U.S. patent application number 11/784976 was filed with the patent office on 2008-03-13 for calcium-sensing receptor 2 (car2) and methods for using.
Invention is credited to George Mbella Ekema, Pieter W. Faber, Benjamin Philip Faga, Gregory B. Foust, John J. Harrington, Paul David Jackson, Robert W. Mays.
Application Number | 20080064764 11/784976 |
Document ID | / |
Family ID | 32109840 |
Filed Date | 2008-03-13 |
United States Patent
Application |
20080064764 |
Kind Code |
A1 |
Ekema; George Mbella ; et
al. |
March 13, 2008 |
Calcium-sensing receptor 2 (CAR2) and methods for using
Abstract
The current disclosure provide a novel human calcium sensing
receptor named CaR2 and the nucleotide sequence that encodes the
receptor. The disclosure further provides antibodies specific for
CaR2. Also disclosed are methods of identifying modulators of the
receptor and methods of using the identified modulators to treat
calcium receptor mediated conditions.
Inventors: |
Ekema; George Mbella;
(Lakewood, OH) ; Faber; Pieter W.; (Westlake,
OH) ; Faga; Benjamin Philip; (University Heights,
OH) ; Foust; Gregory B.; (Euclid, OH) ;
Harrington; John J.; (Mentor, OH) ; Jackson; Paul
David; (Shaker Heights, OH) ; Mays; Robert W.;
(Cleveland Heights, OH) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP
ONE POST OFFICE SQUARE
BOSTON
MA
02109-2127
US
|
Family ID: |
32109840 |
Appl. No.: |
11/784976 |
Filed: |
April 10, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10283842 |
Oct 29, 2002 |
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11784976 |
Apr 10, 2007 |
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60421941 |
Oct 28, 2002 |
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Current U.S.
Class: |
514/789 ;
435/320.1; 435/325; 435/6.16; 435/69.1; 435/7.1; 530/350;
530/388.22; 530/389.1; 536/23.5 |
Current CPC
Class: |
G01N 33/5008 20130101;
A61P 19/10 20180101; A61P 19/00 20180101; G01N 33/5091 20130101;
G01N 33/6872 20130101; A61P 35/04 20180101; A61P 13/08 20180101;
A61K 38/00 20130101; A61P 35/00 20180101; C07K 14/705 20130101;
A61P 43/00 20180101; C07H 21/04 20130101; A61P 3/14 20180101; G01N
33/502 20130101; G01N 2500/10 20130101; A61P 1/02 20180101; G01N
33/5044 20130101; A61P 15/08 20180101; A61P 13/12 20180101 |
Class at
Publication: |
514/789 ;
435/320.1; 435/325; 435/006; 435/069.1; 435/007.1; 530/350;
530/388.22; 530/389.1; 536/023.5 |
International
Class: |
A61K 45/00 20060101
A61K045/00; A61P 19/10 20060101 A61P019/10; A61P 35/00 20060101
A61P035/00; C07H 21/04 20060101 C07H021/04; C07K 14/705 20060101
C07K014/705; C07K 16/28 20060101 C07K016/28; C12N 15/85 20060101
C12N015/85; C12N 5/10 20060101 C12N005/10; C12P 21/06 20060101
C12P021/06; C12Q 1/68 20060101 C12Q001/68; G01N 33/53 20060101
G01N033/53 |
Claims
1. An isolated nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence set forth in SEQ ID NO:2.
2. A vector comprising the nucleic acid molecule of claim 1.
3. The vector of claim 2, which is an expression vector.
4. A host cell transfected with the vector of claim 3.
5. An isolated polypeptide comprising the amino acid sequence set
forth as SEQ ID NO:2.
6. The polypeptide of anyone of claim 5, further comprising
heterologous amino acid sequences.
7. An antibody which selectively binds to the polypeptide claim 5,
or a fragment thereof.
8. A method for detecting the presence of a polypeptide in a
sample, said method comprising contacting said sample with an agent
that specifically allows detection of the presence of the
polypeptide in the sample and then detecting the presence of the
polypeptide, wherein said polypeptide is selected from the group
consisting of: (a) The amino acid sequence shown in SEQ ID NO: 2;
(b) The amino acid sequence of an allelic variant of the amino acid
sequence shown in SEQ ID NO: 2; (c) The amino acid sequence of a
sequence variant of the amino acid sequence shown in SEQ ID NO: 2,
wherein the sequence variant is encoded by a nucleic acid molecule
hybridizing to the nucleic acid molecule shown in SEQ ID NO:1 under
stringent conditions; (d) A fragment of the amino acid sequence
shown in SEQ ID NO: 2, wherein the fragment comprises at least 10
contiguous amino acids; (e) The amino acid sequence of an epitope
bearing region of anyone of the polypeptides of (a)-(e); wherein
said sample is derived from cells involved in abnormal cell
proliferation.
9. The method of claim 8, wherein said agent is capable of
selective physical association with said polypeptide.
10. The method of claim 9, wherein said agent binds to said
polypeptide.
11. The method of claim 10, wherein said agent is an antibody.
12. A method for identifying an agent that modulates the level or
activity of a nucleic acid molecule in a cell, wherein said nucleic
acid molecule has a nucleic acid sequence selected from the group
consisting of: (a) The nucleotide sequence shown in SEQ ID NO:1;
(b) A nucleotide sequence encoding the amino acid sequence shown in
SEQ ID NO: 2; (c) A nucleotide sequence complementary to any of the
nucleotide sequences in (a) or (b); (d) A nucleotide sequence
encoding an amino acid sequence of a sequence variant of the amino
acid sequence shown in SEQ ID NO: 2 that hybridizes to the
nucleotide sequence shown in SEQ ID NO:1 under stringent
conditions; (e) A nucleotide sequence encoding a fragment of the
amino acid sequence shown in SEQ ID NO: 2, wherein the fragment
comprises at least 10 contiguous amino acids; said method
comprising contacting said agent with a cell capable of expressing
said nucleic acid molecule such that said nucleic acid molecule
level or activity can be modulated in said cell by said agent and
measuring said nucleic acid molecule level or activity.
