tissue-specific
regulatory sequences)
can be operably linked to the FCTRX transgene to direct expression of FCTRX
protein to
particular cells. Methods for generating transgenic animals via embryo
manipulation and
microinjection, particularly animals such as mice, have become conventional in
the art and are
described, for example, in U.S. Pat. Nos. 4,736,866; 4,870,009; and 4,873,191;
and Hogan
1986, In: MANIPULATING THE MousE EMBRYO, Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, N.Y. Similar methods are used for production of other
transgenic animals. A
transgenic founder animal can be identified based upon the presence of the
FCTRX transgene
in its genome and/or expression of FCTRX mRNA in tissues or cells of the
animals. A
transgenic founder animal can then be used to breed additional animals
carrying the transgene.
Moreover, transgenic animals carrying a transgene encoding a FCTRX can further
be bred to
other transgenic animals carrying other transgenes.
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To create a homologous recombinant animal, a vector is prepared which contains
at
least a portion of a FCTRX gene into which a deletion, addition or
substitution has been ,
introduced to thereby alter, e.g., functionally disrupt, the FCTRX gene. The
FCTRX gene can
be a human gene (e.g., SEQ ID NOS:1, 3, 5, 7, 9 and 11), but more preferably,
is a non-
human homologue of a human FCTRX gene. For example, a mouse homologue of human
FCTRX gene of SEQ ID NOS:1, 3, 5, 7, 9 and 11 can be used to construct a
homologous
recombination vector suitable for altering an endogenous FCTRX gene in the
mouse genome.
In one embodiment, the vector is designed such that, upon homologous
recombination, the
endogenous FCTRX gene is functionally disrupted (i.e., no longer encodes a
functional
protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous
recombination,
the endogenous FCTRX gene is mutated or otherwise altered but still encodes
functional
protein (e.g., the upstream regulatory region can be altered to thereby alter
the expression of
the endogenous FCTRX protein). In the homologous recombination vector, the
altered portion
of the FCTRX gene is flanked at its 5' and 3' ends by additional nucleic acid
of the FCTRX
gene to allow for homologous recombination to occur between the exogenous
FCTRX protein
gene carried by the vector and an endogenous FCTRX protein gene in an
embryonic stem cell.
The additional flanking FCTRX protein nucleic acid is of sufficient length for
successful
homologous recombination with the endogenous gene. Typically, several
kilobases of
flanking DNA (both at the 5' and 3' ends) are included in the vector. See
e.g., Thomas et al.
(1987) Cell 51:503 for a description of homologous recombination vectors. The
vector is
introduced into an embryonic stem cell line (e.g., by electroporation) and
cells in which the
introduced FCTRX protein gene has homologously recombined with the endogenous
FCTRX
protein gene are selected (see e.g., Li et al. (1992) Cell 69:915).
The selected cells are then injected into a blastocyst of an animal (e.g., a
mouse) to
form aggregation chimeras. See e.g., Bradley 1987, In: TERATOCARC1NOMAS AND
EMBRYONIC
STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A
chimeric
embryo can then be implanted into a suitable pseudopregnant female foster
animal and the
embryo brought to term. Progeny harboring the homologously recombined DNA in
their germ
cells can be used to breed animals in which all cells of the animal contain
the homologously
recombined DNA by germline transmission of the transgene. Methods for
constructing
homologous recombination vectors and homologous recombinant animals are
described
further in Bradley (1991) Curr Opin Biotechnol 2:823-829; PCT International
Publication
Nos.: WO 90/1184; WO 91/01140; WO 92/0968; and WO 93/04169.
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In another embodiment, transgenic non-humans animals can be produced that
contain
selected systems that allow for regulated expression of the transgene. One
example of such a
system is the cre/loxP recombinase system of bacteriophage P 1. For a
description of the
cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.
Another
example of a recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al. (1991) Science 251:181-185. If a cre/loxP
recombinase system is
used to regulate expression of the transgene, animals containing transgenes
encoding both the
Cre recombinase and a selected protein are required. Such animals can be
provided through
the construction of "double" transgenic animals, e.g., by mating two
transgenic animals, one
containing a transgene encoding a selected protein and the other containing a
transgene
encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wilmut et al. (1997) Nature 385:810-813.
In brief, a
cell, e.g., a somatic cell, from the transgenic animal can be isolated and
induced to exit the
growth cycle and enter Go phase. The quiescent cell can then be fused, e.g.,
through the use of
electrical pulses, to an enucleated oocyte from an animal of the same species
from which the
quiescent cell is isolated. The reconstructed oocyte is then cultured such
that it develops to
morula or blastocyte and then transferred to pseudopregnant female foster
animal. The
offspring borne of this female foster animal will be a clone of the animal
from which the cell,
e.g., the somatic cell, is isolated.
Pharmaceutical Compositions
The FCTRX nucleic acid molecules, FCTRX proteins, and anti-FCTRX antibodies of
the invention, and derivatives, fragments, analogs and homologs thereof are
designated "active
compounds" or "Therapeutics" herein. Additionally, low molecular weight
compounds which
have the property that they either bind to the FCTRX nucleic acid molecules,
the FCTRX
proteins, and the anti-FCTRX antibodies of the invention, and derivatives,
fragments, analogs
and homologs thereof, or induce pharmacological agonist or antagonist
responses commonly
ascribed to a FCTR.X nucleic acid molecule, a FCTRX protein, and derivatives,
fragments,
analogs and homologs thereof, are also termed "active compounds" or
"Therapeutics" herein.
These Therapeutics can be incorporated into pharmaceutical compositions
suitable for
administration to a subject. Such compositions typically comprise the nucleic
acid molecule,
protein, or antibody and a pharmaceutically acceptable carrier.
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As used herein, "pharmaceutically acceptable Garner" is intended to include
any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and
absorption delaying agents, and the like, compatible with pharmaceutical
administration.
Suitable carriers are described in the most recent edition of Remington's
Pharmaceutical
Sciences, a standard reference text in the field, which is incorporated herein
by reference.
Preferred examples of such Garners or diluents include, but are not limited
to, water, saline,
Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes
and non-
aqueous vehicles such as fixed oils may also be used. The use of such media
and agents for
pharmaceutically active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active compound, use
thereof in the
compositions is contemplated. Supplementary active compounds can also be
incorporated into
the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with its
intended route of administration. Examples of routes of administration include
parenteral,
e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions used for
parenteral,
intradermal, or subcutaneous application can include the following components:
a sterile
diluent such as water for injection, saline solution, fixed oils, polyethylene
glycols, glycerine,
propylene glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;
chelating agents such
as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or
phosphates, and agents
for the adjustment of tonicity such as sodium chloride or dextrose. The pH can
be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral
preparation can be enclosed in ampoules, disposable syringes or multiple dose
vials made of
glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous
administration,
suitable Garners include physiological saline, bacteriostatic water, Cremophor
ELTM (BASF,
Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the
composition must be
sterile and should be fluid to the extent that easy syringeability exists. It
must be stable under
the conditions of manufacture and storage and must be preserved against the
contaminating
action of microorganisms such as bacteria and fungi. The carrier can be a
solvent or
dispersion medium containing, for example, water, ethanol, polyol (for
example, glycerol,
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propylene glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating
such as lecithin, by
the maintenance of the required particle size in the case of dispersion and by
the use of
surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic
acid, thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents,
for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride
in the
composition. Prolonged absorption of the injectable compositions can be
brought about by
including in the composition an agent which delays absorption, for example,
aluminum
monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g.,
a FCTRX protein or anti-FCTRX protein antibody) in the required amount in an
appropriate
solvent with one or a combination of ingredients enumerated above, as
required, followed by
filtered sterilization. Generally, dispersions are prepared by incorporating
the active
1 S compound into a sterile vehicle that contains a basic dispersion medium
and the required other
ingredients from those enumerated above. In the case of sterile powders for
the preparation of
sterile injectable solutions, methods of preparation are vacuum drying and
freeze-drying that
yields a powder of the active ingredient plus any additional desired
ingredient from a
previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be
enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic
administration, the active compound can be incorporated with excipients and
used in the form
of tablets, troches, or capsules. Oral compositions can also be prepared using
a fluid carrier
for use as a mouthwash, wherein the compound in the fluid Garner is applied
orally and
swished and expectorated or swallowed. Pharmaceutically compatible binding
agents, and/or
adjuvant materials can be included as part of the composition. The tablets,
pills, capsules,
troches and the like can contain any of the following ingredients, or
compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient
such as starch or lactose, a disintegrating agent such as alginic acid,
Primogel, or corn starch; a
lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint,
methyl salicylate, or orange flavoring.
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For administration by inhalation, the compounds are delivered in the form of
an
aerosol spray from pressured container or dispenser which contains a suitable
propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For
S transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, detergents, bile salts,
and fusidic acid
derivatives. Transmucosal administration can be accomplished through the use
of nasal sprays
or suppositories. For transdermal administration, the active compounds are
formulated into
ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention enemas
for rectal delivery.
In one embodiment, the active compounds are prepared with Garners that will
protect
1 S the compound against rapid elimination from the body, such as a controlled
release
formulation, including implants and microencapsulated delivery systems.
Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of
such formulations will be apparent to those skilled in the art. The materials
can also be
obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal
suspensions (including liposomes targeted to infected cells with monoclonal
antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers. These can
be prepared
according to methods known to those skilled in the art, for example, as
described in U.S. Pat.
No. 4,522,811.
Sustained-release preparations can be prepared. Suitable examples of sustained-
release
preparations include semipermeable matrices of solid hydrophobic polymers
containing the
antibody, which matrices are in the form of shaped articles, e.g., films, or
microcapsules.
Examples of sustained-release matrices include polyesters, hydrogels (for
example, poly(2-
hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat.
No. 3,773,919),
copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-
vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT TM
(injectable microspheres composed of lactic acid-glycolic acid copolymer and
leuprolide
acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl
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acetate and lactic acid-glycolic acid enable release of molecules for over 100
days, certain
hydrogels release pharmaceutical active agents over shorter time periods.
It is especially advantageous to formulate oral or parenteral compositions in
dosage
unit form for ease of administration and uniformity of dosage. Dosage unit
form as used
herein refers to physically discrete units suited as unitary dosages for the
subject to be treated;
each unit containing a predetermined quantity of active compound calculated to
produce the
desired therapeutic effect in association with the required pharmaceutical
Garner. The
specification for the dosage unit forms of the invention are dictated by and
directly dependent
on the unique characteristics of the active compound and the particular
therapeutic effect to be
achieved.
The nucleic acid molecules of the invention can be inserted into vectors and
used as
gene therapy vectors. Gene therapy vectors can be delivered to a subject by
any of a number
of routes, e.g., as described in U.S. Patent Nos. 5,703,055. Delivery can thus
also include,
e.g., intravenous injection, local administration (see U.S. Pat. No.
5,328,470) or stereotactic
injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). The pharmaceutical
preparation
of the gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can
comprise a slow release matrix in which the gene delivery vehicle is imbedded.
Alternatively,
where the complete gene delivery vector can be produced intact from
recombinant cells, e.g.,
retroviral vectors, the pharmaceutical preparation can include one or more
cells that produce
the gene delivery system.
The pharmaceutical compositions can be included in a kit, e.g., in a
container, pack, or
dispenser together with instructions for administration.
Also within the invention is the use of a therapeutic in the manufacture of a
medicament for treating a syndrome associated with a human disease, the
disease selected
from a FCTRX-associated disorder, wherein said therapeutic is selected from
the group
consisting of a FCTRX polypeptide, a FCTRX nucleic acid, and an anti-FCTRX
antibody.
Additional Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, and antibodies
described
herein can be used in one or more of the following methods: (a) screening
assays; (b)
detection assays (e.g., chromosomal mapping, cell and tissue typing, forensic
biology), (c)
predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring
clinical trials, and
pharmacogenomics); and (d) methods of treatment (e.g., therapeutic and
prophylactic).
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The isolated nucleic acid molecules of the invention can be used to express a
FCTRX
protein (e.g., via a recombinant expression vector in a host cell in gene
therapy applications),
to detect a FCTRX mRNA (e.g., in a biological sample) or a genetic lesion in a
FCTRX gene,
and to modulate FCTRX activity, as described further below. In addition, the
FCTRX proteins
S can be used to screen drugs or compounds that modulate the FCTRX activity or
expression as
well as to treat disorders characterized by insufficient or excessive
production of the FCTRX
protein, for example proliferative or differentiative disorders, or production
of the FCTRX
protein forms that have decreased or aberrant activity compared to the FCTRX
wild type
protein. In addition, the anti-FCTRX antibodies of the invention can be used
to detect and
isolate FCTRX proteins and modulate FCTRX activity.
This invention further pertains to novel agents identified by the above
described
screening assays and uses thereof for treatments as described herein.
Screening Assays
The invention provides methods (also referred to herein as "screening assays")
for
identifying modulators, i.e., candidate or test compounds or agents (e.g.,
proteins,
polypeptides, nucleic acids or polynucleotides, peptides, peptidomimetics,
small molecules
including agonists or antagonists, or other drugs) that bind to FCTRX proteins
or have a
stimulatory or inhibitory effect on, for example, FCTRX expression or FCTRX
activity. The
candidate or test compounds or agents that may bind to a FCTRX polypeptide may
have a
molecular weight around 50 Da, 100 Da, 150 Da, 300 Da, 330 Da, 350 Da, 400 Da,
500 Da,
750 Da, 1000 Da, 1250 Da, 1500 Da, 1750 Da, 2000 Da, 5000 Da, 10,000 Da,
25,000 Da,
50,000 Da, 75,000 Da, 100,000 Da or more than 100,000 Da. In certain
embodiments, the
candidate substance that binds to a FCTRX polypeptide has a molecular weight
not more than
about 1500 Da.
Details of functional assays are provided herein further below. Any of the
assays
described, as well as additional assays known to practitioners in the fields
of pharmacology,
hematology, internal medicine, oncology and the like, may be employed in order
to screen
candidate substance for their properties as therapeutic agents. As noted, the
therapeutic agents
of the invention encompass proteins, polypeptides, nucleic acids or
polynucleotides, peptides,
peptidomimetics, small molecules including agonists or antagonists, or other
drugs described
herein.
In one embodiment, the invention provides assays for screening candidate or
test
compounds which bind to or modulate the activity of a FCTRX protein or
polypeptide or
biologically active portion thereof. The test compounds of the present
invention can be
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obtained using any of the numerous approaches in combinatorial library methods
known in the
art, including: biological libraries; spatially addressable parallel solid
phase or solution phase
libraries; synthetic library methods requiring deconvolution; the "one-bead
one-compound"
library method; and synthetic library methods using affinity chromatography
selection. The
biological library approach is limited to peptide libraries, while the other
four approaches are
applicable to peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam
(1997) Anticancer Drug Des 12:145).
Examples of methods for the synthesis of molecular libraries can be found in
the art,
for example in: DeWitt et al. (1993) Proc Natl Acad Sci U.S.A. 90:6909; Erb et
al. (1994)
Proc Natl Acad Sci U.S.A. 91:11422; Zuckermann et al. (1994) JMed Chem
37:2678; Cho et
al. (1993) Science 261:1303; Carrell et al. (1994) Angew Chem Int Ed Engl
33:2059; Carell et
al. (1994) Angew Chem Int Ed Engl 33:2061; and Gallop et al. (1994) JMed Chem
37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten (1992)
Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), on chips
(Fodor
(1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores
(Ladner USP
'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on
phage (Scott
and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406;
Cwirla et al.
(1990) Proc Natl Acad Sci U.S.A. 87:6378-6382; Felici (1991) JMoI Biol 222:301-
310;
Ladner above.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses a
membrane-bound form of a FCTRX protein, or a biologically active portion
thereof, on the
cell surface is contacted with a test compound and the ability of the test
compound to bind to a
FCTRX protein determined. The cell, for example, can of mammalian origin or a
yeast cell.
Determining the ability of the test compound to bind to the FCTRX protein can
be
accomplished, for example, by coupling the test compound with a radioisotope
or enzymatic
label such that binding of the test compound to the FCTRX protein or
biologically active
portion thereof can be determined by detecting the labeled compound in a
complex. For
example, test compounds can be labeled with lash 355, ~4C, or 3H, either
directly or indirectly,
and the radioisotope detected by direct counting of radioemission or by
scintillation counting.
Alternatively, test compounds can be enzymatically labeled with, for example,
horseradish
peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label
detected by
determination of conversion of an appropriate substrate to product. In one
embodiment, the
assay comprises contacting a cell which expresses a membrane-bound form of a
FCTRX
protein, or a biologically active portion thereof, on the cell surface with a
known compound
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which binds a FCTRX to form an assay mixture, contacting the assay mixture
with a test
compound, and determining the ability of the test compound to interact with a
FCTRX protein,
wherein determining the ability of the test compound to interact with a FCTRX
protein
comprises determining the ability of the test compound to preferentially bind
to a FCTRX or a
biologically active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a membrane-bound form of a FCTRX protein, or a biologically active
portion
thereof, on the cell surface with a test compound and determining the ability
of the test
compound to modulate (e.g., stimulate or inhibit) the activity of the FCTRX
protein or
biologically active portion thereof. Determining the ability of the test
compound to modulate
the activity of a FCTRX polypeptide or a biologically active portion thereof
can be
accomplished, for example, by determining the ability of the FCTRX protein to
bind to or
interact with a FCTRX target molecule. As used herein, a "target molecule" is
a molecule
with which a FCTRX protein binds or interacts in nature, for example, a
molecule on the
surface of a cell which expresses a FCTRX interacting protein, a molecule on
the surface of a
second cell, a molecule in the extracellular milieu, a molecule associated
with the internal
surface of a cell membrane or a cytoplasmic molecule. A FCTRX target molecule
can be a
non-FCTRX molecule or a FCTRX protein or polypeptide of the present invention.
In one
embodiment, a FCTRX target molecule is a component of a signal transduction
pathway that
facilitates transduction of an extracellular signal (e.g., a signal generated
by binding of a
compound to a membrane-bound FCTRX molecule) through the cell membrane and
into the
cell. The target, for example, can be a second intercellular protein that has
catalytic activity or
a protein that facilitates the association of downstream signaling molecules
with the FCTRX
polypeptide.
Determining the ability of the FCTRX protein to bind to or interact with a
FCTRX
target molecule can be accomplished by one of the methods described above for
determining
direct binding. In one embodiment, determining the ability of the FCTRX
protein to bind to or
interact with a FCTRX target molecule can be accomplished by determining the
activity of the
target molecule. For example, the activity of the target molecule can be
determined by
detecting induction of a cellular second messenger of the target (i.e.
intracellular Ca2+,
diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the
target an appropriate
substrate, detecting the induction of a reporter gene (comprising a FCTRX-
responsive
regulatory element operatively linked to a nucleic acid encoding a detectable
marker, e.g.,
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luciferase), or detecting a cellular response, for example, cell survival,
cellular differentiation,
or cell proliferation.
In yet another embodiment, an assay of the present invention is a cell-free
assay
comprising contacting a FCTRX protein or biologically active portion thereof
with a test
compound and determining the ability of the test compound to bind to the FCTRX
protein or
biologically active portion thereof. Binding of the test compound to the FCTRX
protein can
be determined either directly or indirectly as described above. In one
embodiment, the assay
comprises contacting the FCTRX protein or biologically active portion thereof
with a known
compound which binds FCTRX to form an assay mixture, contacting the assay
mixture with a
test compound, and determining the ability of the test compound to interact
with a FCTRX
protein, wherein determining the ability of the test compound to interact with
a FCTRX
protein comprises determining the ability of the test compound to
preferentially bind to a
FCTRX or biologically active portion thereof as compared to the known
compound.