13. A method for identifying an agent that modulates the level or
activity of a polypeptide in a cell, wherein said polypeptide is
selected from the group consisting of: (a) The amino acid sequence
shown in SEQ ID NO:2; (b) The amino acid sequence of an allelic
variant of the amino acid sequence shown in SEQ ID NO: 2; (c) The
amino acid sequence of a sequence variant of the amino acid
sequence shown in SEQ ID NO: 2, wherein the sequence variant is
encoded by a nucleic acid molecule hybridizing to the nucleic acid
molecule shown in SEQ ID NO:1 under stringent conditions; (d) A
fragment of the amino acid sequence shown in SEQ ID NO.2, wherein
the fragment comprises at least 10 contiguous amino acids; (e) The
amino acid sequence of an epitope bearing region of anyone of the
polypeptides of (a)-(d); said method comprising: contacting said
agent with a cell capable of expressing said polypeptide such that
said polypeptide level or activity can be modulated in said cell by
said agent and measuring said polypeptide level or activity.
14. The method of claim 13 wherein said agent decreases the level
or activity of said polypeptide.
15. A method of treating a subject with a calcium receptor mediated
disorder comprising the step of administering a calcium receptor
modulator identified by the method of claim 12 to said individual
such that the subject is treated.
16. A method of treating a subject with a calcium receptor mediated
disorder comprising the step of administering a calcium receptor
modulator identified by the method of claim 13 to said individual
such that the subject is treated.
17. A method of treating a subject with an osteocalcin mediated
disorder comprising the step of administering a calcium receptor
modulator identified by the method of claim 12 to said individual
such that the subject is treated.
18. A method of treating a subject with an osteocalcin mediated
disorder comprising the step of administering a calcium receptor
modulator identified by the method of claim 13 to said individual
such that the subject is treated.
19. The agent identified by the method of claim 12.
20. The agent identified by the method of claim 13.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 10/283,842, filed Oct. 29, 2002 (currently
pending), which claims the benefit of priority of U.S. Provisional
Patent Application No. 60/421,941, filed on Oct. 28, 2002 and which
is related to U.S. patent application Ser. No. 10/283,656, filed on
Oct. 29, 2002, the entire contents of each of which are
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] Calcium is one of the most important regulatory molecules in
the body because of its diverse intracellular and extracellular
roles (bone mineralization; cofactor for adhesion molecules,
clotting factors and additional proteins) (Brown, E. M., et al.
(1999) Vitamin. Horm. 55, 1-71). For example, intracellular
calcium, particularly the cytosolic free calcium concentration, is
an important second messenger and cofactor for proteins and enzymes
regulating a diverse set of key cellular processes. Intracellular
calcium is utilized in the regulation of neurotransmission,
motility, homornal secretion and cellular proliferation.
Extracellular calcium serves as a cofactor for adhesion molecules,
clotting factors, and other proteins; regulates neuronal
excitability; and is an essential part of the mineral phase of
bone. The skeleton provides a structural framework that protects
critical bodily structures and facilitates locomotion.
[0003] As a consequence of its critical roles the level of
extracellular calcium (Ca.sup.++.sub.0) needs to be precisely
regulated. The essential mechanism through which the metabolism of
Ca.sup.++.sub.0 is maintained is regulation of dietary intake and
absorption, in combination with Ca.sup.++ handling in the
microenvironments of the renal tubules and the skeleton.
[0004] The relatively recent identification of a calcium sensing
receptor from bovine parathyroid (CaR) provided important
information as to the signaling mechanism through which the body
maintains Ca.sup.++ homeostasis. CaR is 1085 amino acid residues in
length and is a member of the G-protein coupled receptor family.
CaR is made up of three distinct domains; an amino terminal
hydrophilic domain; a central core domain, containing 7
transmembrane helices; and a carboxy terminal hydrophobic domain
(for a review see, Chattopadhyah, N. (2000) Int. J. Biochem. &
Cell Biol. 32, 789-804).
[0005] Whereas CaR function can explain most Ca.sup.++.sub.0
sensing (based on an approximate physiological Ca.sup.++
concentration of 1 mM and a CaR EC.sub.50 of 4.1 mM), this molecule
does not adequately explain Ca.sup.++.sub.0-sensing in the
essential kidney and bone environments where local concentrations
can be as high as 40 mM. In kidney, raising peritubular but not
luminal Ca.sup.++.sub.0 diminishes Ca.sup.++ reabsorption in the
thick ascending limb of Henle's loop. In bone, raising Ca.sup.++
stimulates the function of bone-forming osteoblasts (Quarles, L. D.
(1997) J. Bone Miner. Res. 12, 1971-1974) and inhibits bone
resorption by osteoclasts (Zaidi, M., et al. (1989) Biochem.
Biophys. Res. Commun. 183, 1461-1465.
[0006] G-protein-coupled, seven transmembrane receptors (GPCRs or
7.TM. receptors) comprise the largest superfamily of proteins in
the body (approximately 750 human members based on the analysis of
the rough draft of the human genome). The diversity amongst
endogenous ligands is exceptional, including biogenic amines,
peptides, glycoproteins, lipids, nucleotides, ions and proteases.
As a consequence, GPCRs are potential targets for intervention in
many disease areas and, not surprisingly, they represent the most
important target class of proteins for drug discovery to date, with
over 30% of clinically marketed drugs being active at this receptor
family. As these drugs exhibit their activity upon less than 10% of
all known GPCRs, it can be foreseen that this family has the
potential to yield many more clinically relevant targets.
Identification of the expression pattern and correct activating
ligand are crucial in formulating hypotheses about biological
function and pharmacological relevance of novel GPCRs.
[0007] GPCRs can be structurally classified into three major
subfamilies that include the receptors related to the "light
receptor" rhodopsin and the .beta..sub.2-adrenergic receptor
(family A), the receptors related to the glucagon receptor (family
B) and the receptors related to the metabotropic neurotransmitter
receptors (family C). Family C comprises three subgroups of GPCRs,
with Group I including the metabotropic glutamate receptors 1-8,
Group II including the Calcium-sensing receptor (CaR) and a
multigene family of putative pheromone, taste and odorant
receptors, and Group III including GABA.sub.B receptors. Generally,
these subgroups show .gtoreq.20% homology in their
seven-transmembrane regions and posses extracellular ligand-binding
domains (ECD) that are homologous to the bacterial periplasmic
nutrient-binding proteins (PBPs).
[0008] OC is a vitamin K-dependent bone calcium binding protein
also called bone gla protein (BGP). OC is a unique non collagenous
protein of the extra cellular matrix of bone that is synthesized by
the bone forming cells, the osteoblasts. Human OC is a relatively
small protein composed of 49 amino acids and having a molecular
weight of 5,800 daltons. OC was first discovered in the bones of
chicken and bovine. Over 20 years ago, the human OC was isolated
and its amino acid sequence was determined ((1980) The Journal of
Biological Chemistry, Vol 255, No. 18, pp. 8685-8691). OC inhibits
hydroxyapatite formation in vitro and is modulated by the calcium
regulating hormone 1,25-dihydroxyvitamin D, but until the current
study, its precise physiological functions remained unknown.
[0009] In view of the importance of calcium in both normal and
pathological conditions, there is a ongoing need for the
identification and biological characterization of additional
members of the GPCR family in order to elucidate those members
which are valuable drug targets, as well as prognostic and
diagnostic markers for a variety of pathological processes.
SUMMARY OF THE INVENTION
[0010] The present invention is based, at least in part, on the
identification and characterization of a novel human GPCR that
structurally belongs to family C Group II. Following ectopic
expression in mammalian cells, this GPCR was exposed to a wide
variety of potential ligands and found to be activated by
Ca.sup.++. The receptor was termed Calcium-sensing Receptor 2
(CaR2).
[0011] CaR2 has been found to be expressed in environments where
there are high levels of calcium. Immunohistochemical analysis has
shown expression of CaR2 in bone, kidney, prostate, salivary,
glands, testis, thymus, brain, trachea and thyroid. The present
invention shows that OC synergistically activates CaR2.
[0012] Accordingly, OC is a novel drug target for conditions
associated with CaR2. Therefore, the methods disclosed herein are
useful for treatment of conditions associated with the
above-mentioned tissues, including, but not limited to,
extracellular calcium concentration, metabolic conditions
associated with CaR2 or OC, osteoporosis, sperm motility and
viability, regulation of calcium flux in the kidneys, kidney stone
formation, regulation of calcium flux in the prostate, promotion of
osteoblast proliferation, e.g., for the production of osteoblasts
for medical use, metastasis of cancers, cancers, e.g., breast,
renal, prostate and bone cancers, regulation of bone
mineralization, bone overgrowth modulation of bone healing, e.g,
dental caries, osteoporosis, and other bone formation diseases, and
detection of a subset of cells, e.g., for forensic analysis.
[0013] The invention is also based, at least in part, on the
discovery that CaR2 is modulated by OC(OC). OC is the most abundant
non-collagenous protein of the extracellular matrix and is
synthesized primarily by osteoblasts. Accordingly, CaR2 is a target
for conditions associated with the formation and breakdown of the
extracellular matrix, and conditions associated with aberrant
expression of OC.
[0014] Accordingly, in one aspect, this invention provides isolated
nucleic acid molecules encoding CaR2 polypeptides or biologically
active portions thereof, as well as nucleic acid fragments suitable
as primers or hybridization probes for the detection of
CaR2-encoding nucleic acids.
[0015] In one embodiment, the invention features an isolated
nucleic acid molecule that includes the nucleotide sequence set
forth in SEQ ID NO:1. In another embodiment, the invention features
an isolated nucleic acid molecule that encodes a polypeptide
including the amino acid sequence set forth in SEQ ID NO:2.
[0016] In still other embodiments, the invention features isolated
nucleic acid molecules including nucleotide sequences that are
substantially identical (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% identical) to the nucleotide sequence set forth as SEQ ID
NO:1. The invention further features isolated nucleic acid
molecules including at least 50, 100, 150, 200, 300, 400, 500, 600,
700, 800, 900, 1000, 1500. 2000, 2500, 2600, 2700, 2800, or 2900
contiguous nucleotides of the nucleotide sequence set forth as SEQ
ID NO:1. In another embodiment, the invention features isolated
nucleic acid molecules which encode a polypeptide including an
amino acid sequence that is substantially identical (e.g., 60%
identical) to the amino acid sequence set forth as SEQ ID NO:2. The
present invention also features nucleic acid molecules which encode
allelic variants of the polypeptide having the amino acid sequence
set forth as SEQ ID NO:2. In addition to isolated nucleic acid
molecules encoding full-length polypeptides, the present invention
also features nucleic acid molecules which encode fragments, for
example, biologically active or antigenic fragments, of the
full-length polypeptides of the present invention (e.g., fragments
including at least 10 contiguous amino acid residues of the amino
acid sequence of SEQ ID NO:2). In still other embodiments, the
invention features nucleic acid molecules that are complementary
to, antisense to, or hybridize under stringent conditions to the
isolated nucleic acid molecules described herein.
[0017] In another aspect, the invention provides vectors including
the isolated nucleic acid molecules described herein (e.g.,
CaR2-encoding nucleic acid molecules). Such vectors can optionally
include nucleotide sequences encoding heterologous polypeptides.