In another embodiment, an assay is a cell-free assay comprising contacting a
FCTRX
protein or biologically active portion thereof with a test compound and
determining the ability
of the test compound to modulate (e.g., stimulate or inhibit) the activity of
the FCTRX protein
or biologically active portion thereof. Determining the ability of the test
compound to
modulate the activity of a FCTRX polypeptide can be accomplished, for example,
by
determining the ability of the FCTRX protein to bind to a FCTRX target
molecule by one of
the methods described above for determining direct binding. In an alternative
embodiment,
determining the ability of the test compound to modulate the activity of a
FCTRX polypeptide
can be accomplished by determining the ability of the FCTRX protein further
modulate a
FCTRX target molecule. For example, the catalytic/enzymatic activity of the
target molecule
on an appropriate substrate can be determined as previously described.
In yet another embodiment, the cell-free assay comprises contacting the FCTRX
protein or biologically active portion thereof with a known compound which
binds a FCTRX
polypeptide to form an assay mixture, contacting the assay mixture with a test
compound, and
determining the ability of the test compound to interact with a FCTRX protein,
wherein
determining the ability of the test compound to interact with a FCTRX protein
comprises
determining the ability of the FCTRX protein to preferentially bind to or
modulate the activity
of a FCTRX target molecule.
The cell-free assays of the present invention are amenable to use of both a
soluble form
or a membrane-bound form of a FCTRX polypeptide. In the case of cell-free
assays
comprising the membrane-bound form of a FCTRX polypeptide, it may be desirable
to utilize
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a solubilizing agent such that the membrane-bound form of a FCTRX polypeptide
is
maintained in solution. Examples of such solubilizing agents include non-ionic
detergents
such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-
methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X-114,
Thesit~,
Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonio-1-
propane
sulfonate, 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS),
or 3-(3-
cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
It may be desirable to immobilize either a FCTRX polypeptide or its target
molecule to
facilitate separation of complexed from uncomplexed forms of one or both of
the proteins, as
well as to accommodate automation of the assay. Binding of a test compound to
a FCTRX
polypeptide, or interaction of a FCTRX polypeptide with a target molecule in
the presence and
absence of a candidate compound, can be accomplished in any vessel suitable
for containing
the reactants. Examples of such vessels include microtiter plates, test tubes,
and micro-
centrifuge tubes. In one embodiment, a fusion protein can be provided that
adds a domain that
allows one or both of the proteins to be bound to a matrix. For example, GST-
FCTRX
polypeptide fusion proteins or GST-target fusion proteins can be adsorbed onto
glutathione
sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized
microtiter plates,
that are then combined with the test compound or the test compound and either
the non-
adsorbed target protein or a FCTRX protein, and the mixture is incubated under
conditions
conducive to complex formation (e.g., at physiological conditions for salt and
pH). Following
incubation, the beads or microtiter plate wells are washed to remove any
unbound
components, the matrix immobilized in the case of beads, complex determined
either directly
or indirectly, for example, as described above. Alternatively, the complexes
can be
dissociated from the matrix, and the level of a FCTRX binding or activity
determined using
standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
screening assays of the invention. For example, either the FCTRX polypeptide
or its target
molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated
FCTRX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-
succinimide) using techniques well known in the art (e.g., biotinylation kit,
Pierce Chemicals,
Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well
plates (Pierce
Chemical). Alternatively, antibodies reactive with FCTRX protein or target
molecules, but
which do not interfere with binding of the FCTRX protein to its target
molecule, can be
derivatized to the wells of the plate, and unbound target or FCTRX protein
trapped in the wells
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by antibody conjugation. Methods for detecting such complexes, in addition to
those
described above for the GST-immobilized complexes, include immunodetection of
complexes
using antibodies reactive with the FCTRX protein or target molecule, as well
as enzyme-
linked assays that rely on detecting an enzymatic activity associated with the
FCTRX protein
or target molecule.
In another embodiment, modulators of a FCTRX expression are identified in a
method
wherein a cell is contacted with a candidate compound and the expression of a
FCTRX mRNA
or protein in the cell is determined. The level of expression of a FCTRX mRNA
or protein in
the presence of the candidate compound is compared to the level of expression
of a FCTRX
mRNA or protein in the absence of the candidate compound. The candidate
compound can
then be identified as a modulator of a FCTRX expression based on this
comparison. For
example, when expression of a FCTRX mRNA or protein is greater (statistically
significantly
greater) in the presence of the candidate compound than in its absence, the
candidate
compound is identified as a stimulator of a FCTRX mRNA or protein expression.
Alternatively, when expression of a FCTRX mRNA or protein is less
(statistically
significantly less) in the presence of the candidate compound than in its
absence, the candidate
compound is identified as an inhibitor of a FCTRX mRNA or protein expression.
The level of
a FCTRX mRNA or protein expression in the cells can be determined by methods
described
herein for detecting FCTRX mRNA or protein.
In yet another aspect of the invention, the FCTRX proteins can be used as
"bait
proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat.
No. 5,283,317;
Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem
268:12046-12054;
Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene
8:1693-1696;
and Brent W094/10300), to identify other proteins that bind to or interact
with the FCTRX
("FCTRX-binding proteins" or "FCTRX-by") and modulate FCTRX activity. Such
FCTRX-
binding proteins are also likely to be involved in the propagation of signals
by the FCTRX
proteins as, for example, upstream or downstream elements of the FCTRX
pathway.
The two-hybrid system is based on the modular nature of most transcription
factors,
which consist of separable DNA-binding and activation domains. Briefly, the
assay utilizes
two different DNA constructs. In one construct, the gene that codes for a
FCTRX is fused to a
gene encoding the DNA binding domain of a known transcription factor (e.g.,
GAL-4). In the
other construct, a DNA sequence, from a library of DNA sequences, that encodes
an
unidentified protein ("prey" or "sample") is fused to a gene that codes for
the activation
domain of the known transcription factor. If the "bait" and the "prey"
proteins are able to
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interact, in vivo, forming a FCT1RX-dependent complex, the DNA-binding and
activation
domains of the transcription factor are brought into close proximity. This
proximity allows
transcription of a reporter gene (e.g., LacZ) that is operably linked to a
transcriptional
regulatory site responsive to the transcription factor. Expression of the
reporter gene can be
detected and cell colonies containing the functional transcription factor can
be isolated and
used to obtain the cloned gene that encodes the protein which interacts with
the FCT1RX.
Screening can also be performed in vivo. For example, in one embodiment, the
invention includes a method for screening for a modulator of activity or of
latency or
predisposition to a FCTItX-associated disorder by administering a test
compound or to a test
animal at increased risk for a FCTItX-associated disorder. In some
embodiments, the test
animal recombinantly expresses a FCTItX polypeptide. Activity of the
polypeptide in the test
animal after administering the compound is measured, and the activity of the
protein in the
test animal is compared to the activity of the polypeptide in a control animal
not administered
said polypeptide. A change in the activity of said polypeptide in said test
animal relative to
. the control animal indicates the test compound is a modulator of latency of
or predisposition to
a FCT1ZX-associated disorder.
In some embodiments, the test animal is a recombinant test animal that
expresses a test
protein transgene or expresses the transgene under the control of a promoter
at an increased
level relative to a wild-type test animal. Preferably, the promoter is not the
native gene
promoter of the transgene.
This invention further pertains to novel agents identified by the above-
described
screening assays and uses thereof for treatments as described herein.
Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the
corresponding
complete gene sequences) can be used in numerous ways as polynucleotide
reagents. For
example, these sequences can be used to: (i) map their respective genes on a
chromosome;
and, thus, locate gene regions associated with genetic disease; (ii) identify
an individual from a
minute biological sample (tissue typing); and (iii) aid in forensic
identification of a biological
sample.
The FCTRX sequences of the present invention can also be used to identify
individuals
from minute biological samples. In this technique, an individual's genomic DNA
is digested
with one or more restriction enzymes, and probed on a Southern blot to yield
unique bands for
identification. The sequences of the present invention are useful as
additional DNA markers
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for RFLP ("restriction fragment length polymorphisms," described in U.S. Pat.
No.
5,272,057).
Furthermore, the sequences of the present invention can be used to provide an
alternative technique that determines the actual base-by-base DNA sequence of
selected
portions of an individual's genome. Thus, the FCTRX sequences described herein
can be used
to prepare two PCR primers from the 5' and 3' ends of the sequences. These
primers can then
be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this
manner,
can provide unique individual identifications, as each individual will have a
unique set of such
DNA sequences due to allelic differences. The sequences of the present
invention can be used
to obtain such identification sequences from individuals and from tissue. The
FCTRX
sequences of the invention uniquely represent portions of the human genome.
Allelic
variation occurs to some degree in the coding regions of these sequences, and
to a greater
degree in the noncoding regions. It is estimated that allelic variation
between individual
~ humans occurs with a frequency of about once per each 500 bases. Much of the
allelic
variation is due to single nucleotide polymorphisms (SNPs), which include
restriction
fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a
standard
against which DNA from an individual can be compared for identification
purposes. Because
greater numbers of polymorphisms occur in the noncoding regions, fewer
sequences are
necessary to differentiate individuals. The noncoding sequences of SEQ ID
NOS:1, 3, 5, 7, 9
and' 11, as described above, can comfortably provide positive individual
identification with a
panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified
sequence of 100
bases. If predicted coding sequences are used, a more appropriate number of
primers for
positive individual identification would be 500-2,000.
Use Of Partial FCT1:ZX Sequences In Forensic Biology
DNA-based identification techniques based on FCTRX nucleic acid sequences or
polypeptide sequences can also be used in forensic biology. Forensic biology
is a scientific
field employing genetic typing of biological evidence found at a crime scene
as a means for
positively identifying, for example, a perpetrator of a crime. To make such an
identification,
PCR technology can be used to amplify DNA sequences taken from very small
biological
samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at
a crime scene. The amplified sequence can then be compared to a standard,
thereby allowing
identification of the origin of the biological sample.
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The sequences of the present invention can be used to provide polynucleotide
reagents,
e.g., PCR primers, targeted to specific loci in the human genome, that can
enhance the
reliability of DNA-based forensic identifications by, for example, providing
another
"identification marker" (i.e. another DNA sequence that is unique to a
particular individual).
As mentioned above, actual base sequence information can be used for
identification as an
accurate alternative to patterns formed by restriction enzyme generated
fragments. Sequences
targeted to noncoding regions of SEQ ID NOS:1, 3, 5, 7, 9 and 11 are
particularly appropriate
for this use as greater numbers of polymorphisms occur in the noncoding
regions, making it
easier to differentiate individuals using this technique. Examples of
polynucleotide reagents
include the FCTRX sequences or portions thereof, e.g., fragments derived from
the noncoding
regions of one or more of SEQ ID NOS:1, 3, S, 7, 9 and 11, having a length of
at least 20
bases, preferably at least 30 bases.
The FCTRX sequences described herein can further be used to provide
polynucleotide
reagents, e.g., labeled or label-able probes that can be used, for example, in
an in situ
hybridization technique, to identify a specific tissue, e.g., brain tissue,
etc. This can be very
useful in cases where a forensic pathologist is presented with a tissue of
unknown origin.
Panels of such FCTRX probes can be used to identify tissue by species and/or
by organ type.
In a similar fashion, these reagents, e.g., FCTRX primers or probes can be
used to
screen tissue culture for contamination (i.e. screen for the presence of a
mixture of different
types of cells in a culture).
Predictive Medicine
The present invention also pertains to the field of predictive medicine in
which
diagnostic assays, prognostic assays, pharmacogenomics, and monitoring
clinical trials are
used for prognostic (predictive) purposes to thereby treat an individual
prophylactically.
Accordingly, one aspect of the present invention relates to diagnostic assays
for determining a
FCTRX protein and/or nucleic acid expression as well as FCTRX activity, in the
context of a
biological sample (e.g., blood, serum, cells, tissue) to thereby determine
whether an individual
is afflicted with a disease or disorder, or is at risk of developing a
disorder, associated with
aberrant FCTRX expression or activity. The invention also provides for
prognostic (or
predictive) assays for determining whether an individual is at risk of
developing a disorder
associated with a FCTRX protein, nucleic acid expression or activity. For
example, mutations
in a FCTRX gene can be assayed in a biological sample. Such assays can be used
for
prognostic or predictive purpose to thereby prophylactically treat an
individual prior to the
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onset of a disorder characterized by or associated with FCTRX protein, nucleic
acid
expression or activity.
Another aspect of the invention provides methods for determining FCTRX
protein,
nucleic acid expression or FCTRX activity in an individual to thereby select
appropriate
therapeutic or prophylactic agents for that individual (referred to herein as
"pharmacogenomics"). Pharmacogenomics allows for the selection of agents
(e.g., drugs) for
therapeutic or prophylactic treatment of an individual based on the genotype
of the individual
(e.g., the genotype of the individual examined to determine the ability of the
individual to
respond to a particular agent.)
Yet another aspect of the invention pertains to monitoring the influence of
agents (e.g.,
drugs, compounds) on the expression or activity of a FCTRX in clinical trials.
These and other agents are described in further detail in the following
sections.
Diagnostic Assays
Other conditions in which proliferation of cells plays a role include tumors,
restenosis,
1 S psoriasis, Dupuytren's contracture, diabetic complications, Kaposi's
sarcoma and rheumatoid
arthritis.
A FCTRX polypeptide may be used to identify an interacting polypeptide a
sample or
tissue. The method comprises contacting the sample or tissue with the FCTRX,
allowing
formation of a complex between the FCTRX polypeptide and the interacting
polypeptide, and
detecting the complex, if present.
The proteins of the invention may be used to stimulate production of
antibodies
specifically binding the proteins. Such antibodies may be used in
immunodiagnostic
procedures to detect the occurrence of the protein in a sample. The proteins
of the invention
may be used to stimulate cell growth and cell proliferation in conditions in
which such growth
would be favorable. An example would be to counteract toxic side effects of
chemotherapeutic agents on, for example, hematopoiesis and platelet formation,
linings of the
gastrointestinal tract, and hair follicles. They may also be used to stimulate
new cell growth in
neurological disorders including, for example, Alzheimer's disease.
Alternatively,
antagonistic treatments may be administered in which an antibody specifically
binding the
FCTRX-like proteins of the invention would abrogate the specific growth-
inducing effects of
the proteins. Such antibodies may be useful, for example, in the treatment of
proliferative
disorders including various tumors and benign hyperplasias.
Polynucleotides or oligonucleotides corresponding to any one portion of the
FCTRX
nucleic acids of SEQ ID NOS:1, 3, 5, 7, 9 and 11 may be used to detect DNA
containing a
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corresponding ORF gene, or detect the expression of a corresponding FCTRX
gene, or
FCTRX-like gene. For example, a FCTRX nucleic acid expressed in a particular
cell or tissue,
as noted in Table 3, can be used to identify the presence of that particular
cell type.
An exemplary method for detecting the presence or absence of a FCTRX
polypeptide
in a biological sample involves obtaining a biological sample from a test
subject and
contacting the biological sample with a compound or an agent capable of
detecting a FCTRX
protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes a FCTRX protein
such that
the presence of a FCTRX polypeptide is detected in the biological sample. An
agent for
detecting a FCTRX mRNA or genomic DNA is a labeled nucleic acid probe capable
of
hybridizing to a FCTRX mRNA or genomic DNA. The nucleic acid probe can be, for
example, a full-length FCTRX nucleic acid, such as the nucleic acid of SEQ ID
NOS:1, 3, 5,
7, 9 and 1 l, or a portion thereof, such as an oligonucleotide of at least 15,
30, 50, 100, 250 or
500 nucleotides in length and sufficient to specifically hybridize under
stringent conditions to
a FCTRX mRNA or genomic DNA, as described above. Other suitable probes for use
in the
diagnostic assays of the invention are described herein.
An agent for detecting a FCTRX protein is an antibody capable of binding to a
FCTRX
protein, preferably an antibody with a detectable label. Antibodies can be
polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab
or F(ab')2) can be
used. The term "labeled", with regard to the probe or antibody, is intended to
encompass
direct labeling of the probe or antibody by coupling (i.e., physically
linking) a detectable
substance to the probe or antibody, as well as indirect labeling of the probe
or antibody by
reactivity with another reagent that is directly labeled. Examples of indirect
labeling include
detection of a primary antibody using a fluorescently labeled secondary
antibody and end-
labeling of a DNA probe with biotin such that it can be detected with
fluorescently labeled
streptavidin. The term "biological sample" is intended to include tissues,
cells and biological
fluids isolated from a subject, as well as tissues, cells and fluids present
within a subject. That
is, the detection method of the invention can be used to detect a FCTRX mRNA,
protein, or
genomic DNA in a biological sample in vitro as well as in vivo. For example,
in vitro
techniques for detection of a FCTRX mRNA include Northern hybridizations and
in situ
hybridizations. In vitro techniques for detection of a FCTRX protein include
enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of a FCTRX genomic DNA
include
Southern hybridizations. Furthermore, in vivo techniques for detection of a
FCTRX protein
include introducing into a subject a labeled anti-FCTRX antibody. For example,
the antibody
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can be labeled with a radioactive marker whose presence and location in a
subject can be
detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the
test
subject. Alternatively, the biological sample can contain mRNA molecules from
the test
subject or genomic DNA molecules from the test subject. A preferred biological
sample is a
peripheral blood leukocyte sample isolated by conventional means from a
subject.
In another embodiment, the methods further involve obtaining a control
biological
sample from a control subject, contacting the control sample with a compound
or agent
capable of detecting a FCTRX protein, mRNA, or genomic DNA, such that the
presence of a
FCTRX protein, mRNA or genomic DNA is detected in the biological sample, and
comparing
the presence of a FCTRX protein, mRNA or genomic DNA in the control sample
with the
presence of a FCTRX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of a FCTRX
polypeptide in a biological sample. For example, the kit can comprise: a
labeled compound or
agent capable of detecting a FCTRX protein or mlZNA in a biological sample;
means for
determining the amount of a FCTRX polypeptide in the sample; and means for
comparing the
amount of a FCTRX polypeptide in the sample with a standard. The compound or
agent can
be packaged in a suitable container. The kit can further comprise instructions
for using the kit
to detect a FCTRX protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant FCTRX
polypeptide expression or activity. For example, the assays described herein,
such as the
preceding diagnostic assays or the following assays, can be utilized to
identify a subject
having or at risk of developing a disorder associated with a FCTRX protein,
nucleic acid
expression or activity in, e.g., proliferative or differentiative disorders
such as hyperplasias,
tumors, restenosis, psoriasis, Dupuytren's contracture, diabetic
complications, or rheumatoid
arthritis, etc.; and glia-associated disorders such as cerebral lesions,
diabetic neuropathies,
cerebral edema, senile dementia, Alzheimer's disease, etc. Alternatively, the
prognostic
assays can be utilized to identify a subject having or at risk for developing
a disease or
disorder. Thus, the present invention provides a method for identifying a
disease or disorder
associated with aberrant FCTRX expression or activity in which a test sample
is obtained from
a subject and a FCTRX protein or nucleic acid (e.g., mRNA, genomic DNA) is
detected,
wherein the presence of a FCTRX protein or nucleic acid is diagnostic for a
subject having or
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at risk of developing a disease or disorder associated with aberrant FCTRX
expression or
activity. As used herein, a "test sample" refers to a biological sample
obtained from a subject
of interest. For example, a test sample can be a biological fluid (e.g.,
serum), cell sample, or
tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether
a subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein,
peptide, nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder
associated with aberrant FCTRX expression or activity. For example, such
methods can be
used to determine whether a subject can be effectively treated with an agent
for a disorder,
such as a proliferative disorder, differentiative disorder, glia-associated
disorders, etc. Thus,
the present invention provides methods for determining whether a subject can
be effectively
treated with an agent for a disorder associated with aberrant FCTRX expression
or activity in
which a test sample is obtained and a FCTRX protein or nucleic acid is
detected (e.g., wherein
the presence of a FCTRX protein or nucleic acid is diagnostic for a subject
that can be
administered the agent to treat a disorder associated with aberrant FCTRX
expression or
activity.)
The methods of the invention can also be used to detect genetic lesions in a
FCTRX
gene, thereby determining if a subject with the lesioned gene is at risk for,
or suffers from, a
proliferative disorder, differentiative disorder, glia-associated disorder,
etc. In various
embodiments, the methods include detecting, in a sample of cells from the
subject, the
presence or absence of a genetic lesion characterized by at least one of an
alteration affecting
the integrity of a gene encoding a FCTRX protein, or the mis-expression of the
FCTRX gene.
For example, such genetic lesions can be detected by ascertaining the
existence of at least one
of (1) a deletion of one or more nucleotides from a FCTRX gene; (2) an
addition of one or
more nucleotides to a FCTRX gene; (3) a substitution of one or more
nucleotides of a FCTRX
gene, (4) a chromosomal rearrangement of a FCTRX gene; (5) an alteration in
the level of a
messenger RNA transcript of a FCTRX gene, (6) aberrant modification of a FCTRX
gene,
such as of the methylation pattern of the genomic DNA, (7) the presence of a
non-wild type
splicing pattern of a messenger RNA transcript of a FCTRX gene, (8) a non-wild
type level of
a protein, (9) allelic loss of a FCTRX gene, and (10) inappropriate post-
translational
modification of a FCTRX protein. As described herein, there are a large number
of assay
techniques known in the art which can be used for detecting lesions in a FCTRX
gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by
conventional
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means from a subject. However, any biological sample containing nucleated
cells may be
used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a
probe/primer in a
polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and
4,683,202), such as
anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g.,
Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS
91:360-
364), the latter of which can be particularly useful for detecting point
mutations in the FCTRX
gene (see Abravaya et al. (1995) Nucl Acids Res 23:675-682). This method can
include the
steps of collecting a sample of cells from a patient, isolating nucleic acid
(e.g., genomic,
mRNA or both) from the cells of the sample, contacting the nucleic acid sample
with one or
more primers that specifically hybridize to a FCTRX gene under conditions such
that
hybridization and amplification of the FCTRX gene (if present) occurs, and
detecting the
presence or absence of an amplification product, or detecting the size of the
amplification
product and comparing the length to a control sample. It is anticipated that
PCR and/or LCR
may be desirable to use as a preliminary amplification step in conjunction
with any of the
techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication
(Guatelli et al., 1990, Proc Natl Acad Sci USA 87:1874-1878), transcriptional
amplification
system (Kwoh, et al., 1989, Proc Natl Acad Sci USA 86:1173-1177), Q-Beta
Replicase
(Lizardi et al, 1988, BioTechnology 6:1197), or any other nucleic acid
amplification method,
followed by the detection of the amplified molecules using techniques well
known to those of
skill in the art. These detection schemes are especially useful for the
detection of nucleic acid
molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in a FCTRX gene from a sample cell can
be
identified by alterations in restriction enzyme cleavage patterns. For
example, sample and
control DNA is isolated, amplified (optionally), digested with one or more
restriction
endonucleases, and fragment length sizes are determined by gel electrophoresis
and compared.
Differences in fragment length sizes between sample and control DNA indicates
mutations in
the sample DNA. Moreover, the use of sequence specific ribozymes (see, for
example, U.S.
Pat. No. 5,493,531) can be used to score for the presence of specific
mutations by
development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in a FCTRX nucleic acid of the
invention can
be identified by hybridizing a sample and control nucleic acids, e.g., DNA or
RNA, to high
density arrays containing hundreds or thousands of oligonucleotides probes
(Cronin et al.
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(1996) Human Mutation 7: 244-255; Kozal et al. (1996) Nature Medicine 2: 753-
759). For
example, genetic mutations in a FCTRX of the invention can be identified in
two dimensional
arrays containing light-generated DNA probes as described in Cronin et al.
above. Briefly, a
first hybridization array of probes can be used to scan through long stretches
of DNA in a
sample and control to identify base changes between the sequences by making
linear arrays of
sequential overlapping probes. This step allows the identification of point
mutations. This
step is followed by a second hybridization array that allows the
characterization of specific
mutations by using smaller, specialized probe arrays complementary to all
variants or
mutations detected. Each mutation array is composed of parallel probe sets,
one
complementary to the wild-type gene and the other complementary to the mutant
gene.
In yet another embodiment, any of a variety of sequencing reactions known in
the art
can be used to directly sequence the FCTRX gene and detect mutations by
comparing the
sequence of the sample FCTRX gene with the corresponding wild-type (control)
sequence.
Examples of sequencing reactions include those based on techniques developed
by Maxim and
Gilbert (1977) PNAS 74:560 or Sanger (1977) PNAS 74:5463. It is also
contemplated that any
of a variety of automated sequencing procedures can be utilized when
performing the
diagnostic assays (Naeve et al., (1995) Biotechniques 19:448), including
sequencing by mass
spectrometry (see, e.g., PCT International Publ. No. WO 94/16101; Cohen et al.
(1996) Adv
Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol
38:147-159).
Other methods for detecting mutations in the FCTRX gene include methods in
which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or
RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the
art
technique of "mismatch cleavage" starts by providing heteroduplexes of formed
by
hybridizing (labeled) RNA or DNA containing the wild-type FCTRX sequence with
potentially mutant RNA or DNA obtained from a tissue sample. The double-
stranded
duplexes are treated with an agent that cleaves single-stranded regions of the
duplex such as
which will exist due to basepair mismatches between the control and sample
strands. For
instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids
treated with
S 1 nuclease to enzymatically digesting the mismatched regions. In other
embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium
tetroxide
and with piperidine in order to digest mismatched regions. After digestion of
the mismatched
regions, the resulting material is then separated by size on denaturing
polyacrylamide gels to
determine the site of mutation. See, for example, Cotton et al (1988) Proc
Natl Acad Sci USA
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85:4397; Saleeba et al (1992) Methods Enzymol 217:286-295. In an embodiment,
the control
DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more
proteins that recognize mismatched base pairs in double-stranded DNA (so
called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point
mutations in
FCTRX cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli
cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells
cleaves T
at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According
to an
exemplary embodiment, a probe based on a FCTRX sequence, e.g., a wild-type
FCTRX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is
treated with a DNA mismatch repair enzyme, and the cleavage products, if any,
can be
detected from electrophoresis protocols or the like. See, for example, U.S.
Pat. No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify
mutations in FCTRX genes. For example, single strand conformation polymorphism
(SSCP)
may be used to detect differences in electrophoretic mobility between mutant
and wild type
nucleic acids (Orita et al. (1989) Proc Natl Acad Sci USA: 86:2766, see also
Cotton (1993)
Mutat Res 285:125-144; Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-
stranded
DNA fragments of sample and control a FCTRX nucleic acids will be denatured
and allowed
to renature. The secondary structure of single-stranded nucleic acids varies
according to
sequence, the resulting alteration in electrophoretic mobility enables the
detection of even a
single base change. The DNA fragments may be labeled or detected with labeled
probes. ~ The
sensitivity of the assay may be enhanced by using RNA, rather than DNA, in
which the
secondary structure is more sensitive to a change in sequence. In one
embodiment, the subject
method utilizes heteroduplex analysis to separate double stranded heteroduplex
molecules on
the basis of changes in electrophoretic mobility. See, e.g., Keen et al.
(1991) Trends Genet
7:5.
In yet another embodiment the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing gradient
gel electrophoresis (DGGE). See, e.g., Myers et al (1985) Nature 313:495. When
DGGE is
used as the method of analysis, DNA will be modified to insure that it does
not completely
denature, for example by adding a GC clamp of approximately 40 by of high-
melting GC-rich
DNA by PCR. In a further embodiment, a temperature gradient is used in place
of a
denaturing gradient to identify differences in the mobility of control and
sample DNA. See,
e.g., Rosenbaum and Reissner (1987) Biophys Chem 265:12753.
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Examples of other techniques for detecting point mutations include, but are
not limited
to, selective oligonucleotide hybridization, selective amplification, or
selective primer
extension. For example, oligonucleotide primers may be prepared in which the
known
mutation is placed centrally and then hybridized to target DNA under
conditions that permit
hybridization only if a perfect match is found. See, e.g., Saiki et al. (1986)
Nature 324:163);
Saiki et al. (1989) Proc Natl Acad. Sci USA 86:6230. Such allele specific
oligonucleotides
are hybridized to PCR amplified target DNA or a number of different mutations
when the
oligonucleotides are attached to the hybridizing membrane and hybridized with
labeled target
DNA.
Alternatively, allele specific amplification technology that depends on
selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as
primers for specific amplification may carry the mutation of interest in the
center of the
molecule (so that amplification depends on differential hybridization) (Gibbs
et al. (1989)
Nucleic Acids Res 17:2437-2448) or at the extreme 3' end of one primer where,
under
appropriate conditions, mismatch can prevent, or reduce polymerase extension
(Prossner
(1993) Tibtech 11:238). In addition it may be desirable to introduce a novel
restriction site in
the region of the mutation to create cleavage-based detection. See, e.g.,
Gasparini et al (1992)
Mol Cell Probes 6:1. It is anticipated that in certain embodiments
amplification may also be
performed using Taq ligase for amplification. See, e.g., Barany (1991) Proc
Natl Acad Sci
USA 88:189. In such cases, ligation will occur only if there is a perfect
match at the 3' end of
the 5' sequence, making it possible to detect the presence of a known mutation
at a specific site
by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre
packaged diagnostic kits comprising at least one probe nucleic acid or
antibody reagent
described herein, which may be conveniently used, e.g., in clinical settings
to diagnose
patients exhibiting symptoms or family history of a disease or illness
involving a FCTRX
gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes,
in which a
FCTRX of the invention is expressed may be utilized in the prognostic assays
described
herein. However, any biological sample containing nucleated cells may be used,
including, for
example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on FCTRX
activity
(e.g., FCTRX gene expression), as identified by a screening assay described
herein can be
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administered to individuals to treat (prophylactically or therapeutically)
disorders (e.g.,
neurological, cancer-related or gestational disorders) associated with
aberrant FCTRX activity.
In conjunction with such treatment, the pharmacogenomics (i.e., the study of
the relationship
between an individual's genotype and that individual's response to a foreign
compound or
S drug) of the individual may be considered. Differences in metabolism of
therapeutics can lead
to severe toxicity or therapeutic failure by altering the relation between
dose and blood
concentration of the pharmacologically active drug. Thus, the pharmacogenomics
of the
individual permits the selection of effective agents (e.g., drugs) for
prophylactic or therapeutic
treatments based on a consideration of the individual's genotype. Such
pharmacogenomics can
further be used to determine appropriate dosages and therapeutic regimens.
Accordingly, the
activity of a FCTRX protein, expression of a FCTRX nucleic acid, or mutation
content of a
FCTRX genes in an individual can be determined to thereby select appropriate
agents) for
therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected persons. See
e.g., Eichelbaum, 1996, Clin Exp Pharmacol Physiol, 23:983-985 and Linden
1997, Clin
Chem, 43:254-266. In general, two types of pharmacogenetic conditions can be
differentiated.
Genetic conditions transmitted as a single factor altering the way drugs act
on the body
(altered drug action) or genetic conditions transmitted as single factors
altering the way the
body acts on drugs (altered drug metabolism). These pharmacogenetic conditions
can occur
either as rare defects or as polymorphisms. For example, glucose-6-phosphate
dehydrogenase
(G6PD) deficiency is a common inherited enzymopathy in which the main clinical
complication is haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides,
analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major
determinant of both the intensity and duration of drug action. The discovery
of genetic
polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and
cytochrome P450 enzymes CYP2D6 and CYP2C 19) has provided an explanation as to
why
some patients do not obtain the expected drug effects or show exaggerated drug
response and
serious toxicity after taking the standard and safe dose of a drug. These
polymorphisms are
expressed in two phenotypes in the population, the extensive metabolizer (EM)
and poor
metabolizer (PM). The prevalence of PM is different among different
populations. For
example, the gene coding for CYP2D6 is highly polymorphic and several
mutations have been
identified in PM, which all lead to the absence of functional CYP2D6. Poor
metabolizers of
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CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and
side
effects when they receive standard doses. If a metabolite is the active
therapeutic moiety, PM
show no therapeutic response, as demonstrated for the analgesic effect of
codeine mediated by
its CYP2D6-formed metabolite morphine. The other extreme are the so called
ultra-rapid
metabolizers who do not respond to standard doses. Recently, the molecular
basis of ultra-
rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of a FCTRX protein, expression of a FCTRX nucleic acid, or
mutation content of a FCTRX genes in an individual can be determined to
thereby select
appropriate agents) for therapeutic or prophylactic treatment of the
individual. In addition,
pharmacogenetic studies can be used to apply genotyping of polymorphic alleles
encoding
drug-metabolizing enzymes to the identification of an individual's drug
responsiveness
phenotype. This knowledge, when applied to dosing or drug selection, can avoid
adverse
reactions or therapeutic failure and thus enhance therapeutic or prophylactic
efficiency when
treating a subject with a FCTRX modulator, such as a modulator identified by
one of the
exemplary screening assays described herein.
Monitoring Clinical Efficacy
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity of a FCTRX (e.g., the ability to modulate aberrant cell proliferation
and/or
differentiation) can be applied in basic drug screening and in clinical
trials. For example, the
effectiveness of an agent determined by a screening assay as described herein
to increase
FCTRX gene expression, protein levels, or upregulate FCTRX activity, can be
monitored in
clinical trials of subjects exhibiting decreased FCTRX gene expression,
protein levels, or
downregulated FCTRX activity. Alternatively, the effectiveness of an agent
determined by a
screening assay to decrease FCTRX gene expression, protein levels, or
downregulate FCTRX
activity, can be monitored in clinical trials of subjects exhibiting increased
FCTRX gene
expression, protein levels, or upregulated FCTRX activity. In such clinical
trials, the
expression or activity of a FCTRX and, preferably, other genes that have been
implicated in,
for example, a proliferative or neurological disorder, can be used as a "read
out" or marker of
the responsiveness of a particular cell. Other FCTRX-associated disorders
include, e.g.,
cancers, cell proliferation disorders, anxiety disorders; CNS disorders;
diabetes; obesity; and
infectious disease.
For example, genes, including genes encoding a FCTRX of the invention, that
are
modulated in cells by treatment with an agent (e.g., compound, drug or small
molecule) that
modulates a FCTRX activity (e.g., identified in a screening assay as described
herein) can be
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identified. Thus, to study the effect of agents on cellular proliferation
disorders, for example,
in a clinical trial, cells can be isolated and RNA prepared and analyzed for
the levels of
expression of a FCTRX and other genes implicated in the disorder. The levels
of gene
expression (i.e., a gene expression pattern) can be quantified by Northern
blot analysis or RT-
PCR, as described herein, or alternatively by measuring the amount of protein
produced, by
one of the methods as described herein, or by measuring the levels of activity
of a gene or
other genes. In this way, the gene expression pattern can serve as a marker,
indicative of the
physiological response of the cells to the agent. Accordingly, this response
state may be
determined before, and at various points during, treatment of the individual
with the agent.
In one embodiment, the invention provides a method for monitoring the
effectiveness
of treatment of a subject with an agent (e.g., an agonist, antagonist,
protein, peptide, nucleic
acid, peptidomimetic, small molecule, or other drug candidate identified by
the screening
assays described herein) comprising the steps of (i) obtaining a pre-
administration sample
from a subject prior to administration of the agent; (ii) detecting the level
of expression of a
FCTRX protein, mRNA, or genomic DNA in the preadministration sample; (iii)
obtaining one
or more post-administration samples from the subject; (iv) detecting the level
of expression or
activity of the FCTRX protein, mRNA, or genomic DNA in the post-administration
samples;
(v) comparing the level of expression or activity of the FCTRX protein, mRNA,
or genomic
DNA in the pre-administration sample with the FCTRX protein, mRNA, or genomic
DNA in
the post administration sample or samples; and (vi) altering the
administration of the agent to
the subject accordingly. For example, increased administration of the agent
may be desirable
to increase the expression or activity of a FCTRX to higher levels than
detected, i.e., to
increase the effectiveness of the agent. Alternatively, decreased
administration of the agent
may be desirable to decrease expression or activity of a FCTRX to lower levels
than detected,
i.e., to decrease the effectiveness of the agent.
Methods of Treatment
The present invention provides for both prophylactic and therapeutic methods
of
treating a subject at risk of (or susceptible to) a disorder or having a
disorder associated with
aberrant FCTRX expression or activity.
Diseases and disorders that are characterized by increased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics
that antagonize
activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may
be utilized include, but are not limited to, (i) a FCTRX polypeptide, or
analogs, derivatives,
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fragments or homologs thereof; (ii) antibodies to a FCTRX peptide; (iii)
nucleic acids
encoding a FCTRX peptide; (iv) administration of antisense nucleic acid and
nucleic acids that
are "dysfunctional" (i.e., due to a heterologous insertion within the coding
sequences of
coding sequences to a FCTRX polypeptide) that are utilized to "knockout"
endogenous
function of a FCTRX polypeptide by homologous recombination (see, e.g.,
Capecchi, 1989,
Science 244: 1288-1292); or (v) modulators (i.e., inhibitors, agonists and
antagonists,
including additional peptide mimetic of the invention or antibodies specific
to a peptide of the
invention) that alter the interaction between a FCTRX peptide and its binding
partner.
Diseases and disorders that are characterized by decreased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that
upregulate activity
may be administered in a therapeutic or prophylactic manner. Therapeutics that
may be
utilized include, but are not limited to, a polypeptide, a peptide, or
analogs, derivatives,
fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide
and/or
RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and
assaying it in vitro for
RNA or polypeptide levels, structure and/or activity of the expressed
polypeptides (or mRNAs
encoding. a FCTRX polypeptide). Methods that are well-known within the art
include, but are
not limited to, immunoassays (e.g., by Westein blot analysis,
immunoprecipitation followed
by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis,
immunocytochemistry,
etc.) and/or hybridization assays to detect expression of mRNAs (e.g.,
Northern assays, dot
blots, in situ hybridization, etc.).
In one aspect, the invention provides a method for preventing, in a subject, a
disease or
condition associated with aberrant FCTRX expression or activity, by
administering to the
subject an agent that modulates FCTRX expression or at least one FCTRX
activity. Subjects
at risk for a disease that is caused or contributed to by aberrant FCTRX
expression or activity
can be identified by, for example, any or a combination of diagnostic or
prognostic assays as
described herein. Administration of a prophylactic agent can occur prior to
the manifestation
of symptoms characteristic of the FCTRX aberrancy, such that a disease or
disorder is
prevented or, alternatively, delayed in its progression. Depending on the type
of a FCTRX
aberrancy, for example, a FCTRX agonist or FCTRX antagonist agent can be used
for treating
the subject. The appropriate agent can be determined based on screening assays
described
herein.
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Another aspect of the invention pertains to methods of modulating FCT1ZX
expression
or activity for therapeutic purposes. The modulatory method of the invention
involves
contacting a cell with an agent that modulates one or more of the activities
of a FCTRX
protein activity associated with the cell. An agent that modulates a FCTRX
protein activity
can be an agent as described herein, such as a nucleic acid or a protein, a
naturally-occurring
cognate ligand of a FCTRX protein, a peptide, a FCTRX peptidomimetic, or other
small
molecule. In one embodiment, the agent stimulates one or more a FCT1RX protein
activity.
Examples of such stimulatory agents include active a FCTRX protein and a
nucleic acid
molecule encoding a FCTRX polypeptide that has been introduced into the cell.
In another
embodiment, the agent inhibits one or more a FCTRX protein activity. Examples
of such
inhibitory agents include antisense a FCTRX nucleic acid molecules and anti-
FCTItX
antibodies. These modulatory methods can be performed in vitro (e.g., by
culturing the cell
with the agent) or, alternatively, in vivo (e.g., by administering the agent
to a subject). As
such, the present invention provides methods of treating an individual
afflicted with a disease
or disorder characterized by aberrant expression or activity of a FCTRX
protein or nucleic
acid molecule. In one embodiment, the method involves administering an agent
(e.g., an agent
identified by a screening assay described herein), or combination of agents
that modulates
(e.g., upregulates or downregulates) FCTRX expression or activity. In another
embodiment,
the method involves administering a FCT1RX protein or nucleic acid molecule as
therapy to
compensate for reduced or aberrant FCTRX expression or activity.