Also featured are host cells including such vectors (e.g., host
cells including vectors suitable for producing CaR2 nucleic acid
molecules and polypeptides).
[0018] The invention further provides nucleic acid constructs
comprising the nucleic acid molecules described herein. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence.
[0019] In another aspect, the invention features isolated CaR2
polypeptides and/or biologically active or antigenic fragments
thereof. Exemplary embodiments feature a polypeptide including the
amino acid sequence set forth as of SEQ ID NO:2, a polypeptide
including an amino acid sequence at least 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%
identical to the amino acid sequence set forth as of SEQ ID NO:2, a
polypeptide encoded by a nucleic acid molecule including a
nucleotide sequence at least 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% 97%, 98%, 99%% identical to
the nucleotide sequence set forth as SEQ ID NO:1. Also featured are
fragments of the full-length polypeptides described herein (e.g.,
fragments including at least 10, 25, 50, 100, 150, 200, 250, 300,
350, 400, 450, 500, 600, 700, 800, or 900 contiguous amino acid
residues of the sequence set forth as of SEQ ID NO:2) as well as
allelic variants of the polypeptide having the amino acid sequence
set forth as SEQ ID NO:2.
[0020] The CaR2 polypeptides and/or biologically active or
antigenic fragments thereof, are useful, for example, as reagents
or targets in assays applicable to treatment and/or diagnosis of
calcium associated conditions. In one embodiment, a CaR2
polypeptide or fragment thereof, has a CaR2 activity.
[0021] Accordingly, it is an object of the invention to provide
methods wherein CaR2 polypeptides are useful as reagents or targets
in calcium sensing receptor assays that are applicable to treatment
and diagnosis of conditions related to calcium flux, or related to,
CaR2.
[0022] It is a further object of the invention to provide methods
wherein polynucleotides corresponding to the CaR2 polypeptides are
useful as probes, targets or reagents that are applicable to
treatment and diagnosis of conditions related CaR2.
[0023] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression or
activity of CaR2 in cells or tissues. Such compounds can be used to
prevent, or treat conditions mediated by, or related to, CaR2.
[0024] Accordingly, in one aspect the invention provides methods of
screening for compounds that modulate expression or activity of the
CaR2 polypeptides or nucleic acids (RNA or DNA) in cells or
tissues. In certain embodiments, the cells or tissues are derived
from cells or tissues in which CaR2 expression or activity has been
altered, e.g., from animals or individuals having a disorder
mediated by or related to CaR2.
[0025] A further object of the invention is to provide compounds
that modulate expression or activity of CaR2 for treatment and
diagnosis of conditions mediated by or related to CaR2, such as the
conditions disclosed herein.
[0026] The invention also utilizes vectors and host cells that
express CaR2 and provides methods for expressing CaR2 nucleic acid
molecules and polypeptides in cells, and particularly recombinant
vectors and host cells.
[0027] The invention also utilizes methods of making the vectors
and host cells and provides methods for using them to assay
expression and cellular effects of expression of the CaR2 nucleic
acid molecules and polypeptides in specific cell types and
conditions.
[0028] The invention also utilizes antibodies or antigen-binding
fragments thereof that selectively bind the CaR2 polypeptides and
fragments.
[0029] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression or
activity of CaR2 in cells or tissues. Such compounds can be used to
alter conditions mediated by, or related to, CaR2. Accordingly, in
one aspect the invention provides methods of screening for
compounds that modulate expression or activity of the CaR2
polypeptides or nucleic acids (RNA or DNA) in cells or tissues. In
certain embodiments, the cells or tissues are derived from cells or
tissues in which CaR2 expression or activity has been altered,
e.g., from animals or individuals having a disorder mediated by or
related to CaR2.
[0030] It is further an object of the invention to provide
compounds that modulate the ability of CaR2 to bind OC for the
treatment of conditions mediated by, or related to CaR2, e.g.,
metastasis of cancer.
[0031] A further object of the invention is to provide compounds
that modulate expression or activity of CaR2 for treatment and
diagnosis of conditions mediated by or related to CaR2, such as the
conditions disclosed herein.
[0032] The invention also provides a process for modulating CaR2
polypeptides or nucleic acid expression or activity, especially
using the screened compounds. Modulation can be used to treat
conditions related to aberrant activity or expression of the CaR2
polypeptides or nucleic acids.
[0033] The invention further provides assays for determining the
activity of, or the presence or absence of CaR2 polypeptides or
nucleic acid molecules in biological samples, including for
diagnosing conditions disclosed herein.
[0034] The invention also provides assays for determining the
presence of a mutation in CaR2 polypeptides or nucleic acid
molecules, including for diagnosing conditions disclosed
herein.
[0035] The invention utilizes isolated CaR2 polypeptides, including
a polypeptide having the amino acid sequence shown in SEQ ID NO
2.
[0036] The invention also utilizes isolated CaR2 nucleic acid
molecule having the sequence shown in SEQ ID NO:1 or a complement
thereof.
[0037] The invention also utilizes variant polypeptides having an
amino acid sequence that is substantially homologous to the amino
acid sequence shown in SEQ ID NO:2.
[0038] The invention also utilizes variant nucleic acid sequences
that are substantially homologous to the nucleotide sequence shown
in SEQ ID NO 1.
[0039] The invention also utilizes fragments of the polypeptide
shown in SEQ ID NO:2 and nucleotide sequence shown in SEQ ID NO:1,
complements of the nucleotide sequence shown in SEQ ID NO 1, as
well as substantially homologous fragments of the polypeptide or
nucleic acid.
[0040] The invention further utilizes nucleic acid constructs
comprising the nucleic acid molecules described herein. In certain
embodiments, the nucleic acid molecules of the invention are
operatively linked to a regulatory sequence.
[0041] The invention also utilizes vectors and host cells that
express CaR2 and provides methods for expressing CaR2 nucleic acid
molecules and polypeptides in cells, and particularly recombinant
vectors and host cells.
[0042] The invention also utilizes methods of making the vectors
and host cells and provides methods for using them to assay
expression and cellular effects of expression of the CaR2 nucleic
acid molecules and polypeptides in specific cell types and
conditions.
[0043] The invention also utilizes antibodies or antigen-binding
fragments thereof that selectively bind the CaR2 polypeptides and
fragments.
DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 shows the nucleotide (SEQ ID NO:1) and the deduced
amino acid sequence (SEQ ID NO:2) of CaR2.
[0045] FIG. 2 shows recombinant CaR2 expressed in HEK 293 cells is
activated by high concentrations of Ca.sup.++.
[0046] FIG. 3 shows OC(OC) dependent potentiation of the activation
of CaR2 by Ca.sup.++.
[0047] FIG. 4 shows CaR2 activation by Ca.sup.++. CaR2 is robustly
activated by 40 mM Ca.sup.++, whereas there is modest activation by
10 mM Ca.sup.++. OC(OC) activates CaR2 when pre-incubated with 10
mM Ca.sup.++ in a dose-dependent manner. OC activation of CaR2 is
reversed when OC and Ca.sup.2+ are pre-incubated with Pb.sup.2+,
which is known to prevent the formation of OC/Ca.sup.++
complexes.
[0048] FIGS. 5 A-B show activation of CaR2 by bone morphogenic
peptide (BMP-2) and calcitonin (CT). FIG. 5A shows multiple well
overlay showing activation by BMP-2 and CT of recombinant CaR2.
FIG. 5B shows activation by BMP-2 and CT is delayed, and falls out
of the range of direct GPCR activation as recorded by FLIPR.
[0049] FIG. 6 shows a structural, hydrophobicity, hydrophilicity,
amphipathic region, antigenic index, and surface probability
prediction for SEQ ID NO:2.
DETAILED DESCRIPTION OF THE INVENTION
[0050] Applicants have discovered a novel calcium-sensing receptor,
designated CaR2, that is pharmacologically distinct from previously
identified calcium receptors. CaR2 localizes to bone (osteoblasts),
kidney (apical side of epithelial cells in the thick ascending limb
of Henle's loop), and prostate (epithelial duct lining cells), as
determined immunohistochemically. OC(OC) increases the sensitivity
of CaR2 to Ca.sup.++, while OC in the absence of calcium does not
activate CaR2.
[0051] CaR2 is expressed prominently in kidney and prostate,
tissues that show a high frequency of skeletal metastases (Brage M
E and Simon M A. (1992) Orthopedics 15(5):589-96; Mine A, et al.
(1998) J Surg Oncol (4):255-60). Unique among these metastatic
tumors, skeletal metastasis of primary prostate cancers display
osteoblastic, rather than osteolytic, functions and this
osteoblastic character has been hypothesized to result from tumor
expression of bone regulatory molecules (e.g. BMP6). CaR2 and OC
expressing osteoblastic cells have a survival and proliferative
advantage when challenged with the high concentration of free
Ca.sup.++ that activates CaR2 and that is normally present in
mineralizing bone. It is reasonably predictable that CaR2
expression in cells from other tissues confers on those cells a
preference for and a survival advantage in high calcium
environments. For example, the CaR2 expression that is seen in
kidney and prostate epithelial cells would be expected to allow
cancer cells from these epithelia a similar preference for and
survival advantage in the bone environment, and this could explain
the reported propensity to bone metastases for these primary
cancers. In addition, the OC/Ca.sup.++ interaction with CaR2 could
serve a chemoattractive function, fomenting the attraction of
metastasizing kidney and prostate cancer cells toward the high
OC/Ca.sup.++ signals that originate from bone.
[0052] CaR2 has been found to be expressed in environments where
there are high levels of calcium. Immunohistochemical analysis has
shown expression of CaR2 in bone, kidney, prostate, salivary,
glands, testis, thymus, brain, trachea and thyroid.
[0053] The identification of CaR2 as a high concentration calcium
sensor that is expressed in the epithelial ducts in kidney and
prostate, and in bone tissue, allows for the physiological
consequences expected to result from CaR2 hypo- and hyper-activity
to be monitored. Normocalcemia in mammals and other tetrapods is
believed to be maintained primarily through actions of CaR.
Specific activating and inactivating mutations in CaR lead to
hyper- and hypocalcaemic disease states. In analogy with CaR, such
mutations in CaR2 would be expected to strongly influence its
specific microenvironments, such as kidney and bone. In kidney,
CaR2 should respond to the Ca.sup.++ concentration in fluids
passing through the kidney tubules, resulting in either
reabsorbtion of Ca.sup.++ or its secretion in urine. Dysregulation
leads to excessive calcium loss or the formation of kidney stones.
Similarly, osteoblast CaR2 activation would be expected to affect
bone formation and bone mass. Accordingly, using drugs to modulate
CaR2 activity would be expected to have therapeutic benefit for
conditions such as the conditions disclosed herein.
[0054] CaR2 dysfunction in prostate disrupts fertility by leading
to abnormal Ca.sup.++ levels in seminal fluid. Changes in CaR2
function also have pathological consequences in tissues in which
CaR2 is not expressed. For example, CaR2 dysfunction leads to
global disruption of Ca.sup.++.sub.0 homeostasis and influence the
overall health and physiology of the organism by causing secondary
pathology in distant organs and cells.
[0055] Activation of CaR2 by Ca.sup.++ and the modulatory effect of
OC, in combination with its cellular expression pattern, indicate a
unique role for CaR2 in the pharmacology of Ca.sup.++ homeostasis
in microenvironments where Ca.sup.++ levels are high. This role is
expected to provide novel approaches for therapeutic intervention
in key pathological processes. Moreover, this finding indicates a
role of CaR2 in the metastasis of cancers to bone.