Determination of the Biological Effect of a Therapeutic
In various embodiments of the present invention, suitable in vitro or in vivo
assays are
utilized to determine the effect of a specific Therapeutic and whether its
administration is
indicated for treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with
representative
cells of the types) involved in the patient's disorder, to determine if a
given Therapeutic exerts
the desired effect upon the cell type(s). Compounds for use in therapy may be
tested in
suitable animal model systems including, but not limited to rats, mice,
chicken, cows,
monkeys, rabbits, and the like, prior to testing in human subjects. Similarly,
for in vivo testing,
any of the animal model system known in the art may be used prior to
administration to human
subj ects.
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Malignancies
Some FCTRX polypeptides are expressed in cancerous cells and are therefore
implicated in the regulation of cell proliferation. Accordingly, Therapeutics
of the present
invention may be useful in the therapeutic or prophylactic treatment of
diseases or disorders
that are associated with cell hyperproliferation and/or loss of control of
cell proliferation (e.g.,
cancers, malignancies and tumors). For a review of such hyperproliferation
disorders, see e.g.,
Fishman, et al., 1985. MEDICINE, 2nd ed., J.B. Lippincott Co., Philadelphia,
PA.
Therapeutics of the present invention may be assayed by any method known
within the
art for efficacy in treating or preventing malignancies and related disorders.
Such assays
include, but are not limited to, in vitro assays utilizing transformed cells
or cells derived from
the patient's tumor, as well as in vivo assays using animal models of cancer
or malignancies.
Potentially effective Therapeutics are those that, for example, inhibit the
proliferation of
tumor-derived or transformed cells in culture or cause a regression of tumors
in animal
models, in comparison to the controls.
In the practice of the present invention, once a malignancy or cancer has been
shown to
be amenable to treatment by modulating (i.e., inhibiting, antagonizing or
agonizing) activity,
that cancer or malignancy may subsequently be treated or prevented by the
administration of a
Therapeutic that serves to modulate protein function.
Premalignant Conditions
The Therapeutics of the present invention that are effective in the
therapeutic or
prophylactic treatment of cancer or malignancies may also be administered for
the treatment of
pre-malignant conditions and/or to prevent the progression of a pre-malignancy
to a neoplastic
or malignant state. Such prophylactic or therapeutic use is indicated in
conditions known or
suspected of preceding progression to neoplasia or cancer, in particular,
where non-neoplastic
cell growth consisting of hyperplasia, metaplasia or, most particularly,
dysplasia has occurred.
For a review of such abnormal cell growth see e.g., Robbins & Angell, 1976.
BASIC
PATHOLOGY, 2nd ed., W.B. Saunders Co., Philadelphia, PA.
Hyperplasia is a form of controlled cell proliferation involving an increase
in cell
number in a tissue or organ, without significant alteration in its structure
or function. For
example, it has been demonstrated that endometrial hyperplasia often precedes
endometrial
cancer. Metaplasia is a form of controlled cell growth in which one type of
mature or fully
differentiated cell substitutes for another type of mature cell. Metaplasia
may occur in
epithelial or connective tissue cells. Dysplasia is generally considered a
precursor of cancer,
and is found mainly in the epithelia. Dysplasia is the most disorderly form of
non-neoplastic
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cell growth, and involves a loss in individual cell uniformity and in the
architectural
orientation of cells. Dysplasia characteristically occurs where there exists
chronic irritation or
inflammation, and is often found in the cervix, respiratory passages, oral
cavity, and gall
bladder. .
Alternatively, or in addition to the presence of abnormal cell growth
characterized as
hyperplasia, metaplasia, or dysplasia, the presence of one or more
characteristics of a
transformed or malignant phenotype displayed either in vivo or in vitro within
a cell sample
derived from a patient, is indicative of the desirability of
prophylactic/therapeutic
administration of a Therapeutic that possesses the ability to modulate
activity of An
aforementioned protein. Characteristics of a transformed phenotype include,
but are not
limited to: (i) morphological changes; (ii) looser substratum attachment;
(iii) loss of cell-to-
cell contact inhibition; (iv) loss of anchorage dependence; (v) protease
release; (vi) increased
sugar transport; (vii) decreased serum requirement; (viii) expression of fetal
antigens, (ix)
disappearance of the 250 kDa cell-surface protein, and the like. See e.g.,
Richards, et al., 1986.
MOLECULAR PATHOLOGY, W.B. Saunders Co., Philadelphia, PA.
In a specific embodiment of the present invention, a patient that exhibits one
or more
of the following predisposing factors for malignancy is treated by
administration of an
effective amount of a Therapeutic: (i) a chromosomal translocation associated
with a
malignancy (e.g., the Philadelphia chromosome (bcrlabl) for chronic
myelogenous leukemia
and t(14;20) for follicular lymphoma, etc.); (ii) familial polyposis or
Gardner's syndrome
(possible forerunners of colon cancer); (iii) monoclonal gammopathy of
undetermined
significance (a possible precursor of multiple myeloma) and (iv) a first
degree kinship with
persons having a cancer or pre-cancerous disease showing a Mendelian (genetic)
inheritance
pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary
exostosis,
polyendocrine adenomatosis, Peutz-Jeghers syndrome, neurofibromatosis of Von
Recklinghausen, medullary thyroid carcinoma with amyloid production and
pheochromocytoma, retinoblastoma, carotid body tumor, cutaneous
melanocarcinoma,
intraocular melanocarcinoma, xeroderma pigmentosum, ataxia telangiectasia,
Chediak-Higashi
syndrome, albinism, Fanconi's aplastic anemia and Bloom's syndrome).
In another embodiment, a Therapeutic of the present invention is administered
to a
human patient to prevent the progression to breast, colon, lung, pancreatic,
or uterine cancer,
or melanoma or sarcoma.
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Hyperproliferative And Dysproliferative Disorders
In one embodiment of the present invention, a Therapeutic is administered in
the
therapeutic or prophylactic treatment of hyperproliferative or benign
dysproliferative
disorders. The efficacy in treating or preventing hyperproliferative diseases
or disorders of a
Therapeutic of the present invention may be assayed by any method known within
the art.
Such assays include in vitro cell proliferation assays, in vitro or in vivo
assays using animal
models of hyperproliferative diseases or disorders, or the like. Potentially
effective
Therapeutics may, for example, promote cell proliferation in culture or cause
growth or cell
proliferation in animal models in comparison to controls.
Specific embodiments of the present invention are directed to the treatment or
prevention of cirrhosis of the liver (a condition in which scarring has
overtaken normal liver
regeneration processes); treatment of keloid (hypertrophic scar) formation
causing disfiguring
of the skin in which the scarring process interferes with normal renewal;
psoriasis (a common
skin condition characterized by excessive proliferation of the skin and delay
in proper cell fate
determination); benign tumors; fibrocystic conditions and tissue hypertrophy
(e.g., benign
prostatic hypertrophy).
Neurodegenerative Disorders
Some a FCTRX proteins are found in cell types have been implicated in the
deregulation of cellular maturation and apoptosis, which are both
characteristic of
neurodegenerative disease. Accordingly, Therapeutics of the invention,
particularly but not
limited to those that modulate (or supply) activity of an aforementioned
protein, may be
effective in treating or preventing neurodegenerative disease. Therapeutics of
the present
invention that modulate the activity of an aforementioned protein involved in
neurodegenerative disorders can be assayed by any method known in the art for
efficacy in
treating or preventing such neurodegenerative diseases and disorders. Such
assays include in
vitro assays for regulated cell maturation or inhibition of apoptosis or in
vivo assays using
animal models of neurodegenerative diseases or disorders, or any of the assays
described
below. Potentially effective Therapeutics, for example but not by way of
limitation, promote
regulated cell maturation and prevent cell apoptosis in culture, or reduce
neurodegeneration in
animal models in comparison to controls.
Once a neurodegenerative disease or disorder has been shown to be amenable to
treatment by modulation activity, that neurodegenerative disease or disorder
can be treated or
prevented by administration of a Therapeutic that modulates activity. Such
diseases include all
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degenerative disorders involved with aging, especially osteoarthritis and
neurodegenerative
disorders.
Disorders Related To Organ Transplantation
Some FCT1RX proteins can be associated with disorders related to organ
transplantation, in particular but not limited to organ rejection.
Therapeutics of the invention,
particularly those that modulate (or supply) activity, may be effective in
treating or preventing
diseases or disorders related to organ transplantation. Therapeutics of the
invention
(particularly Therapeutics that modulate the levels or activity of an
aforementioned protein)
can be assayed by any method known in the art for efficacy in treating or
preventing such
diseases and disorders related to organ transplantation. Such assays include
in vitro assays for
using cell culture models as described below, or in vivo assays using animal
models of
diseases and disorders related to organ transplantation, see e.g., below.
Potentially effective
Therapeutics, for example but not by way of limitation, reduce immune
rejection responses in
animal models in comparison to controls.
Accordingly, once diseases and disorders related to organ transplantation are
shown to
be amenable to treatment by modulation of activity, such diseases or disorders
can be treated
or prevented by administration of a Therapeutic that modulates activity.
Cardiovascular Disease
Proteins related to FCTRX proteins have been implicated in cardiovascular
disorders,
including in atherosclerotic plaque formation. Diseases such as cardiovascular
disease,
including cerebral thrombosis or hemorrhage, ischemic heart or renal disease,
peripheral
vascular disease, or thrombosis of other major vessel, and other diseases,
including diabetes
mellitus, hypertension, hypothyroidism, cholesterol ester storage disease,
systemic lupus
erythematosus, homocysteinemia; and familial protein or lipid processing
diseases, and the
like, are either directly or indirectly associated with atherosclerosis.
Accordingly, Therapeutics
of the invention, particularly those that modulate (or supply) activity or
formation may be
effective in treating or preventing atherosclerosis-associated diseases or
disorders.
Therapeutics of the invention (particularly Therapeutics that modulate the
levels or activity)
can be assayed by any method known in the art, including those described
below, for efficacy
in treating or preventing such diseases and disorders.
A vast array of animal and cell culture models exist for processes involved in
atherosclerosis. A limited and non-exclusive list of animal models includes
knockout mice for
premature atherosclerosis (Kurabayashi and Yazaki, 1996, Int. Angiol. 15: 187-
194),
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transgenic mouse models of atherosclerosis (Kappel et al., 1994, FASEB J. 8:
583-592),
antisense oligonucleotide treatment of animal models (Callow, 1995, Curr.
Opin. Cardiol. 10:
569-576), transgenic rabbit models for atherosclerosis (Taylor, 1997, Ann.
N.Y. Acad. Sci
811: 146-152), hypercholesterolemic animal models (Rosenfeld, 1996, Diabetes
Res. Clin.
Pract. 30 Suppl.: 1-11), hyperlipidemic mice (Paigen et al., 1994, Curr. Opin.
Lipidol. 5: 258-
264), and inhibition of lipoxygenase in animals (Sigal et al., 1994, Ann. N.Y.
Acad. Sci. 714:
211-224). In addition, in vitro cell models include but are not limited to
monocytes exposed to
low density lipoprotein (Frostegard et al., 1996, Atherosclerosis 121: 93-
103), cloned vascular
smooth muscle cells (Suttles et al., 1995, Exp. Cell Res. 218: 331-338),
endothelial cell-
derived chemoattractant exposed T cells (Katz et al., 1994, J. Leukoc. Biol.
S5: 567-573),
cultured human aortic endothelial cells (Farber et al., 1992, Am. J. Physiol.
262: H1088-
1085), and foam cell cultures (Libby et al., 1996, Curr Opin Lipidol 7: 330-
335). Potentially
effective Therapeutics, for example but not by way of limitation, reduce foam
cell formation
in cell culture models, or reduce atherosclerotic plaque formation in
hypercholesterolemic
mouse models of atherosclerosis in comparison to controls.
Accordingly, once an atherosclerosis-associated disease or disorder has been
shown to
be amenable to treatment by modulation of activity or formation, that disease
or disorder can
be treated or prevented by administration of a Therapeutic that modulates
activity.
Cytokine and Cell Proliferation/Differentiation Activity
A FCTRX protein or a cognate Therapeutic of the present invention may exhibit
cytokine, cell proliferation (either inducing or inhibiting) or cell
differentiation (either
inducing or inhibiting) activity or may induce production of other cytokines
in certain cell
populations. Many protein factors discovered to date, including all known
cytokines, have
exhibited activity in one or more factor dependent cell proliferation assays,
and hence the
assays serve as a convenient confirmation of cytokine activity. The activity
of a protein of the
present invention is evidenced by any one of a number of routine factor
dependent cell
proliferation assays for cell lines including, without limitation, 32D, DA2,
DA1G, T10, B9,
B9/11, BaF3, MC9/G, M+ (preB M+ ), 2E8, RBS, DA1, 123, T1165, HT2, CTLL2, TF-
1,
Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured
by the
following methods: Assays for T-cell or thymocyte proliferation include
without limitation
those described in: CURRENT PROTOCOLS IN IMMUNOLOGY, Ed by Coligan et al.,
Greene
Publishing Associates and Wiley-Interscience (Chapter 3 and Chapter 7); Takai
et al., J
Immunol 137:3494-3500, 1986; Bertagnoili et al., Jlmmunol 145:1706-1712, 1990;
99
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Bertagnolli et al., Cell Immunol 133:327-341, 1991; Bertagnolli, et al.,
Jlmmunol 149:3778-
3783, 1992; Bowman et al., Jlmmunol 152:1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node cells or
thymocytes include, without limitation, those described by Kruisbeek and
Shevach, In:
S CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al., eds. Vol 1, pp. 3.12.1-14,
John Wiley
and Sons, Toronto 1994; and by Schreiber, In: CURRENT PROTOCOLS IN IMMUNOLOGY.
Coligan eds. Vol 1 pp. 6.8.1-8, John Wiley and Sons, Toronto 1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic cells
include, without limitation, those described by Bottomly et al., In: CURRENT
PROTOCOLS IN
1O IMMUNOLOGY. Coligan et al., eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and
Sons, Toronto 1991;
deVries et al., JExp Med 173:1205-1211, 1991; Moreau et al., Nature 336:690-
692, 1988;
Greenberger et al., Proc Natl Acad Sci U.S.A. 80:2931-2938, 1983; Nordan, In:
CURRENT
PROTOCOLS IN IMMUNOLOGY. Coligan et al., eds. Vol 1 pp. 6.6.1-5, John Wiley
and Sons,
Toronto 1991; Smith et al., Proc Natl Acad Sci U.S.A. 83:1857-1861, 1986;
Measurement of
15 human Interleukin 11-Bennett, et al. In: CURRENT PROTOCOLS IN IMMUNOLOGY.
Coligan et al.,
eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto 1991; Ciarletta, et al.,
In: CURRENT
PROTOCOLS IN IMMUNOLOGY. Coligan et al., eds. Vol 1 pp. 6.13.1, John Wiley and
Sons,
Toronto 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
20 proteins that affect APC-T cell interactions as well as direct T-cell
effects by measuring
proliferation and cytokine production) include, without limitation, those
described In:
CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al., eds., Greene Publishing
Associates and
Wiley-Interscience (Chapter 3Chapter 6, Chapter 7); Weinberger et al., Proc
Natl Acad Sci
USA 77:6091-6095, 1980; Weinberger et al., Eur Jlmmun 11:405-411, 1981; Takai
et al., J
25 Immunol 137:3494-3500, 1986; Takai et al., Jlmmunol 140:508-512, 1988.
Immune Stimulating or Suppressing Activity
A FCTRX protein or a cognate Therapeutic of the present invention may also
exhibit
immune stimulating or immune suppressing activity, including without
limitation the activities
for which assays are described herein. A protein may be useful in the
treatment of various
30 immune deficiencies and disorders (including severe combined
immunodeficiency (SCID)),
e.g., in regulating (up or down) growth and proliferation of T and/or B
lymphocytes, as well as
effecting the cytolytic activity of NK cells and other cell populations. These
immune
deficiencies may be genetic or be caused by vital (e.g., HIV) as well as
bacterial or fungal
infections, or may result from autoimmune disorders. More specifically,
infectious diseases
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causes by vital, bacterial, fungal or other infection may be treatable using a
protein of the
present invention, including infections by HIV, hepatitis viruses, herpes
viruses, mycobacteria,
Leishmania species., malaria species. and various fungal infections such as
candidiasis. Of
course, in this regard, a protein of the present invention may also be useful
where a boost to
the immune system generally may be desirable, i. e., in the treatment of
cancer.
Autoimmune disorders which may be treated using a protein or a cognate
Therapeutic
of the present invention include, for example, connective tissue disease,
multiple sclerosis,
systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary
inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes
mellitus,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease. Such
a protein of the present invention may also to be useful in the treatment of
allergic reactions
and conditions, such as asthma (particularly allergic asthma) or other
respiratory problems.
Other conditions, in which immune suppression is desired (including, for
example, organ
transplantation), may also be treatable using a protein of the present
invention.
Using a protein or a cognate Therapeutic of the invention it may also be
possible to
modulate immune responses, in a number of ways. Down regulation may be in the
form of
inhibiting or blocking an immune response already in progress or may involve
preventing the
induction of an immune response. The functions of activated T cells may be
inhibited by
suppressing T cell responses or by inducing specific tolerance in T cells, or
both.
Immunosuppression of T cell responses is generally an active, non-antigen-
specific, process
which requires continuous exposure of the T cells to the suppressive agent.
Tolerance, which
involves inducing non-responsiveness or energy in T cells, is distinguishable
from
immunosuppression in that it is generally antigen-specific and persists after
exposure to the
tolerizing agent has ceased. Operationally, tolerance can be demonstrated by
the lack of a T
cell response upon re-exposure to specific antigen in the absence of the
tolerizing agent.
Down regulating or preventing ode or more antigen functions (including without
limitation B lymphocyte antigen functions (such as, for example, B7)), e.g.,
preventing high
level lymphokine synthesis by activated T cells, will be useful in situations
of tissue, skin and
organ transplantation and in graft-versus-host disease (GVHD). For example,
blockage of T
cell function should result in reduced tissue destruction in tissue
transplantation. Typically, in
tissue transplants, rejection of the transplant is initiated through its
recognition as foreign by T
cells, followed by an immune reaction that destroys the transplant. The
administration of a
molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with
its natural
ligand(s) on immune cells (such as a soluble, monomeric form of a peptide
having B7-2
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activity alone or in conjunction with a monomeric form of a peptide having an
activity of
another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), prior
to transplantation
can lead to the binding of the molecule to the natural ligand(s) on the immune
cells without
transmitting the corresponding costimulatory signal. Blocking B lymphocyte
antigen function
in this matter prevents cytokine synthesis by immune cells, such as T cells,
and thus acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient
to energize
the T cells, thereby inducing tolerance in a subject. Induction of long-term
tolerance by B
lymphocyte antigen-blocking reagents may avoid the necessity of repeated
administration of
these blocking reagents. To achieve sufficient immunosuppression or tolerance
in a subject, it
may also be necessary to block the function of B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or
GVHD can be assessed using animal models that are predictive of efficacy in
humans.