[0056] CaR2 is pharmacologically distinct from the previously
identified Calcium-sensing Receptor (CaR) (for recent reviews see,
Brown, E. M., et al., (1993) Nature 366, 575-580; Chattopadhyay N.
(2000) Int. J. Biochem. & Cell Biol. 32, 789-804; Brown, E. M.
(2000) Annu. Rev. Nutr. 20, 507-533) with an EC.sub.50 value for
Ca.sup.++ (85 mM) that is much greater than that of CaR (4 mM).
Consistent with a functional role in high Ca.sup.++ environments,
CaR2 localizes to bone (osteoblasts), kidney (apical side of
epithelial cells in the thick ascending limb of Henle's loop), and
prostate (epithelial duct lining cells), as determined
immunohistochemically. Prior to the studies presented herein, OC
thought to be only a structural protein. The present invention, for
the first time, identifies the receptor associated with OC. OC
increases the sensitivity of CaR2 to Ca.sup.++, while OC in the
absence of calcium does not activate CaR2. Genetic studies have
indicated that OC functions as an inhibitor of osteoblast function,
but no experimental evidence had implicated OC in signal
transduction prior to the present invention.
[0057] The ability of OC to stimulate CaR2 signal transduction was
unexpected and surprising. OC is a protein that is secreted by
osteoblasts, and that has been identified as a structural component
of the extracellular bone matrix (osteoid) and of mineralized bone.
Secreted OC is thought to play an integral part in organizing the
formation of osteoid and mineralized bone, both as a structural
component of osteoid and as a nucleus for mineralization. In this
role, OC remains in the osteoid matrix during bone mineralization
to become an integral part of the mineralized bone matrix. However,
OC is asymmetrically distributed in bone, with enrichment in highly
mineralized and metabolically less active cortical bone, while
trabecular bone is enriched in osteonectin. This asymmetry in the
distribution of OC and other noncollagenous proteins has led to
speculation that these proteins in bone might have regulatory
functions in addition to their functions in bone structure and
mineralization.
[0058] In addition to the likely influence of CaR2 on bone, kidney
and prostate function, the receptor may also act as a survival
factor in high calcium environments. In this role, CaR2 influences
the cell cycle in response to Ca.sup.++ or to other signals (e.g.,
protein ligands like OC). CaR2 regulation of cell proliferation,
differentiation and cell survival in response to extracellular
Ca.sup.++ and/or protein ligands could help determine the
tumorigenic potential of prostate, kidney or bone cells. Bone
marrow is known to be a major homing site for metastasis of primary
epithelial cancers of breast, colon, kidney and prostate. Clear
cell renal carcinomas and epithelial prostate cancers show a
particularly efficient ability to colonize bone. Numerous
explanations for the bone tropism of these metastasis have been
advanced, including suggestions that tumor cell survival rather
than attraction is promoted by the environment in target tissues,
that tumor cells can be trapped by favorable cell adhesive
interactions within target tissues, and that chemokines could
actively attract cancer cells to bone and other preferred
metastases tissue destinations. CaR2 is expressed prominently in
kidney and prostate, tissues that show a high frequency of skeletal
metastasis. Unique among these metastatic tumors, skeletal
metastasis of primary prostate cancers display osteoblastic, rather
than osteolytic, functions.
[0059] Accordingly, in one aspect, the invention pertains to
compositions that are able to inhibit CaR2 mediated conditions,
e.g., the metastasis of tumors by interacting with CaR2. These
compounds can be, for example, small molecules, peptides, e.g.,
calcium analogs, OC, a fragment of OC, or peptidomimetics.
[0060] In one aspect, the invention provides methods and reagents
for diagnosing conditions associated with calcium receptors such as
the conditions disclosed herein. The diagnostic and prognostic
assays of this invention include methods involving antibody-based
detection of CaR2 polypeptides, and nucleic acid-based detection of
CaR2 mRNA and DNA.
[0061] In one embodiment, this invention provides a method for
identifying conditions associated with CaR2 such as the conditions
disclosed herein, comprising: assessing the level of CaR2 in a
biological sample from the subject, wherein an elevation or
reduction in the level of CaR2 is indicative of a disorder related
to aberrant expression or activity of a calcium receptor.
[0062] In one embodiment, this invention provides a method for
treating subjects with conditions caused by aberrant expression of
OC. Since OC potentiates the response of CaR2 to Ca.sup.++, the
current invention provides methods for treating individuals with
conditions caused by aberrant expression of OC by modulating
CaR2.
[0063] In another embodiment, this invention provides a method for
identifying conditions associated with calcium receptors, such as
the conditions disclosed herein, comprising: assessing the level of
CaR2 in a biological sample from the subject, wherein an elevation
in the level of CaR2 is indicative of a disorder related to calcium
receptors.
[0064] In a further related embodiment, this invention provides a
method for identifying metastatic cancer in a subject, comprising:
assessing the level of CaR2 in a biological sample from the
subject, wherein an elevation in the level of CaR2 is indicative of
cancer, and wherein the subject was previously identified as having
cancer, e.g., prostate or kidney cancer.
[0065] Further, provided by this invention are the above methods,
wherein assessing the level of CaR2 in a biological sample from the
subject includes contacting the biological sample with an antibody
to CaR2 or a fragment thereof; determining the amount of binding of
the antibody to the biological sample; and comparing the amount of
antibody bound to the biological sample to a predetermined base
level. The amount of binding of the antibody to the biological
sample can be determined by the intensity of the signal emitted by
the labeled antibody and/or by the number cells in the biological
sample bound to the labeled antibody.
[0066] Also encompassed by this invention are the above methods
wherein the level of CaR2 is assessed by detecting a level of CaR2
nucleic acid in a biological sample; and comparing the level of
CaR2 in the biological sample with a level of CaR2 in a control
sample. For example, in certain embodiments CaR2 nucleic acid is
detected using hybridization probes and/or nucleic acid
amplification methods.
[0067] The diagnostic and prognostic assays of this invention can
be further used in combination with other methods of diagnosing
conditions disclosed herein. Examples of diagnostic methods that
can be used in combination with the assays of the invention
include, but are not limited to, current diagnostic methods known
to medical practitioners skilled in the art such as ultrasonography
or magnetic resonance imaging (MRI), bone scanning, X-rays,
skeletal survey, intravenous pyelography, CAT-scan, and biopsy.
[0068] The present invention also includes methods of determining
whether a subject is likely to respond to a treatment regimen
comprising agents, or modulators which have a stimulatory or
inhibitory effect on CaR2 activity (e.g., CaR2 gene expression or
enzyme activity). For example, CaR2 inhibitors can be administered
to individuals, such as those identified using the diagnostic and
prognostic methods of the invention as having elevated levels of
CaR2, to treat (prophylactically or therapeutically) conditions
associated with aberrant CaR2 activity or level.
[0069] The invention also provides methods for diagnosing active
conditions, or predisposition to conditions, in a patient having a
variant CaR2. Thus, CaR2 can be isolated from a biological sample
and assayed for the presence of a genetic mutation that results in
an aberrant protein. This includes amino acid substitution,
deletion, insertion, rearrangement, (as the result of aberrant
splicing events), and inappropriate post-translational
modification. Analytic methods include altered electrophoretic
mobility, altered tryptic peptide digest, altered activity in
cell-based or cell-free assay, alteration in ligand binding, or
antibody-binding pattern, altered isoelectric point, direct amino
acid sequencing, and any other of the known assay techniques useful
for detecting mutations in a protein in general or in a calcium
sensing receptor specifically. Mutations resulting in aberrant
levels of CaR2 expression can be further identified using standard
nucleic acid detection techniques such as those described herein.
Mutations resulting in aberrant CaR2 protein activity can be
further identified by assays measuring the response of a cell to
calcium, such as, but not limited to those described herein.
[0070] The invention also encompasses kits for detecting the
presence of a CaR2 polypeptide or nucleic acid in a biological
sample according to the methods described herein. For example, the
kit can comprise a labeled compound or agent capable of detecting
CaR2 polypeptide or an mRNA encoding a CaR2 in a biological sample
and means for determining the amount of the CaR2 polypeptide or
CaR2 mRNA in the sample (e.g., an antibody which binds the
polypeptide or an oligonucleotide probe which binds to DNA or mRNA
encoding CaR2). Kits can also include immunomagnetic beads that can
be used to facilitate serum assays. Kits can further include
instructions for carrying out the methods of the invention and/or
for interpreting the results obtained from using the kit.
[0071] For example, antibody-based kits can comprise: (1) a first
antibody (e.g., attached to a solid support) which binds to a CaR2
polypeptide; and, optionally, (2) a second, different antibody
which binds to either the CaR2 polypeptide or the first antibody
and is conjugated to a detectable label. Nucleic acid-based-kits
can comprise, for example: (1) an oligonucleotide, e.g., a
detectably labeled oligonucleotide, which hybridizes to a nucleic
acid encoding CaR2 or (2) a pair of primers useful for amplifying a
nucleic acid molecule encoding CaR2. Kits can also comprise a
buffering agent, a preservative, or a protein stabilizing agent,
components necessary for detecting the detectable label (e.g., an
enzyme or a substrate), a control sample or a series of control
samples which can be assayed and compared to the biological sample.
Each component of the kit can also be enclosed within an individual
container, and all of the various containers can be within a single
package, along with instructions for interpreting the results of
the assays performed using the kit.
[0072] In another aspect the invention provides methods for
identifying modulators of CaR2 protein activity or CaR2 gene
expression. These modulators can be used in the treatment of CaR2
and OC related conditions such as those described herein.
[0073] Accordingly, in certain embodiments, the invention provides
methods for identifying agents that interact with the CaR2 protein.
This interaction can be detected by functional assays, such as
assays for calcium receptor activity. Determining the ability of
the test compound to interact with CaR2 can also comprise
determining the ability of the test compound to preferentially bind
to the polypeptide as compared to the ability of a known binding
molecule to bind the polypeptide.
[0074] In related embodiments, the invention provides methods to
identify agents that modulate calcium receptor activity. Such
agents, for example, can increase or decrease affinity or rate of
binding to substrate, e.g., OC, calcium, G-protein, for binding to
the CaR2, or displace the substrate bound to the calcium receptor.
For example, both CaR2 and appropriate variants and fragments can
be used in high-throughput screens to assay candidate compounds for
the ability to bind to the receptor. These compounds can be further
screened against a functional CaR2 to determine the effects of the
compound on the receptor activity. Compounds can be identified that
activate (agonist) or inactivate (antagonist) the receptor to a
desired degree. 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). The subject can be
a human subject, for example, a subject in a clinical trial or
undergoing treatment or diagnosis, or a non-human transgenic
subject, such as a transgenic animal or a non-human non-transgenic
animal.
[0075] Accordingly, the invention provides methods to screen a
compound for the ability to stimulate or inhibit interaction
between the receptor protein and a target molecule that normally
interacts with the receptor protein. The assay includes the steps
of combining the receptor protein with a candidate compound under
conditions that allow the receptor or fragment to interact with the
target molecule, and to detect the formation of a complex between
the receptor and the target, or to detect the biochemical
consequence of the interaction with the receptor and the
target.
[0076] In further related embodiments, the invention provides drug
screening assays, in cell-based or cell-free systems. Cell-based
systems can be native, i.e., cells that normally express the CaR2,
as a biopsy, or expanded in cell culture. In one embodiment,
cell-based assays involve recombinant host cells expressing CaR2.