Examples of appropriate systems which can be used include allogeneic cardiac
grafts in rats
and xenogeneic pancreatic islet cell grafts in mice, both of which have been
used to examine
the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described
in Lenschow
et al., Science 257:789-792 (1992) and Turka et al., Proc Natl Acad Sci USA,
89:11102-11105
(1992). In addition, marine models of GVHD (see Paul ed., FUNDAMENTAL
IMMUNOLOGY,
Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect
of blocking B
lymphocyte antigen function in vivo on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune
diseases. Many autoimmune disorders are the result of inappropriate activation
of T cells that
are reactive against self tissue and which promote the production of cytokines
and auto-
antibodies involved in the pathology of the diseases. Preventing the
activation of autoreactive
T cells may reduce or eliminate disease symptoms. Administration of reagents
which block
costimulation of T cells by disrupting receptor:ligand interactions of B
lymphocyte antigens
can be used to inhibit T cell activation and prevent production of auto-
antibodies or T cell-
derived cytokines which may be involved in the disease process. Additionally,
blocking
reagents may induce antigen-specific tolerance of autoreactive T cells which
could lead to
long-term relief from the disease. The efficacy of blocking reagents in
preventing or
alleviating autoimmune disorders can be determined using a number of well-
characterized
animal models of human autoimmune diseases. Examples include marine
experimental
autoimmune encephalitis, systemic lupus erythematosis in MRL/lpr/lpr mice or
NZB hybrid
mice, marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and
BB rats, and
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murine experimental myasthenia gravis (see Paul ed., FUNDAMENTAL IMMUNOLOGY,
Raven
Press, New York, 1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen
function), as a
means of up regulating immune responses, may also be useful in therapy.
Upregulation of
immune responses may be in the form of enhancing an existing immune response
or eliciting
an initial immune response. For example, enhancing an immune response through
stimulating
B lymphocyte antigen function may be useful in cases of viral infection. In
addition, systemic
vital diseases such as influenza, the common cold, and encephalitis might be
alleviated by the
administration of stimulatory forms of B lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient by
removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-pulsed
APCs either expressing a peptide of the present invention or together with a
stimulatory form
of a soluble peptide of the present invention and reintroducing the in vitro
activated T cells
into the patient. Another method of enhancing anti-vital immune responses
would be to isolate
1 S infected cells from a patient, transfect them with a nucleic acid encoding
a protein of the
present invention as described herein such that the cells express all or a
portion of the protein
on their surface, and reintroduce the transfected cells into the patient. The
infected cells would
now be capable of delivering a costimulatory signal to, and thereby activate,
T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B
lymphocyte antigen function) may be useful in the induction of tumor immunity.
Tumor cells
(e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma)
transfected with a
nucleic acid encoding at least one peptide of the present invention can be
administered to a
subject to overcome tumor-specific tolerance in the subject. If desired, the
tumor cell can be
transfected to express a combination of peptides. For example, tumor cells
obtained from a
patient can be transfected ex vivo with an expression vector directing the
expression of a
peptide having B7-2-like activity alone, or in conjunction with a peptide
having B7-1-like
activity and/or B7-3-like activity. The transfected tumor cells are returned
to the patient to
result in expression of the peptides on the surface of the transfected cell.
Alternatively, gene
therapy techniques can be used to target a tumor cell for transfection in
vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
costimulation
signal to T cells to induce a T cell mediated immune response against the
transfected tumor
cells. In addition, tumor cells which lack MHC class I or MHC class II
molecules, or which
fail to reexpress sufficient amounts of MHC class I or MHC class II molecules,
can be
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transfected with nucleic acid encoding all or a portion of (e.g., a
cytoplasrriic-domain truncated
portion) of an MHC class I a chain protein and (32 microglobulin protein or an
MHC class II a
chain protein and an MHC class II ~i chain protein to thereby express MHC
class I or MHC
class II proteins on the cell surface. Expression of the appropriate class I
or class II MHC in .
conjunction with a peptide having the activity of a B lymphocyte antigen
(e.g., B7-1, B7-2,
B7-3) induces a T cell mediated immune response against the transfected tumor
cell.
Optionally, a gene encoding an antisense construct which blocks expression of
an MHC class
II associated protein, such as the invariant chain, can also be cotransfected
with a DNA
encoding a peptide having the activity of a B lymphocyte antigen to promote
presentation of
tumor associated antigens and induce tumor specific immunity. Thus, the
induction of a T cell
mediated immune response in a human subject may be sufficient to overcome
tumor-specific
tolerance in the subject.
The activity of a protein or a cognate Therapeutic of the invention may, among
other
means, be measured by the following methods: Suitable assays for thymocyte or
splenocyte
cytotoxicity include, without limitation, those described In: CURRENT
PROTOCOLS nr
IMMUNOLOGY. Coligan et al., eds. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, Chapter 7); Hemnann et al., Proc Natl Acad Sci USA 78:2488-2492,
1981;
Hemnann et al., Jlmmunol 128:1968-1974, 1982; Handa et al., Jlmmunol 20:1564-
1572,
1985; Takai et al., Jlmmunol 137:3494-3500, 1986; Takai et al., Jlmmunol
140:508-512,
1988; Herrmann et al., Proc Natl Acad Sci USA 78:2488-2492, 1981; Hemnann et
al., J
Immunol 128:1968-1974, 1982; Handa et al., Jlmmunol 18:1564-1572, 1985; Takai
et al., J
Immunol 137:3494-3500, 1986; Bowman et al., J Virology 61:1992-1998; Takai et
al., J
Immunol 140:508-512, 1988; Bertagnolli et al., Cell Immunol 133:327-341, 1991;
Brown et
al., Jlmmunol 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which
will identify, among others, proteins that modulate T-cell dependent antibody
responses and
that affect Thl/Th2 profiles) include, without limitation, those described in:
Maliszewski, J
Immunol 144:3028-3033, 1990; and Mond and Brunswick In: CURRENT PROTOCOLS IN
IMMUNOLOGY. Coligan et al., (eds.) Vol 1 pp. 3.8.1-3.8.16, John Wiley and
Sons, Toronto
1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins
that generate predominantly Thl and CTL responses) include, without
limitation, those
described In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al., eds. Greene
Publishing
Associates and Wiley-Interscience (Chapter 3, Chapter 7); Takai et al.,
Jlmmunol 137:3494-
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3500, 1986; Takai et al., Jlmmunol 140:508-512, 1988; Bertagnolli et al.,
Jlmmunol
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed
by dendritic cells that activate naive T-cells) include, without limitation,
those described in:
Guery et al., Jlmmunol 134:536-544, 1995; Inaba et al., JExp Med 173:549-559,
1991;
Macatonia et al., Jlmmunol 154:5071-5079, 1995; Porgador et al., JExp Med
182:255-260,
1995; Nair et al., J Virol 67:4062-4069, 1993; Huang et al., Science 264:961-
965, 1994;
Macatonia et al., JExp Med 169:1255-1264, 1989; Bhardwaj et al., J Clin
Investig 94:797-
807, 1994; and Inaba et al., JExp Med 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins
that prevent apoptosis after superantigen induction and proteins that regulate
lymphocyte
homeostasis) include, without limitation, those described in: Darzynkiewicz et
al., Cytometry
13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al.,
Cancer Res
53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Jlmmunol
145:4037-
4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al.,
Internat J Oncol
1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
development
include, without limitation, those described in: Antica et al., Blood 84:111-
117, 1994; Fine et
al., Cell Immunol 155: 111-122, 1994; Galy et al., Blood 85:2770-2778, 1995;
Toki et al.,
Proc Nat Acad Sci USA 88:7548-7551, 1991.
Hematopoiesis Regulating Activity
A FCTRX protein or a cognate Therapeutic of the present invention may be
useful in
regulation of hematopoiesis and, consequently, in the treatment of myeloid or
lymphoid cell
deficiencies. Even marginal biological activity in support of colony forming
cells or of factor-
dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
in supporting the
growth and proliferation of erythroid progenitor cells alone or in combination
with other
cytokines, thereby indicating utility, for example, in treating various
anemias or for use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid precursors
and/or erythroid cells; in supporting the growth and proliferation of myeloid
cells such as
granulocytes and monocytes/macrophages (i.e., traditional CSF activity)
useful, for example,
in conjunction with chemotherapy to prevent or treat consequent myelo-
suppression; in
supporting the growth and proliferation of megakaryocytes and consequently of
platelets
thereby allowing prevention or treatment of various platelet disorders such as
thrombocytopenia, and generally for use in place of or complimentary to
platelet transfusions;
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and/or in supporting the growth and proliferation of hematopoietic stem cells
which are
capable of maturing to any and all of the above-mentioned hematopoietic cells
and therefore
find therapeutic utility in various stem cell disorders (such as those usually
treated with
transplantation, including, without limitation, aplastic anemia and paroxysmal
nocturnal
hemoglobinuria), as well as in repopulating the stem cell compartment post
irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with
bone marrow
transplantation or with peripheral progenitor cell transplantation (homologous
or
heterologous)) as normal cells or genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the
following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines are
cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without limitation,
those described in: Johansson et al. Cellular Biology 15:141-1 S 1, 1995;
Keller et al., Mol.
Cell. Biol. 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among
others,
proteins that regulate lympho-hematopoiesis) include, without limitation,
those described in:
Methylcellulose colony forming assays, Freshney, In: CULTURE OF HEMATOPOIETIC
CELLS.
Freshney, et al. (eds.) Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y 1994;
Hirayama et
al., Proc Natl Acad Sci USA 89:5907-5911, 1992; McNiece and Briddeli, In:
CULTURE OF
HEMATOPOIETIC CELLS. Freshney, et al. (eds.) Vol pp. 23-39, Wiley-Liss, Inc.,
New York,
N.Y. 1994; Neben et al., Exp Hematol 22:353-359, 1994; Ploemacher, In: CULTURE
of
HEMATOPOIETIC CELLS. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New
York, N.Y.
1994; Spoonceret al., In: CULTURE of HEMATOPOIETIC CELLS. Freshhey, et al.,
(eds.) Vol pp.
163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Sutherland, In: CULTURE of
HEMATOPOIETIC CELLS. Freshney, et al., (eds.) Vol pp. 139-162, Wiley-Liss,
Inc., New York,
N.Y. 1994.
Tissue Growth Activity
A FCTRX protein or a cognate Therapeutic of the present invention also may
have
utility in compositions used for bone, cartilage, tendon, ligament and/or
nerve tissue growth or
regeneration, as well as for wound healing and tissue repair and replacement,
and in the
treatment of burns, incisions and ulcers.
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A protein or a cognate Therapeutic of the present invention, which induces
cartilage
and/or bone growth in circumstances where bone is not normally formed, has
application in
the healing of bone fractures and cartilage damage or defects in humans and
other animals.
Such a preparation employing a protein of the invention may have prophylactic
use in closed
as well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of congenital,
trauma induced, or oncologic resection induced craniofacial defects, and also
is useful in
cosmetic plastic surgery.
A protein or a cognate Therapeutic of this invention may also be used in the
treatment
of periodontal disease, and in other tooth repair processes. Such agents may
provide an
environment to attract bone-forming cells, stimulate growth of bone-forming
cells or induce
differentiation of progenitors of bone-forming cells. A protein of the
invention may also be
useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation of bone
and/or cartilage repair or by blocking inflammation or processes of tissue
destruction
(collagenase activity, osteoclast activity, etc.) mediated by inflammatory
processes.
Another category of tissue regeneration activity that may be attributable to
the protein
of the present invention is tendon/ligament formation. A protein of the
present invention,
which induces tendon/ligament-like tissue or other tissue formation in
circumstances where
such tissue is not normally formed, has application in the healing of tendon
or ligament tears,
deformities and other tendon or ligament defects in humans and other animals.
Such a
preparation employing a tendon/ligament-like tissue inducing protein may have
prophylactic
use in preventing damage to tendon or ligament tissue, as well as use in the
improved fixation
of tendon or ligament to bone or other tissues, and in repairing defects to
tendon or ligament
tissue. De novo tendon/ligament-like tissue formation induced by a composition
of the present
invention contributes to the repair of congenital, trauma induced, or other
tendon or ligament
defects of other origin, and is also useful in cosmetic plastic surgery for
attachment or repair of
tendons or ligaments. The compositions of the present invention may provide an
environment
to attract tendon- or ligament-forming cells, stimulate growth of tendon- or
ligament-forming
cells, induce differentiation of progenitors of tendon- or ligament-forming
cells, or induce
growth of tendon/ligament cells or progenitors ex vivo for return in vivo to
effect tissue repair.
The compositions of the invention may also be useful in the treatment of
tendonitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also include an
appropriate matrix and/or sequestering agent as a career as is well known in
the art.
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A protein or a cognate Therapeutic of the present invention may also be useful
for
proliferation of neural cells and for regeneration of nerve and brain tissue,
i.e. for the treatment
of central and peripheral nervous system diseases and neuropathies, as well as
mechanical and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the peripheral
nervous system, such as peripheral nerve injuries, peripheral neuropathy and
localized
neuropathies, and central nervous system diseases, such as Alzheimer's,
Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.
Further
conditions which may be treated in accordance with the present invention
include mechanical
and traumatic disorders, such as spinal cord disorders, head trauma and
cerebrovascular
diseases such as stroke. Peripheral neuropathies resulting from chemotherapy
or other medical
therapies may also be treatable using a protein of the invention.
Proteins of the invention may also be useful to promote better or faster
closure of non-
healing wounds, inciading without limitation pressure ulcers, ulcers
associated with vascular
insufficiency, surgical and traumatic wounds, ar~ithe like.
It is expected that a protein of the present invention may also exhibit
activity for
generation or regeneration of other tissues, such as organs (including, for
example, pancreas,
liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or
cardiac) and vascular
(including vascular endothelium) tissue, or for promoting the growth of cells
comprising such
tissues. Part of the desired effects may be by inhibition or modulation of
fibrotic scarring to
allow normal tissue to regenerate. A protein of the invention may also exhibit
angiogenic
activity.
A protein of the present invention may also be useful for gut protection or
regeneration
and treatment of lung or liver fibrosis, reperfusion injury in various
tissues, and conditions
resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells;
or.for inhibiting the
growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the
following methods:
Assays for tissue generation activity include, without limitation, those
described in:
International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International
Patent Publication No. W095/05846 (nerve, neuronal); International Patent
Publication No.
W091/07491 (skin, endothelium).
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Assays for wound healing activity include, without limitation, those described
in:
Winter, EPIDERMAL WOUND HEALING, pp. 71-112 (Maibach and Rovee, eds.), Year
Book
Medical Publishers, Inc., Chicago, as modified by Eaglstein and Menz, J.
Invest. Dermatol
71:382-84 (1978).
Activin/Inhibin Activity
A FCTRX protein or a cognate Therapeutic of the present invention may also
exhibit
activin- or inhibin-related activities. Inhibins are characterized by their
ability to inhibit the
release of follicle stimulating hormone (FSH), while activins and are
characterized by their
ability to stimulate the release of follicle stimulating hormone (FSH). Thus,
a protein of the
present invention, alone or in heterodimers with a member of the inhibin a
family, may be
useful as a contraceptive based on the ability of inhibins to decrease
fertility in female
mammals and decrease spermatogenesis in male mammals. Administration of
sufficient
amounts of other inhibins can induce infertility in these mammals.
Alternatively, the protein of
the invention, as a homodimer or as a heterodimer with other protein subunits
of the inhibin-b
group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
U.S. Pat. No. 4,798,885. A protein of the invention may also be useful for
advancement of the
onset of fertility in sexually immature mammals, so as to increase the
lifetime reproductive
performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured
by the
following methods:
Assays for activin/inhibin activity include, without limitation, those
described in: Vale
et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986;
Vale et al.,
Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et
al., Proc Natl
Acad Sci USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein or a cognate Therapeutic of the present invention may have
chemotactic or
chemokinetic activity (e.g., act as a chemokine) for mammalian cells,
including, for example,
monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils,
epithelial and/or
endothelial cells. Chemotactic and chemokinetic proteins can be used to
mobilize or attract a
desired cell population to a desired site of action. Chemotactic or
chemokinetic proteins
provide particular advantages in treatment of wounds and other trauma to
tissues, as well as in
treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
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neutrophils to tumors or sites of infection may result in improved immune
responses against
the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it can
stimulate, directly or indirectly, the directed orientation or movement of
such cell population.
Preferably, the protein or peptide has the ability to directly stimulate
directed movement of
cells. Whether a particular protein has chemotactic activity for a population
of cells can be
readily determined by employing such protein or peptide in any known assay for
cell
chemotaxis.
The activity of a protein of the invention may, among other means, be measured
by
following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
chemotaxis) consist of assays that measure the ability of a protein to induce
the migration of
cells across a membrane as well as the ability of a protein to induce the
adhesion of one cell
population to another cell population. Suitable assays for movement and
adhesion include,
without limitation, those described in: CURRENT PROTOCOLS IN IMMUNOLOGY,
Coligan et al.,
eds. (Chapter 6.12, MEASUREMENT OF ALPHA AND BETA CHEMOKINES 6.12.1-6.12.28);
Taub et
al. JClin Invest 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995;
Muller et al., Eur
Jlmmunol 25: 1744-1748; Gruberet al. Jlmmunol 152:5860-5867, 1994; Johnston et
al., J
Immunol 153: 1762-1768, 1994.
Hemostatic and Thrombolytic Activity
A protein or a cognate Therapeutic of the invention may also exhibit
hemostatic or
thrombolytic activity. As a result, such a protein is expected to be useful in
treatment of
various coagulation disorders (including hereditary disorders, such as
hemophiliac) or to
enhance coagulation and other hemostatic events in treating wounds resulting
from trauma,
surgery or other causes. A protein of the invention may also be useful for
dissolving or
inhibiting formation of thromboses and for treatment and prevention of
conditions resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels (e.g.,
stroke).
The activity of a protein of the invention may, among other means, be measured
by the
following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al., Thrombosis
Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474, 1988.
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Receptor/Ligand Activity
A protein or a cognate Therapeutic of the present invention may also
demonstrate
activity as receptors, receptor ligands or inhibitors or agonists of
receptor/ligand interactions.
Examples of such receptors and ligands include, without limitation, cytokine
receptors and
their ligands, receptor kinases and their ligands, receptor phosphatases and
their ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
cellular adhesion molecules (such as selectins, integrins and their ligands)
and receptor/ligand
pairs involved in antigen presentation, antigen recognition and development of
cellular and
humoral immune responses). Receptors and ligands are also useful for screening
of potential
peptide or small molecule inhibitors of the relevant receptor/ligand
interaction. A protein of
the present invention (including, without limitation, fragments of receptors
and ligands) may
themselves be useful as inhibitors of receptor/ligand interactions.
The activity of a protein of the invention may, among other means, be measured
by the
following methods:
Suitable assays for receptor-ligand activity include without limitation those
described
in: CURRENT PROTOCOLS 1N IMMUNOLOGY, Ed by Coligan, et al., Greene Publishing
Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular
Adhesion under
static conditions 7.28.1-7.28.22), Takai et al., Proc Natl Acad Sci USA
84:6864-6868, 1987;
Bierer et al., J .. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med. 169:149-160
1989; Stoltenborg et al., Jlmmunol Methods 175:59-68, 1994; Stitt et al., Cell
80:661-670,
1995.
Anti-Inflammatory Activity
Proteins or cognate Therapeutics of the present invention may also exhibit
anti-
inflammatory activity. The anti-inflammatory activity may be achieved by
providing a
stimulus to cells involved in the inflammatory response, by inhibiting or
promoting cell-cell
interactions (such as, for example, cell adhesion), by inhibiting or promoting
chemotaxis of
cells involved in the inflammatory process, inhibiting or promoting cell
extravasation, or by
stimulating or suppressing production of other factors which more directly
inhibit or promote
an inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without limitation
inflammation associated with infection (such as septic shock, sepsis or
systemic inflammatory
response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality,
arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-
induced lung
injury, inflammatory bowel disease, Crohn's disease or resulting from over
production of
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cytokines such as TNF or IL-1. Proteins of the invention may also be useful to
treat
anaphylaxis and hypersensitivity to an antigenic substance or material.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention
of tumors, a protein of the invention may exhibit other anti-tumor activities.
A protein may
inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
A protein may
exhibit its tumor inhibitory activity by acting on tumor tissue or tumor
precursor tissue, by
inhibiting formation of tissues necessary to support tumor growth (such as,
for example, by
inhibiting angiogenesis), by causing production of other factors, agents or
cell types which
inhibit tumor growth, br by suppressing, eliminating or inhibiting factors,
agents or cell types
which promote tumor growth.
Other Activities
A protein or a cognate Therapeutic of the invention may also exhibit one or
more of
the following additional activities or effects: inhibiting the growth,
infection or function of, or
killing, infectious agents, including, without limitation, bacteria, viruses,
fungi and other
parasites; effecting (suppressing or enhancing) bodily characteristics,
including, without
limitation, height, weight, hair color, eye color, skin, fat to lean ratio or
other tissue
pigmentation, or organ or body part size or shape (such as, for example,
breast augmentation
or diminution, change in bone form or shape); effecting biorhythms or
circadian cycles or
rhythms; effecting the fertility of male or female subjects; effecting the
metabolism,
catabolism, anabolism, processing, utilization, storage or elimination of
dietary fat, lipid,
protein, carbohydrate, vitamins, minerals, cofactors or other nutritional
factors or
component(s); effecting behavioral characteristics, including, without
limitation, appetite,
libido, stress, cognition (including cognitive disorders), depression
(including depressive
disorders) and violent behaviors; providing analgesic effects or other pain
reducing effects;
promoting differentiation and growth of embryonic stem cells in lineages other
than
hematopoietic lineages; hormonal or endocrine activity; in the case of
enzymes, correcting
deficiencies of the enzyme and treating deficiency-related diseases; treatment
of
hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-
like activity
(such as, for example, the ability to bind antigens or complement); and the
ability to act as an
antigen in a vaccine composition to raise an immune response against such
protein or another
material or entity which is cross-reactive with such protein.
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Neural disorders in general include Parkinson's disease, Alzheimer's disease,
Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS),
peripheral
neuropathy, tumors of the nervous system, exposure to neurotoxins, acute brain
injury,
peripheral nerve trauma or injury, and other neuropathies, epilepsy, and/or
tremors.
The invention will be further illustrated in the following non-limiting
examples.
EXAMPLES
Example 1. Molecular cloning of a mature form (30664188Øm99) polypeptide
from
clone 30664188Ø99
A mature form of clone 30664188Ø99, coding for residues 24 to 370 of the
amino
acid sequence of SEQ ID N0:2, was cloned. This fragment was designated
30664188Øm99
and corresponds to the polypeptide sequence remaining after a signal peptide
predicted to be
cleaved between residues 23 and 24 has been removed. The following
oligonucleotide
primers were designed to PCR amplify the predicted mature form of
30664188Ø99.
30664188 Eco Forward:
CTCGTC GAATTC ACC CCG CAG AGC GCA TCC ATC AAA GC (SEQ ID N0:25)
3066418 Xho Reverse:
CTCGTC CTC GAG TCG AGG TGG TCT TGA GCT GCA GAT ACA (SEQ ID
N0:26)
The forward primer included an in frame EcoRI restriction site, and the
reverse primer
included an XhoI restriction site. The EcoRI/XhoI fragment is compatible with
the pET28a
E.coli expression vector and with the pMelVSHis baculovirus expression vector.
PCR reactions were set up using 5 ng human spleen and fetal lung cDNA
templates.
The reaction mixtures contained 1 microM of each of the 30664188 Eco Forward
and 3066418
Xho Reverse primers, S micromoles dNTP (Clontech Laboratories, Palo Alto CA)
and 1
microliter of SOxAdvantage-HF 2 polymerise (Clontech Laboratories, Palo Alto
CA) in 50
microliter volume. The following reaction conditions were used:
a) 96°C 3 minutes
b) 96°C 30 seconds denaturation
c) 70°C 30 seconds, primer annealing. This temperature was gradually
decreased by 1°C/cycle
d) 72°C 1 minute extension.
Repeat steps b-d 10 times
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e) 96°C 30 seconds denaturation
f) 60°C 30 seconds annealing
g) 72°C 1 minute extension
Repeat steps e-g 25 times
h) 72°C 5 minutes final extension
The amplified product expected to have 1041 by was detected by agarose gel
electrophoresis in both samples. The fragments were purified from agarose gel
and ligated to
pCR2.l vector (Invitrogen, Carlsbad, CA). The cloned inserts were sequenced
using M13
Forward, M13 Reverse and the following gene specific primers:
3066418 S 1: GGA CGA TGG TGT GGA CAC AAG (SEQ ID N0:27),
3066418 S2: CTT GTG TCC ACA CCA TCG TCC (SEQ ID N0:28),
3066418 S3: TAT CGA GGC AGG TCA TAC CAT (SEQ ID N0:29) and
3066418 S4: ATG GTA TGA CCT GCC TCG ATA (SEQ ID N0:30).
The cloned inserts were verified as an open reading frame coding for the
predicted
mature form of 30664188Ø99. The construct derived from fetal lung, called
30664188-
S311a, was used for further subcloning into expression vectors (see below).
The nucleotide
sequence of 30664188-Slla within the restriction sites was found to be 100%
identical to the
corresponding fragment in the ORF of 30664188Ø99 (Table. 1; SEQ ID NO:1).
Example 2. Preparation of mammalian expression vector pCEP4/Sec.
FCTRX nucleic acids were expressed in mammalian cells in a vector named
pCEP4/SEC. The vector was prepared using the oligonucleotide primers,
pSec-VS-His Forward
CTCGTCCTCGAGGGTAAGCCTATCCCTAAC (SEQ ID N0:31) and
pSec-VS-His Reverse
CTCGTCGGGCCCCTGATCAGCGGGTTTAAAC (SEQ ID N0:32),
These primers were designed to amplify a fragment from the pcDNA3.1-VSHis
(Invitrogen, Carlsbad, CA) expression vector that includes VS and His6. The
PCR product
was digested with XhoI and ApaI and ligated into the XhoI/ApaI digested
pSecTag2 B vector
harboring an Ig kappa leader sequence (Invitrogen, Carlsbad CA). The correct
structure of the
resulting vector, pSecVSHis, including an in-frame Ig-kappa leader and VS-His6
was verified
by DNA sequence analysis. The vector pSecVSHis was digested with PmeI and NheI
to
provide a fragment retaining the above elements in the correct frame. The PmeI-
NheI
fragment was ligated into the BamHI/Klenow and NheI treated vector pCEP4
(Invitrogen,
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Carlsbad, CA). The resulting vector was named pCEP4/Sec and includes an in-
frame Ig kappa
leader, a site for insertion of a clone of interest, V5 and 6xHis under
control of the PCMV
and/or the PT7 promoter. pCEP4/Sec is an expression vector that allows
heterologous protein
expression and secretion by fusing any protein to the Ig Kappa chain signal
peptide. Detection
and purification of the expressed protein are aided by the presence of the V5
epitope tag and
6xHis tag at the C-terminus (Invitrogen, Carlsbad, CA).
Example 3. Expression of 30664188.m99 polypeptide in E. coli
The vector pRSETA (InVitrogen Inc., Carlsbad, CA) was digested with XhoI and
NcoI
restriction enzymes. Oligonucleotide linkers
CATGGTCAGCCTAC (SEQ ID N0:33); and
TCGAGTAGGCTGAC (SEQ ID N0:34)
were annealed at 37 degrees Celsius and ligated into the XhoI-NcoI treated
pRSETA. The
resulting vector was confirmed by restriction analysis and sequencing and was
named
pETMY. The BamHI-XhoI fragment containing the 30664188 sequence ( Example 3)
was
ligated into BamHI-XhoI digested pETMY. The resulting expression vector was
named
pETMY-30664188. In this vector, 30664188 is fused to the T7 epitope and a
6xHis tag at its
N-terminus The plasmid pETMY-30664188 was then transfected into the E. coli
expression
host BL21(DE3, pLys) (Novagen, Madison, WI) and expression of the protein was
induced
according to the manufacturer's instructions. After induction, the E. coli
cells were harvested,
and proteins were analyzed by Western blotting using anti-His6Gly antibody
(Invitrogen,
Carlsbad, CA). FIG. 2 shows 30664188.m99 was expressed as a protein of
apparent
molecular weight 40 kDa. This approximates the molecular weight expected for
the
30664188.m99 sequence.
Example 4. Expression of 30664188.m99,polypeptide in human embryonic kidney
293
cells.
The EcoRI-XhoI fragment containing the 30664188.m99 sequence was isolated from
30664188-S311a (Example 1) and subcloned into the vector pE28a (Novagen,
Madison, WI)
to give the plasmid pET28a-30664188. Subsequently, pET28a-30664188 was
partially
digested with BamHI restriction enzyme, and then completely digested with
XhoI. A
fragment of 1.1 kb was isolated and ligated into BamHI-XhoI digested pCEP4/Sec
(Example
2) to generate expression vector pCEP4/Sec-30664188. The pCEP4/Sec-30664188
vector was
transfected into human embryonic kidney 293 cells (ATCC No. CRL-1573,
Manassas, VA)
using the LipofectaminePlus reagent following the manufacturer's instructions
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(GibcoBRL/Life Technologies, Rockville, MD). The cell pellet and supernatant
were
harvested 72 hours after transfection and examined for expression of the
30664188.m99
protein by Western blotting of an SDS-PAGE run under reducing conditions using
an anti-VS
antibody. FIG. 3 shows that 30664188.m99 is expressed as three discrete
protein bands of
apparent molecular weight S0, 60, and 98 kDa secreted by 293 cells. The 50 kDa
band
migrated at a sized expected for a monomer glycosylated form of 30664188.m99,
and the 98
kDa band migrated at a sized consistent with a dimer of the monomer form.
Example 5. Radiation Hybrid Mapping of 30664188Ø99.
Radiation hybrid mapping using human chromosome markers was carned out for
clone
30664188Ø99. The procedure used to obtain these results is analogous to that
described in
Steen, RG et al. (A High-Density Integrated Genetic Linkage and Radiation
Hybrid Map of
the Laboratory Rat, Genome Research 1999 (Published Online on May 21,
1999)Vol. 9, AP1-
APB, 1999). A panel of 93 cell clones containing the randomized radiation-
induced human
chromosomal fragments was screened in 96 well plates using PCR primers
designed to
identify the sought clones in a unique fashion. Clone 30664188Ø99 was found
to be located
on chromosome 11, 3.1 cR from marker WI-9345 and 1.7 cR from marker
CHLC.GATA6C 11.
Example 6. Expression and Purification of 30664188.m99 protein
The segment representing the mature protein cloned in Example 1 was excised
and
subcloned into the vector pCEP4/Sec (Example 2) suitable for transfection of
HEK 293 cells
under the control of the pCEP4 promoter. The resulting vector was named
pCEP4/Sec/30664188.
HEK 293 cells were grown in Dulbecco's modified eagle's medium (DMEM)/10%
fetal bovine serum medium to 90 % confluence. The cells were transfected with
pCEP4sec or
pCEP4sec/30664188.m99 using Lipofectamine 2000 according to the manufacturer's
specifications (GibcoBRL/Life Technologies, Rockville, MD). Transfected cells
were
incubated for 2 days with DMEM and conditioned medium was prepared by
collection of cell
supernatants. The conditioned medium was enriched by Talon metal affinity
chromatography
(Clontech, Palo Alto, CA). Briefly, 7 ml of conditioned medium was incubated
with 1 ml of
Talon metal affinity resin in spin columns. The spin columns were washed twice
with one ml
of PBS. The columns were then eluted twice with 0.65 ml of PBS/O.SM imidazole
pH 8.0 and
the eluates pooled. Imidazole was removed by buffer exchange dialysis into PBS
using
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Microcon centrifugal filter devices (Millipore Corp., Bedford, MA). The
enriched gene
products were stored at 4°C.
The purified protein obtained was subjected to SDS-PAGE under reducing
conditions
and probed with an anti-VS antibody, which was detected with an enzyme label.
The results
of two separate transfection and purification runs are shown in the gels. They
show that the
product is a mixture of VS-containing polypeptides. The largest has an
apparent molecular
weight of about 50 kDa (FIG. 4). The program ProSite predicts one N-
glycosylation site in
the mature protein. Glycosylation may explain the apparent molecular weight
found. Thus
the SOkDa band is consistent with the length expected for full length gene
product. Other
bands, preponderantly having apparent molecular weights of about 20-25 kDa
also arise.
These are presumed to be the result of proteolysis occurnng either
intracellularly within the
293 cells or extracellularly after secretion from them.
Example 7. Real time tissue expression profiling of sequence 30664188 by
quantitative
PCR.
Real time PCR was followed for multiple tissue or cell samples by monitoring
release
of a 5' fluorogenic label from a specific oligonucleotide probe bearing a 3'
quencher. The
target sequence specific for the 30664188 transcript is detected and monitored
in real time, as
the PCR takes place using the fluorogenic 5' nuclease assay performed with the
TaqMan~
PCR Reagent Kit (Roche Molecular Systems, Inc.) and the Perkin-Elmer
Biosystems ABI
PRISM~ 7700 Sequence Detection System.
Probes and primers were designed according to Perkin Elmer Biosystem's Primer
Express Software package (version I for Apple Computer's Macintosh Power PC)
using the
sequence of 30664188 as input. Default settings were used for reaction
conditions and the
following parameters were set before selecting primers: primer concentration =
250 nM,
primer melting temperature (Tm) range = 58°-60° C, primer
optimal Tm = 59° C, maximum
primer difference = 2°C, probe does not have 5' G, probe Tm must be
10°C greater than primer
Tm, amplicon size 75 by to 100 bp. Three sets of primers and probe were
synthesized by
Synthegen (Houston, TX, USA), and were HPLC purified twice to remove uncoupled
dye.
Mass spectroscopy was used to verify efficient coupling of reporter and
quencher dyes to the
5' and 3' ends of the probe, respectively.
PCR preparation and conditions included the following steps: Sample RNA from
each
tissue (poly A+ RNA, 2.8 pg) and the cell lines (total RNA, 70 ng) was spotted
in each well of
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a 96 well PCR plate (Perkin Elmer Biosystems). A panel of 41 normal human
tissues and 55
human cancer cell lines was employed
Table 7. Results of Real Time TaqManTM Tissue Profiling
Normal & Tumor Tissues Relative
Expression
(%)
Ag33 Ag66 Ag168
1 Endothelial cells 1.66 1.23 0.00
2 Endothelial cells (treated)2.80 1.51 0.00
3 Pancreas 36.35 28.72 37.89
4 Pancreatic ca. CAPAN 2 1.05 0.46 0.00
Adipose 10.37 30.57 54.34
6 Adrenal gland 100.00 100.00 0.00
7 Thyroid 20.45 8.19 1.42
8 Salivary gland 6.52 6.75 0.19
9 Pituitary gland 5.83 4.01 0.00
Brain (fetal) 2.16 2.32 0.00
11 Brain (whole) 3.54 2.66 0.00
12 Brain (amygdala) 1.29 0.85 0.05
13 Brain (cerebellum) 1.30 1.02 0.00
14 Brain (hippocampus) 3.26 1.88 0.00
Brain (hypothalamus) 42.93 37.11 46.98
16 Brain (substantia nigra) 2.05 0.00 0.00
17 Brain (thalamus) 0.39 0.25 0.00
18 Spinal cord 4.58 2.78 0.00
19 CNS ca. (glio/astro) U87-MG0.00 0.00 0.00
CNS ca. (glio/astro) U-118-MG0.00 0.07 0.00
21 CNS ca. (astro) SW1783 1.94 1.49 0.00
22 CNS ca.* (neuro; met ) 2.05 1.04 0.00
SK-N-AS
23 CNS ca. (astro) SF-539 0.32 0.13 0.00
24 CNS ca. (astro) SNB-75 5.29 5.26 0.00
CNS ca. (glio) SNB-19 3.85 3.64 0.03
26 CNS ca. (glio) U251 2.82 1.67 0.00
27 CNS ca. (glio) SF-295 82.36 53.59 100.00
28 Heart 14.66 13.58 1.42
29 Skeletal muscle 1.29 0.96 0.00
Bone marrow 1.23 0.69 0.00
31 Thymus 6.04 2.78 0.00
32 Spleen 2.24 1.78 0.00
33 Lyrnph node 5.79 3.74 0.03
34 Colon (ascending) 2.06 3.61 0.01
Stomach 24.66 26.06 15.07
36 Small intestine 5.95 5.11 0.02
37 Colon ca. 5W480 0.00 0.00 0.00
38 Colon ca.* (5W480 met)SW6200.00 0.00 0.00
39 Colon ca. HT29 0.00 0.02 0.00
Colon ca. HCT-116 0.00 0.00 0.00
41 Colon ca. CaCo-2 0.01 0.03 0.00
42 Colon ca. HCT-15 0.00 0.00 0.00
43 Colon ca. HCC-2998 0.00 0.00 0.00
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44 Gastric ca.* (liver met) 0.00 0.00 0.00
NCI-N87
45 Bladder 2.92 13.21 0.00
46 Trachea 24.49 15.82 17.43
47 Kidney 5.40 4.09 0.23
48 Kidney (fetal) 14.16 10.08 0.00
49 Renal ca. 786-0 0.00 0.00 0.00
50 Renal ca. A498 0.82 0.55 0.00
51 Renal ca. RXF 393 0.08 0.06 0.00
52 Renal ca. ACHN 0.69 0.44 0.00
53 Renal ca. U0-31 0.12 0.09 0.00
54 Renal ca. TK-10 1.50 0.57 0.00
55 Liver 5.37 4.45 1.75
56 Liver (fetal) 1.56 1.12 0.00
57 Liver ca. (hepatoblast) 0.00 0.00 0.00
HepG2
58 Lung 0.34 1.30 0.00
59 Lung (fetal) 2.68 1.62 0.00
60 Lung ca. (small cell) 0.00 0.00 0.00
LX-1
61 Lung ca. (small cell) 0.63 0.44 0.00
NCI-H69
62 Lung ca. (s.cell var.) 0.00 0.00 0.01
SHP-77
63 Lung ca. (large cell)NCI-H4600.63 0.48 0.00
64 Lung ca. (non-sm. cell) 6.98 6.12 0.00
A549
65 Lung ca. (non-s.cell) 0.22 0.12 0.00
NCI-H23
66 Lung ca (non-s.cell) HOP-622.78 2.03 0.00
67 Lung ca. (non-s.cl) NCI-H5220.03 0.01 0.00
68 Lung ca. (squam.) SW 900 11.50 11.19 2.40
69 Lung ca. (squam.) NCI-H5964.97 4.09 0.00
70 Mammary gland 32.76 31.43 24.32
71 Breast ca.* (p1. effusion)0.00 0.00 0.00
MCF-7
72 Breast ca.* (pl.ef) MDA-MB-2310.00 0.01 0.00
73 Breast ca.* (p1. effusion)0.00 0.11 0.00
T47D
74 Breast ca. BT-549 7.59 7.38 0.00
75 Breast ca. MDA-N 0.00 0.02 0.00
76 Ovary 9.61 11.03 0.00
77 Ovarian ca. OVCAR-3 0.84 0.22 0.00
78 Ovarian ca. OVCAR-4 0.31 0.20 0.00
79 Ovarian ca. OVCAR-5 81.79 78.46 93.95
80 Ovarian ca. OVCAR-8 2.08 1.54 0.00
81 Ovarian ca. IGROV-1 3.00 2.05 0.00
82 Ovarian ca.* (ascites) 0.12 0.05 0.00
SK-OV-3
83 Myometrium 5.08 7.38 0.26
84 Uterus 8.30 4.94 0.20
85 Placenta 7.33 5.79 0.29
86 Prostate 5.56 4.01 0.04
87 Prostate ca.* (bone met)PC-319.75 9.47 0.00
88 Testis 20.88 21.46 6.89
89 Melanoma Hs688(A).T 0.89 0.45 0.00
90 Melanoma* (met) Hs688(B).T0.91 0.46 0.00
91 Melanoma UACC-62 0.21 0.13 0.00
92 Melanoma M 14 0.68 ~ 0.20 ~ 0.00
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93 Melanoma LOX IMVI 1.57 0.99 0.00
94 Melanoma* (met) SK-MEL-5 1.47 0.50 0.00
95 Melanoma SK-MEL-28 5.95 4.45 0.00
96 Melanoma UACC-257 3.69 3.21 1.99
In Table 7, the following abbreviations are used:
ca. = carcinoma,
* = established from metastasis,
met = metastasis,
s cell var= small cell variant,
non-s = non-sm =non-small,
squam = squamous,
p1. eff = p1 effusion = pleural effusion,
glio = glioma,
astro = astrocytoma, and
neuro = neuroblastoma.
PCR cocktails including two sets primers and probes (a 30664188-specific and a
reference gene-specific probe, commonly (3-actin and/or GAPDH, multiplexed
with the
30664188 probe) were set up using 1X TaqManTM PCR Master Mix for the PE
Biosystems
7700, with 5 mM MgCl2, dNTPs (dA, dG, dC, dU at 1:1:1:2 ratios), 0.25 U/ml
AmpliTaq
GoIdTM (PE Biosystems), and 0.4 U/p,l RNase inhibitor, and 0.25 U/pl reverse
transcriptase.
Reverse transcription was performed at 48° C for 30 minutes followed by
amplification/PCR
cycles as follows: 95° C 10 min, then 40 cycles of 95° C for 15
seconds, 60° C for 1 minute.
The TaqMan probes and primers used were:
Ag33(F): 5'-CGCTTGGCATCATCATTGAG-3' (SEQ ID N0:35),
Ag33(R): 5'-CGGTATCGAGGCAGGTCATAC-3' (SEQ ID N0:36), and
Ag33(P): TET-5'-TCCAGGTCAACTTTTGACTTCCGGTCA-3'-TAMRA (SEQ ID N0:37);
Ag66(R): 5'-CACAAGGAAGTTCCTCCAAGGATA-3' (SEQ ID N0:38),
Ag66(F): 5'-AATCCAGGTTTAGCCACAAAGTAGTC (SEQ ID N0:39), and
Ag66(P): FAM-5'-AGAACGAACCAAATTAAAATCACATTCAAGTCCGA-TAMRA (SEQ ID
N0:40); and
Ag 168 (F): 5'-GCATGTGCAGGACCTCCAGT-3' (SEQ ID N0:41),
Ag 168 (R): 5'-TCCACGTTGCCTCCTCGT-3' (SEQ ID N0:42), and
Ag 168 (P): TET-5'-CAGTTCCACAGCCACAATTTCCTCCAC-3'-TAMRA (SEQ ID
N0:43).
Among normal tissues examined, clone 30664188 is highly expressed in pancreas,
adrenal gland, adipose tissue, stomach, trachea, mammary gland and testis.
Among various
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cancer cell lines, the clone is strongly expressed specifically in CNS cancer
(CNS ca. (glio)
SF-295), lung cancer (squamous cells, SW 900) and ovarian cancer (ovarian ca.
OVCAR-5).
Example 8. The clone 30664188Øm99 protein induces cellular DNA synthesis
Human CCD-1070 fibroblast cells (ATCC No. CRL-2091, Manassas, VA) or murine
NIH 3T3 (ATCC No. CRL-1658, Manassas, VA) fibroblast cells were cultured in
DMEM
supplemented with 10% fetal bovine serum or 10% calf serum respectively.
Fibroblasts were
grown to confluence at 37°C in 10% COZ/air. Cells were then starved in
DMEM for 24 h.
pCEP4/Sec ( Example 2) or pCEP4/Sec/30664188.m99 ( Example 6) enriched
conditioned
medium was added (10 microL/100 microL of culture ) for 18 h. BrdU (10 uM) was
then
added and incubated with the cells for 5 h. BrdU incorporation was assayed by
colorimetric
immunoassay according to the manufacturer's specifications (Boehringer
Mannheim,
Indianapolis, III.
FIG. 5 demonstrates that 30664188.m99 induced an approximate four- to five-
fold
increase in BrdU incorporation in either cell type compared to cells treated
with control
conditioned medium or untreated cells. The proliferative increase observed was
similar to the
increase in BrdU incorporation induced by platelet derived FCTRX (PDGF), basic
fibroblast
growth factor (bFGF), or serum treatment. Additionally, 30664188.m99 partially
purified
conditioned medium did not induce BrdU incorporation in human MG-63 epithelial
cells or
CCD1106 keratinocytes (data not shown). These results suggest that 30664188
selectively
induces DNA synthesis in human and mouse fibroblasts, but not in epithelial
cell lines.
In separate experiments, CCD-1070 cells and MG-63 osteosarcoma cells (ATCC
Cat.
No. CRL-1427) treated with pCEP4/Sec/30664188 each incorporated BrdU in a dose-
dependent fashion, with 1 ug/mL providing the full effect (approximately 2.5-
to 3-fold
increase over control), 100 ng/mL providing slightly less than one-half the
effect, and 10 and 1
ng/mL providing approximately control levels of incorporation. Furthermore,
the dose
response of NIH 3T3 cells shows that a 50% response occurs between doses of 10
and 50
ng/mL of pCEP4/Sec/30664188 (FIG. 6).
Example 9. Induction of Proliferation of NIH 3T3 cells by 30664188.m99
Murine NIH 3T3 fibroblasts were plated at 40% confluency and cultured in DMEM
supplemented with 10% fetal bovine serum or 10% calf serum for 24 hrs. The
culture medium
was removed and replaced with an equivalent volume of pCEP4/Sec (Example 2) or
pCEP4/Sec/30664188 (Example 6) conditioned medium. After 48 h, cells were
photographed
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with a Zeiss Axiovert 100. Cell numbers were determined by trypsinization
followed by
counting using a Coulter Z 1 Particle Counter.
Treatment of NIH 3T3 fibroblasts with conditioned medium from 30664188
transfected HEK293 kidney epithelial cells resulted in a 6 to 8 fold increase
in cell number
over a two day period (Fig. 7). Cells treated with control conditioned medium
from HEK293
cells transfected with the pCEP4/Sec vector alone demonstrated little or no
growth (Fig. 7).
To determine whether 30664188.m99 conditioned medium was able to induce
phenotypic changes characteristic of cellular transformation, cells treated
with either
30664188 conditioned medium or mock conditioned medium were examined by light
microscopy. FIG. 8 shows that NIH 3T3 cells treated with 30664188.m99, but not
control
treated NIH 3T3 cells, showed a marked increase in cell number, as well as
refractile
properties. Loss of contact inhibition of growth was evident. The cobblestone
appearance
characteristic of confluent NIH 3T3 cells was lost and density independent
growth was
evident. The latter was also suggested by the more rounded appearance of the
NIH 3T3 cells
1 S due to subtle retraction. Transfection of pCEP4/Sec/30664188.m99 also
showed nearly
identical potency in transformation potential 2 to 5 days in culture. After 7
to 10 days in
culture, however, the morphologically transformed phenotype appeared to
revert.
Example 10. Induction of proliferation of human primary osteoblast cells by
the
30664188 protein
In an experiment similar to that described in Example 9, human primary
osteoblast cells
(NHost; Clonetics)also underwent a dose-dependent increase in cell number by 3-
to 4-fold
(Fig. 9). The dose required to elicit a 50% response in Fig. 9 is below 100
ng/mL of
pCEP4/Sec/30664188.m99. In addition, Jurkat cells contacted with partially
purified
conditioned
medium containing the 30664188 gene product exhibited a doubling of BrdU
uptake
compared to the medium from mock transfection, whereas the same cells
contacted with 13
other CuraGen Corporation gene products thought to have growth promoting
activity elicited
no effect.
In summary, the observations that the 30664188 protein induces DNA synthesis
(Example 8), cell growth (Examples 9 and 10), and morphological transformation
(Example 9)
indicate that the protein possesses transforming properties.
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Example 11. Induction of tumor formation by the 30664188 protein
NIH 3T3 cells with treated conditioned medium from cells transfected with
pCEP4/Sec
or pCEP4/Sec/30664188 were cultured as described above. 106 cells in O.lmL PBS
were then
injected subcutaneously into the lateral subcutis of female nude mice (Charles
River
Laboratory), n=S per group (termed, e.g., pCEP4/Sec/30664188.m99 mice). After
11 and 14
days, tumor formation was assayed with calipers.
After 11 days, tumor growth was evident in pCEP4/Sec/30664188.m99 mice.
pCEP4/Sec/30664188.m99 mice (5/5) were positive for tumor formation with tumor
size
measuring 6.74 ~ 0.58 mm3. After 14 days in culture a noticeable decrease in
tumor size was
evident in pCEP4/Sec/30664188.m99 mice with 3/5 mice positive and average
tumor volume
1.44 ~ 0.88 mm3. Notably, and as a positive control, 5 of S mice treated with
bFGF developed
tumors which increased in volume to 66.56 ~ 13.2 mm3. Control vector mice
(0/5) were
negative for tumor formation. Although these data strongly suggest that
30664188.m99
overexpression induces tumor formation in nude mice, tumors appeared to be
lost as a function
of time. Strikingly, these data parallel the morphological reversion
properties noted in the
NIH 3T3 transformation assay.
Example 12. Purification of Intact and Cleaved Products of the 30664188.m99
Protein.
It was observed that in certain experiments treatment with the vector
pCEP4/Sec/30664188.m99 did not result in DNA synthesis or cell proliferation.
In additional
experiments, medium conditioned with 30664188.m99 was obtained from HEK 293
cells
grown in the presence of serum (Example 6). The 30664188.m99 gene product was
purified
by canon exchange chromatography, followed by nickel affinity chromatography.
The protein
product was run under nonreducing and reducing conditions on SDS-PAGE, and
developed by
Coomassie stain. The results are shown in FIGS. 10A and l OB. In the presence
of serum, the
30664188.m99 gene product appeared as a protein of about 35 kDa under
nonreducing
conditions (FIG. l OB. However, this polypeptide appears as three degraded
bands when run
under reducing conditions. The apparent molecular weights of the two bands
were 22-25 kDa
(band I), about 16 kDa (band II) and about S-6 kDa (band III). N-terminal
amino acid analysis
of these fragments indicates that bands I and II both begin at residue 247 of
the 30664188.m99
amino acid sequence, and that band III begins at residue 339. These results
are consistent with
cleavage of the polypeptide corresponding to band I to provide the fragments
of bands II and
III. It is possible that the 35 kDa band observed under nonreducing conditions
is a dimer
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composed of band I, and/or the bonded polypeptide composed of bands II and
III, observed
under reducing conditions.
Amino terminal analysis indicates that the gene product from
pCEP4sec/30664188.m99-transfected 293 cells grown in the presence of serum,
isolated
according to the procedure described above, is a carboxyl-terminal fragment of
the full length
protein. The 35 kDa band found under nonreducing conditions is termed p35
below.
When 293 cells were cultured in the absence of serum, and the same isolation
and
detection procedure described in the preceding paragraph is followed, a
different gene product
is observed. Under nonreducing conditions a band was found at about 85 kDa
(FIG. 10A).
This protein is termed p85 below. The corresponding gene product observed
under reducing
conditions a major band is found at about 53-54 kDa. N-terminal amino acid
analysis of this
gene product provides the amino acids at the multiple cloning site used in
pCEP4sec/30664188.m99 (Example 6). The residues corresponding to the Ig kappa
leader
sequence, cloned upstream from the multiple cloning site, are absent. These
results indicate
that the gene product obtained in the absence of serum represents the full
amino acid sequence
encoded in pCEP4sec/30664188.m99. The p85 polypeptide is thought to be a dimer
of the 50
kDa species observed on reducing SDS-PAGE.
Example 13. Activity of Intact and Cleaved Fragments of the 30664188.m99
Protein
Purified p85 and p35 FCTRX proteins were separately applied to NIH 3T3 cells
in a
range of concentrations. Incorporation of BrdU was evaluated as described in
Example 8.
The results are shown in Fig. 11. It is seen that p85 has growth-promoting
activity that does
not differ from control levels except at the highest concentration used. p35,
on the other hand,
was at least as active, if not more so, than unfractionated pCEP4/Sec/30664188
conditioned
medium. The concentration of p35 giving 50% of the maximum DNA synthesis falls
between
20 and 50 ng/mL.
These results suggest that the p35 fragment derived from intact 30664188.m99
has
growth-promoting activity but that the intact dimeric form of the .m99
protein, p85, does not.
Therefore, reversion of transformation and tumor formation seen in Examples 9
and 11 may be
the result of the emergence of a species in the culture at such longer times
that inhibits or
prevents formation of a p35-like species from p85.
Example 14. Isolation of murine PDGFD cDNAs
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Marine nucleic acid sequence encoding a PDGFD polypeptide was amplified from a
marine brain library (Clontech) by PCR using the forward primer
5'-CGCGGATCCATGC AACGGCTCGTTTTAGTCTCCATTCTCC-3' (SEQ ID N0:94)
and the reverse primer
S 5'- CGCGGATCCTTATCGAGGTGGTCTTGAGCTGCAGATA CAGTC-3' (SEQ ID N0:45).
The sequences of the marine polynucleotide (SEQ ID NO:S) and the corresponding
polypeptide encoded by it (SEQ ID N0:6) are shown in Table 3.
Example 15 Genomic organization of the PDGFD gene.
Utilizing genomic DNA sequences obtained from GenBank exon/intron organization
of the PDGFD gene was determined. Intron/exon boundaries were deduced using
standard
consensus splicing parameters (1616. S. Mount, Nucleic Acids Res. 10, 459-472.
(1982).).
Phase I genomic DNA sequence reveals the PDGF D gene to be comprised of 7
exons (Fig.
13), similar to PDGF A and PDGF B. BLASTN analysis generated hits (>99%) to
the
following genomic clones: Acc. Nos. AC026640, AC023129, AC024052, and
AC067870. All
clones were mapped to chromosome 11 q23.3-24. Chromosomal location was further
refined
by radiation hybrid analysis.
The initiation codon is located in exon 1 and the TAA termination codon
located in
exon 7. Exon 1 is located on AC023129; whereas exons 2-7 are located on
AC024052. The
clones comprising the majority of the exons (AC023129 and AC024052) are Phase
I
unordered genomic clones so intron sizes could not be determined. For PDGF D,
both the
CUB (exons 2 & 3) and PDGF (exons 6 & 7) domains span two exons. PDGF D lacks
the
COOH terminal retention motif found in the PDGF A exon 6 splice variant and
PDGF B (W.
LaRochelle, M. May-Siroff, K. Robbins, S. Aaronson, Genes Dev. 5, 1191-1199
(1991).). An
in-frame stop codon was found 9 by upstream of the initiator methionine.
Example 16: .Molecular Cloning of Novel Splice Variants of 30664188Ø99
In this example, cloning is described for novel spice variants of clone
30664188.099.
Olignucleotide primers were designed to PCR amplify the sequence, these
primers include:
30664188 TOPO F: CCACC ATG CAC CGG CTC ATC TTT GTC TAC ACT C (SEQ ID
NO: 46), and
30664188 TOPO R: TCG AGG TGG TCT TGA GCT GCA GAT ACA (SEQ ID N0: 47).
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PCR reactions were performed using S ng human pancreas cDNA templates. The
reaction mixtures contained 1 microM of each of the 30664188 Eco Forward and
3066418
Xho Reverse primers, 5 micromoles dNTP (Clontech Laboratories, Palo Alto CA)
and 1
microliter of SOxAdvantage-HF 2 polymerase (Clontech Laboratories, Palo Alto
CA) in 50
microliter volume. The following reaction conditions were used:
a) 96°C 3 minutes
b) 96°C 30 seconds denaturation
c) 70°C 30 seconds, primer annealing. This temperature was gradually
decreased by 1°C/cycle
d) 72°C 1 minute extension.
Repeat steps b-d 10 times
e) 96°C 30 seconds denaturation
f) 60°C 30 seconds annealing
g) 72°C 1 minute extension
Repeat steps e-g 25 times
h) 72°C 5 minutes final extension
In addition to the amplified product predicted for the full length clone of
30664188Ø99, having 1041 bp, two additional bands were detected. These
fragments were
purified from agarose gel and ligated to pCR2.1 vector (Invitrogen, Carlsbad,
CA). The
cloned inserts were sequenced using M13 Forward, M13 Reverse and the four gene
specific
primers presented in Example 1.
Both cloned inserts were sequenced and verified as shorter spice forms of
30664188Ø99.
Example 17. Purification of recombinant PDGF DD. The gene product of PDGFD was
expressed in HEK293 cells grown on porous microcarners (Cultisphere-GL,
Hyclone; Logan,
UT) in 1 L spinner flasks. As noted in Examples 2 and 4, the recombinant PDGF
D gene
includes a 6xHis fusion at the 3' end. Cells were grown in DMEM/F 12 media
containing 1
penicillin/ streptomycin in the presence or absence of 5% fetal bovine serum
(FBS). The
0
conditioned medium was harvested by centrifugation (4000 x g for 1 S minutes
at 4 C) and
loaded onto a POROS HS50 column (PE Biosystems; Foster City, CA), pre-
equilibrated with
20 mM Tris-acetate (pH 7.0). After washing with the equilibration buffer,
bound proteins
were eluted with a NaCI step gradient (0.25 M, 0.5 M, 1.0 M and 2.0 M).
Fractions containing
PDGF DD p35 (1.0 M NaCI step elution) or p85 (0.5 M NaCI step elution) (see
Example 12)
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were pooled and diluted with an equal volume of phosphate-buffered saline
(PBS), pH 8.0
containing 0.5 M NaCI, then loaded onto a POROS MC20 column pre-charged with
nickel
sulfate (PE Biosystems). After washing with PBS/0.5 M NaCI, bound proteins
were eluted
with a linear gradient of imidazole (0 - 0.5 M). Fractions containing PDGF DD
(homodimers
of PDGFD) (100 - 150 mM imidazole) were pooled and dialyzed twice against 1000
volumes
of 20 mM Tris-HCI, pH 7.5, 50 mM NaCI. The protein purity was estimated to be
> 95% by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 4-20%
Tris-glycine
gradient gel; Invitrogen, Carlsbad, CA) analysis (See, for example, the
results in Example 12,
including Fig. 10 A).
Biochemical Properties of PDGF D. To examine the biochemical properties of the
gene product of PDGF D, the cDNA encoding PDGF D protein was subcloned into a
mammalian expression vector, pCEP4/Sec-30664188 (Example 4). This construct
incorporates an epitope tag (V5) and a polyhistidine tag into the COON
terminus of the protein
to aid in its identification and purification (expression vector pCEP4/Sec-
30664188; Example
4).
Following transfection into 293 HEK cells and growth in serum-free culture, a
secreted
polypeptide with an apparent molecular weight of ~49 kDa (p49 species) was
identified by
Western blot analysis under reducing conditions (Fig. 14 A, lane 2). The fact
that the apparent
molecular weight of p49 is greater than the expected value of ~43-kDa may be
attributable to
glycosylation. In contrast, a 20-kD protein was secreted when PDGF D-
transfected cells were
grown in the presence of FBS (Fig. 14 A, lane 3). Conditioned media from mock
transfected
cells did not react with the anti-V5 antibody (Fig. 14 A, lane 1).
In addition, PDGF D was expressed in the presence or absence of FBS and
purified to
>95% homogeneity. As shown in Fig. 14 B (lane 2), expression of PDGF D under
serum-free
conditions resulted in the detection of the expected 49-kD gene product under
reducing
conditions, when the gel was stained using Coomassie Blue. A polypeptide
species with an
apparent molecular weight of about 84 kDa, corresponding to a dimeric p85
species of p49,
was seen under non-reducing conditions (Fig. 14 B, lane 1). When PDGF DD was
purified
from serum-containing conditioned medium and run under nonreducing conditions,
a species
with an apparent molecular weight of about 35 kDa (p35) was observed (Fig.l4
B, lane 3).
Under reducing conditions, p35 was found to yield three bands when visualized
with
Coomassie Blue, which migrate with apparent molecular weights of approximately
20, 14,
and 6 kDa (Fig. 14 B, lane 4).
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Amino terminal sequence analysis of p35 demonstrated proteolytic cleavage
after
8247 or 8249 (Fig. 15). As indicated in Panel A of Fig. 15, two peptides were
found, one
beginning with GlyArg (shown with these two residues underlined), and the
second beginning
with the third residue, Ser. The ratio of these peptides was found to be
SYHDR:GRSYHDR =
4:1. The additional sequencing results in Fig. 15 (Panels B and C) indicate
that further
processing produces the remaining polypeptides seen with Coomassie blue
staining but not
with anti-V5 Westerns, namely the 16 kDa and 6 kDa species shown. These are
joined
together to provide p35.
The results presented in this Example indicate that the PDGF D gene products
are
dimers in both the holoprotein form (p85) and the C-terminal fragment (p35).
The p85 form
appears to be processed in the presence of FBS to provide the p35 form. These
dimeric forms
are designated PDGF DD.
Example 18. Processing of the 30664188 Gene Product in the Presence of Fetal
Bovine
Serum and Calf Serum.
The 30664188 gene product was incubated in the presence of increasing
concentrations
of calf serum (Fig. 16, Panel A) or fetal bovine serum (Panel B). The results
demonstrate that
only fetal bovine serum (Panel B) but not calf serum (Panel A) processes the
p85 form of the
30664188 gene product to provide p35.
Example 19 Induction of DNA synthesis
This example demonstrates the ability of PDGF DD to induce DNA synthesis.
Various cells were cultured in 96-well plates to 100% confluence, washed, fed
with
DMEM and starved for 24 hrs. Recombinant PDGF DD, PDGF AA, or PDGF BB was then
added at the indicated concentration to the cells for 18 hrs. In some
instances, cells were
untreated or treated with 10% FBS. The BrdU assay was performed according to
the
manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN)
using a 5 hr
BrdU incorporation time.
In human CCD1070 foreskin fibroblasts, it was determined that p35 induces DNA
synthesis at a half maximal concentration of ~ 20 ng/ml (Fig. 17A). In
contrast, p85 did not
induce DNA synthesis at concentrations up to 100 ng/ml. Comparatively, PDGF AA
and
PDGF BB induced half maximal DNA synthesis at ~ 5 and 8 ng/ml respectively.
PDGF DD
and PDGF BB induced similar DNA synthesis at maximal doses, while PDGF AA was
four-
fold less potent.
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In NIH 3T3 embryonic lung fibroblasts, p35 induced DNA synthesis at a half
maximal
concentration of approximately 20 ng/ml (Fig. 17 B). In contrast, p85 did not
induce DNA
synthesis incorporation at concentrations up to 1 ug/ml (Fig. 17B), nor did it
block p35 or
PDGF BB-induced DNA synthesis.
p35 also induced DNA synthesis in a variety of human cells including MG-63
osteosarcoma cells and primary smooth muscle cells. This suggest that PDGF DD
is a latent
growth factor whose activity is dependent on proteolytic dissociation of the
PDGF core
domain from the CUB-containing region.
Example 20 Cell Proliferation
This EXAMPLE demonstrates that PDGF DD is able to sustain cell growth. NIH 3T3
fibroblasts were cultured in 6-well plates to ~35% confluence, washed with
DMEM and then
starved 8 hrs. Cells were then treated with DMEM supplemented with either
recombinant
PDGF DD, PDGF AA, or PDGF BB (200 ng/ml) or S % FBS. Growth factors were added
after 24 h and quantitated after trypsinization using a Beckman Coulter Z1
series counter
(Beckman Coulter, Fullerton, CA).
PDGF DD induced a ~2-fold increase in NIH 3T3 cell number after the first day
and a
~4-fold increase after two days relative to untreated cells. The increase in
proliferation was
similar to that of PDGF AA and PDGF BB. (FIG. 17C) PDGF DD was also able to
sustain
the growth of CCD1070 fibroblasts and that of cells from several smooth muscle
types over
several days, as well as slightly enhance the growth rate of NIH-3T3
fibroblasts when used in
combination with PDGF BB
Example 21 PDGF Receptor tyrosine phosphorylation.
This EXAMPLE demonstrates the ability of PDGF D to bind the PDGF receptor.
NIH 3T3 fibroblasts were serum starved, and then treated with 200 ng/ml PDGF
DD,
PDGF AA or PDGF BB for 10 min. Cells were washed once with PBS, 100 uM sodium
orthovanadate. Whole cell lysates were solubilized in RIPA buffer [50 mM Tris-
HCI, pH 7.4,
50 mM NaCI, 1.0% Triton X-100, 5 mM EDTA, 10 mM sodium pyrophosphate, 50 mM
sodium
fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride,
leupeptin (10
ug/ml), pepstatin (10 ug/ml), and aprotinin (1 ug/ml)], sonicated, and
incubated on ice for 30
min. Lysates were clarified by centrifugation at 15,000 x g for 10 min.
Supernatants containing
equivalent amounts of total protein were incubated with anti-a or anti-(3
PDGFR antibody
(Santa Cruz Biotechnology; Santa Cruz, CA, 5 ug) for 2 hrs. Next, 100 u1 of a
1:1 slurry of
Protein G agarose was added for 2 hrs. Immunocomplexes were washed 3X with
RIPA buffer.
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SDS-PAGE sample buffer containing 100 mM dithiothreitol was added, and the
samples were
fractionated on 4-15% SDS-polyacrylamide gels. After electrophoretic transfer
to Immobilon P
membranes ( Millipore; Bedford, MA ), filters were blocked in TTBS (20 mM Tris-
HCI, pH 7.4,
150 mM NaCI, 0.05% Tween 20), 3% nonfat milk. Membranes were then incubated
with anti-a
S or (3 PDGFR antibody (1: S00) or anti-phosphotyrosine monoclonal antibody
(Upstate
Biotechnology Inc.; Lake Placid, NY, 1:1000) for 1-2 hrs in TTBS, 1% BSA, and
washed 4X
with TTBS. Bound antibody was detected after a 1 hr incubation with goat anti-
rabbit IgG
(whole molecule; 1:2,000) or goat anti-mouse IgG (H & L; 1:10,000) conjugated
to horseradish
peroxidase (Boehringer Mannheim, Indianapolis, IN) followed by 4 washes with
TTBS.
Enhanced chemiluminescence (Amersham; Piscataway, NJ) was performed according
to the
manufacturer's protocol.
To investigate the possibility that PDGF DD might signal through a and/or [3
PDGFRs,
PDGFR autophosporylation on tyrosine residues was examined after ligand
treatment. NIH 3T3
fibroblasts were serum starved and stimulated with 100 ng/ml 3066, PDGF AA or
PDGF BB for
10 min. Cells were washed once with PBS, 100 uM sodium orthovanadate. Whole
cell lysates
were prepared by solubilization in RIPA buffer [SO mM Tris pH 7.4, 50 mM NaCI,
1.0% Triton
X-100, 5 mM EDTA, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM
sodium
orthovanadate, 1 mM phenylmethylsulfonylfluoride, leupeptin (10 ug/ml),
pepstatin (10 ug/ml),
and aprotinin (1 ug/ml)], sonication, and incubation on.ice for 30 min.
Lysates were cleared by
centrifugation at 14,000 rpm for 10 min. Lysates containing equivalent amounts
of total protein
were incubated with anti-alpha or beta PDGFR antibody for 2 hr. Next, 100 u1
of a 1:1 slurry of
protein G Sepharose was added for 2 hr. Immunocomplexes were washed three
times with
RIPA buffer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) sample
buffer containing 100 mM dithiothreitol was added, and the samples were
fractionated on 4-15%
SDS-polyacrylamide gels. After electrophoretic transfer to Immobilon P
membranes, filters
were blocked in TTBS (20 mM Tris pH 7.4, 150 mM NaCI, .0S% Tween 20), 3%
nonfat milk.
Membranes were then incubated with anti-alpha or beta PDGFR serum (1:1000) or
anti-
phosphotyrosine (1:1000) for 1-2 hours in TTBS, 1% BSA, and washed four times
with TTBS.
Bound antibody was detected by incubation with anti-rabbit (1:10,000) or anti-
mouse antibody
(1:10,000) conjugated to horseradish peroxidase (Amersham, Arlington Heights,
IL) for 30 min
and subsequently washing four times with TTBS. Enhanced chemiluminescence
(Amersham)
was performed according to the manufacturer's protocol.
As shown in Fig. 17D, a 10 min exposure of NIH 3T3 fibroblasts to PDGF DD
induced the tyrosine phosporylation of both a and (3 PDGFRs. The observed
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phosphorylation was identical to that observed after PDGF BB treatment. As
expected, PDGF
AA induced only a PDGFR phosphorylation, confirming the specificity of the
assay. PDGF
DD, like PDGF BB, but not PDGF AA, was also able to induce the tyrosine
phosphorylation
of (3 PDGFRs in H-157 cells K. Forsberg, J. Bergh, B. Westermark, Int. J.
Cancer 53, 556-560
(1993)) that express only the (3 PDGFR These data show that PDGF DD, like PDGF
BB,
stimulates cell growth and proliferation through activation of both alpha and
beta PDGFRs.
Example 22. Competition of 30664188 p85 with Other Growth Factors that Induce
Growth of NIH/3T3 Cells.
NIH/3T3 cells were incubated with PDGF BB alone, 30664188 p35 alone, p35 in
the
presence of 100-fold increasing concentrations of p85, or PDGF BB in the
presence of 100-
fold increasing concentrations of p85 (from left to right in Fig. 18). Cell
growth was
determined by a BrdU incorporation assay. 30664188 p35 alone and PDGF BB alone
profoundly stimulate the growth of NIH/3T3 cells over that provided by
starving the cells (Fig.
18, left). It is seen that p85 has no effect on the growth induced by either
of these growth
factors, even at the very high concentration of 5000 ng/mL. Thus p85, which is
the dimer of
the full length gene product, has no affinity for the receptor or receptors to
which p35 and
PDGF BB bind. This experiment shows that processing of p85 to provide p35 is a
necessary
requirement for the 30664188 gene product to exert its activity.
Example 23. Differential Gene Expression Induced by Treatment with Growth
Factors.
GeneCallingTM reactions were performed on CCD1070 primary human foreskin
fibroblasts treated for 3 hrs with 200 ng of PDGF DD, PDGF BB, PDGF AA or
control buffer
(20 mM Tris-HCI, pH 7.5, 50 mM NaCI). GeneCallingTM analysis is described
fully in U. S.
Patent No. 5,871,697 and in Shimkets et al., "Gene expression analysis by
transcript profiling
coupled to a gene database query" Nature Biotechnology 17:198-803 (1999),
incorporated
herein by reference in their entireties.
Triplicate samples were prepared for each treatment. Total RNA was isolated
with
Trizol (Life Technologies, Inc.; Rockville MD) and poly(A)+ mRNA was prepared.
cDNAs
were synthesized using Superscript II (Life Technologies, Inc.), and then
digested by 48
distinct pairs of 6-by recognition site restriction endonucleases. The
restriction fragments were
then tagged with both biotin and fluorescent label, and amplified for 20
cycles by PCR. The
resulting product from each individual digestion was separated over a
streptavidin column and
eluted fragments containing both restriction enzyme recognition sites were
resolved by
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capillary electrophoresis on a MegaBace instrument ( Molecular Dynamics;
Sunnyvale, CA ).
Trace data output was analyzed by the Open Genome InitiativeTM software suite
(Shimkets et
al., (1999).) and differentially expressed peaks between each treatment and
the vehicle control
were identified using the GeneScapeTM data analysis suite. Putative gene
assignments for each
S differentially expressed fragment were made by database lookup using the
determined size for
each fragment as well as the 12 by of known sequence pre-determined by the
presence of
terminal restriction sites. Gene assignments were confirmed using
oligonucleotide poisoning,
as previously described. Oligonucleotide poisoning is described fully in U. S.
Patent
Application Serial No. 09/381,779 filed August 7, 1999, and in Shimkets et al.
(1999),
incorporated herein by reference in their entireties.
Fragmentation of cDNAs with 48 pairs of restriction enzymes resulted in a
survey of
approximately 85%, or about 19,000 individual gene fragments (R. Shimkets et
al., (1999)) of
the CCD1070 transcriptome. As shown in Fig. 19A, 301 gene fragments,
representing 1.6%
of all expressed genes, were found to be differentially regulated (greater
than + 2-fold, shaded
or hatched boxes) by at least one of the treatments. PDGF AA demonstrated the
most
restricted activity, changing the expression of only 57 gene fragments ( Fig.
19 A; 0.3% of
expressed fibroblast genes). PDGF DD and PDGF BB modulated 209 (1.1% of
expressed
genes ) and 289 (1.5% of expressed genes ) gene fragments, respectively. All
PDGF proteins
exhibited preferentially inductive effects on transcription since 237 (78.5%)
of all gene
fragments detected were up-regulated in the assayed treatments (Fig. 19 A).
Suprisingly, of the 209 gene fragments modulated by PDGF DD, 199 were
similarly
affected by PDGF BB (Fig. 19A). As shown in Fig. 19B, genes regulated by both
PDGF DD
and BB include secreted cytokines/chemokines (e.g., vascular endothelial cell
growth factor
(VEGF), IL-11, pre-B cell enhancing factor, monocyte chemotactic protein (MCP-
1)),
receptors (e.g., IL-1 receptor), proteases and protease inhibitors
(e.g.,plasminogen activator
inhibitor-1), signaling molecules/transcription factors (e.g., guanylate
binding protein land
adenosylmethionine decarboxylase), and matrix associated proteins. In
addition, PDGF BB
differentially regulated an additional 90 gene fragments not significantly
affected ( < ~ 2-fold)
by PDGF DD. Examples of genes induced preferentially by PDGF BB include, e.g.,
plasminogen activator inhibitor-2, progression associated protein, glycerol
kinase, and
aminopeptidase N/CD13. These results indicate that PDGF DD and PDGF BB share
similar
signaling mechanisms, suggesting that they signal through identical receptors
(Fambrough D,
McClure K, Kazlauskas A, L. ES, Cell 97, 727-741 (1999)).
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Example 24 Competition of Growth of CCD 1070 Cells in Response to Growth
Factors in
the Absence or Presence of Receptor Antibodies.
CCD 1070 cells were incubated in the presence of the purified p35 form of
30664188,
PDGF AA or PDGF BB. In each case the growth factor was incubated by itself, or
with a
nonspecific antibody (Rab) or with an antibody specific for the alpha PDGF
receptor (alpha
Rab), the beta PDGF receptor (beta Rab), or in the presence of both specific
antibodies. The
specific antibodies were from R&D Systems, and were added at 10 ug/ml. The
growth of
the cells was monitored by determining the uptake of BrdU using an ELISA assay
specific for
BrdU incorporation.
The results are shown in Fig. 20. It is seen that in the presence of p35, the
uptake of
BrdU is reduced by coincubation with anti-beta PDGF receptor, or coincubation
with the
mixture of both specific antibodies. The same pattern is observed for the
growth induced by
PDGF BB. With PDGF AA, on the other hand, the growth induced by the growth
factor is
reduced in the presence of anti-alpha PDGF receptor antibody, or in the
presence of the
1 S mixture.
The results of this experiment indicate that the active form of the 30664188
gene
product, p35 binds primarily or exclusively to the PDGF beta receptor, and
minimally or not at
all to the alpha receptor.
Example 25 Stimulation of Growth of Pulmonary Artery Smooth Muscle Cells by
Growth Factors.
This EXAMPLE demonstrates the ability of PDGF DD to stimulate growth of
pulmonary artery smooth muscle cells.
The p35 dimer of 30664188, PDGF AA or PDGF BB were added at various
concentrations to pulmonary artery smooth muscle cells (Clonetics) after being
cultured in 6-
well plates to ~35% confluence, washed with DMEM, and starved overnight. After
18 hrs,
BrdU was added, and S hrs later the cells were analyzed for BrdU incorporation
using a BrdU-
directed ELISA.
The results are shown in Fig. 21 It is seen that the maximal effect achieved
by
treatment with p35 dimer exceeds that given by both PDGF AA and PDGF BB. As
found in
Example 23, it is seen that the effects of p35 dimer and PDGF BB resemble each
other more
closely than the effect obtained with PDGF AA. Of all three growth factors
tested, p35 dimer
induced the greatest growth in smooth muscle cells, as determined by BrdU
incorporation,
with 50% maximal effect obtained at less than 12.5 ng/mL.
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Example 26 Proliferation of Pulmonary Artery Smooth Muscle Cells in Response
to
Various Growth-Promoting Treatments.
This EXAMPLE demonstrates the ability of PDGF DD to stimulate proliferation of
pulmonary artery smooth muscle cells.
Pulmonary artery smooth muscle cells were cultured in 6-well plates to ~35%
confluence, washed with DMEM, and starved overnight. Cells were then fed with
DMEM
supplemented with recombinant 30664188, PDGF AA, or PDGF BB (200 ng/ml) or 10%
FBS
for three days. Culture fluids were removed and replaced with same media for
an additional 2-
3 days. To quantitate the smooth muscle cell growth assay, cells were
trypsinized and counted
with a Beckman Coulter Z1 series counter (Beckman Coulter, Fullerton, CA).
The results are shown in Fig. 22. It is seen that PDGF produces a modest
increase in
cell number, whereas treatment with 30664188 provides an effect, compared with
control, that
is almost double that observed with PDGF. A positive control using treatment
with 10% FBS
gave a very pronounced effect. Treatment of smooth muscle cells with 30664188
and PDGF
BB led to elongated bipolar spindle shaped phenotype in contrast to the flat
club shaped
phenotype observed with serum.
30664188 is an effective stimulant of pulmonary artery smooth muscle cell
proliferation, and suggests that 30664188 and 30664188 antibodies has a
therapeutic use in
wound healing, tissue repair and cartilage repair. Furthermore, antibodies
directed against
30664188 may have therapeutic use in inhibiting or preventing restenosis of
patent
vasculature.
Example 27 Proliferation of Saphenous Vein Cells in Response to Various Growth-
Promoting Treatments.
This EXAMPLE illustrates the ability of PDGF DD to stimulate proliferation in
saphenous vein cells. Saphenous vein cells were treated and analyzed as
described in Example
26. The results are shown in Fig. 23. It is seen that PDGF produces a slightly
lower increase
in cell number than does treatment with 30664188, which provides proliferation
to almost 5
times the cell number seen with the control. A positive control using
treatment with 10% FBS
gave a very pronounced effect. 30664188 is an effective stimulant of saphenous
vein cell
proliferation, and suggests that 30664188 and 30664188 antibodies has a
therapeutic use in
wound healing, tissue repair and cartilage repair. Furthermore, antibodies
directed against
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30664188 may have therapeutic use in inhibiting or preventing restenosis of
patent
vasculature.
Example 28 Inhibition of the Growth of NIH 3T3 Mouse Cells
This EXAMPLE demostrates the ability of an anti-30664188 antibody to inhibit
the
growth of NIH3T3 cells. NIH/3T3 mouse fibroblasts were grown in the presence
30664188
alone, or together with increasing concentrations of antibody. Either a fully
human polyclonal
antibody directed against 30664188, or nonimmune antibody as a control was
used. The
polyclonal antibody was obtained by methods such as those described above in
the Detailed
Description of the Invention in the section on "Antibodies".
The results are shown in Fig. 24. It is seen that the 30664188-specific
antibody
abrogates the growth effect induced by treatment with 30664188 alone.
Treatment with
nonimmune antibody has no effect leading to a decrease in the induced growth.
The specific
antibody has a 50% maximal effect at a concentration of approximately S00
ng/mL. In a
parallel experiment, the anti-30664188 antibody had no effect on the growth of
NIH/3T3 cells
induced by PDGF AA or PDGF BB.
Therapeutic applications for treatment with a 30664188-specific antibody
include for
example, any pathology or disease in which growth that is stimulated by
30664188 would be
beneficially inhibited or prevented. These pathologies include for example,
diseases related to
growth of vasculature, inflammatory disorders, e.g., arthritis , bowel
disease, atherosclerosis,
restenosis of patent vasculature, and various solid tumors.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in
conjunction with
the detailed description thereof, the foregoing description is intended to
illustrate and not limit
the scope of the invention, which is defined by the scope of the appended
claims. Other
aspects, advantages, and modifications are within the scope of the following
claims.
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