Accordingly, cells that are useful in this regard include, but are
not limited to, cells differentially expressing CaR2 (e.g.,
metastasic cells). These include, but are not limited to, cells or
tissues derived from an individual having an CaR2 disorder (e.g.,
cancerous tissue or tumors, bone). Such cells can naturally express
the gene or can be recombinant. Recombinant cells include cells
containing one or more copies of exogenously-introduced CaR2
sequences, or cells that have been genetically modified to modulate
expression of the endogenous CaR2 sequence.
[0077] In these embodiments, the invention particularly relates to
cells derived from subjects with conditions involving the tissues
in which CaR2 is expressed or derived from tissues subject to
conditions including, but not limited to, those disclosed herein.
These conditions may naturally occur, as in populations of human
subjects, or may occur in model systems such as in vitro systems or
in vivo, such as in non-human transgenic organisms, particularly in
non-human transgenic animals.
[0078] In yet another aspect of the invention, the invention
provides methods to identify proteins that interact with the
calcium receptor in the tissues and conditions disclosed. For
example, the proteins of the invention 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. Blod. Chem. 268:12046-12054; Bartel et al.
Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene
8:1693-1696; and Brent, WO 94/10300), to identify other proteins
(captured proteins) which bind to or interact with the proteins of
the invention and modulate their activity.
I. CaR2 Reagents
[0079] A. CaR2 Polypeptides
[0080] "CaR2 polypeptide" or "CaR2 protein" refers to the
polypeptide in SEQ ID NO:2 (FIG. 1). This protein contains 926
amino acids. Alignment of this sequence with sequences of the other
members of the GPCR subfamily C indicates that CaR2 possesses all
of the characteristic domains of the other GPCR subfamily C
proteins including a large N-terminal ECD and a C-terminal
transmembrane domain.
[0081] Accordingly, the term "CaR2 protein" or "CaR2 polypeptide",
further includes fragments derived from the full-length CaR2s
including various domains, as well as the numerous variants
described herein.
[0082] The present invention thus utilizes an isolated or purified
CaR2 polypeptide and variants and fragments thereof. As used
herein, a polypeptide is said to be "isolated" or "purified" when
it is substantially free of cellular material, when it is isolated
from recombinant and non-recombinant cells, or free of chemical
precursors or other chemicals when it is chemically synthesized. A
polypeptide, however, can be joined to another polypeptide with
which it is not normally associated in a cell and still be
considered "isolated" or "purified."
[0083] The CaR2 polypeptides can be purified to homogeneity. It is
understood, however, that preparations in which the polypeptide is
not purified to homogeneity are useful and considered to contain an
isolated form of the polypeptide. The critical feature is that the
preparation allows for the desired function of the polypeptide,
even in the presence of considerable amounts of other components.
Thus, the invention encompasses various degrees of purity.
[0084] In one embodiment, the language "substantially free of
cellular material" includes preparations of CaR2 having less than
about 30% (by dry weight) other proteins (i.e., contaminating
protein), less than about 20% other proteins, less than about 10%
other proteins, or less than about 5% other proteins. When the
polypeptide is recombinantly produced, it can also be substantially
free of culture medium, i.e., culture medium represents less than
about 20%, less than about 10%, or less than about 5% of the volume
of the protein preparation.
[0085] A CaR2 polypeptide is also considered to be isolated when it
is part of a membrane preparation or is purified and then
reconstituted with membrane vesicles or liposomes.
[0086] The language "substantially free of chemical precursors or
other chemicals" includes preparations of CaR2 polypeptide in which
it is separated from chemical precursors or other chemicals that
are involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the polypeptide having less than about 30%
(by dry weight) chemical precursors or other chemicals, less than
about 20% chemical precursors or other chemicals, less than about
10% chemical precursors or other chemicals, or less than about 5%
chemical precursors or other chemicals.
[0087] In one embodiment, the CaR2 polypeptide comprises the amino
acid sequence shown in SEQ ID NO:2. However, the invention also
encompasses sequence variants. Variants include a substantially
homologous protein encoded by the same genetic locus in an
organism, i.e., an allelic variant.
[0088] Variants also encompass proteins derived from other genetic
loci in an organism, but having substantial homology to the CaR2 of
SEQ ID NO:2. Variants also include proteins substantially
homologous to CaR2 but derived from another organism, i.e., an
ortholog. Variants also include proteins that are substantially
homologous to CaR2 that are produced by chemical synthesis.
Variants also include proteins that are substantially homologous to
CaR2 that are produced by recombinant methods. It is understood,
however, that variants exclude any amino acid sequences disclosed
prior to the invention.
[0089] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences are at
least about 70-75%, typically at least about 80-85%, and most
typically at least about 90-95%, 97%, 98% or 99% or more
homologous. A substantially homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic
acid sequence hybridizing to the nucleic acid sequence, or portion
thereof, of the sequence shown in SEQ ID NO:1 under stringent
conditions as more fully described below.
[0090] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% or more of the
length of the reference sequence. 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 identical at that position (as used herein amino acid or
nucleic acid "identity" is equivalent to amino acid or nucleic acid
"homology"). The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
[0091] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by CaR2.
Similarity is determined by conserved amino acid substitution. Such
substitutions are those that substitute a given amino acid in a
polypeptide by another amino acid of like characteristics.
Conservative substitutions are likely to be phenotypically silent.
Typically seen as conservative substitutions are the replacements,
one for another, among the aliphatic amino acids Ala, Val, Leu, and
Ile; interchange of the hydroxyl residues Ser and Thr, exchange of
the acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr. Guidance
concerning which amino acid changes are likely to be phenotypically
silent are found in Bowie et al., Science 247:1306-1310 (1990).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0092] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, HG., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, van Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[0093] A preferred, non-limiting example of such a mathematical
algorithm is described in Karlin et al. (1993) Proc. Natl. Acad.
Sci. USA 90:5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs (version 2.0) as described in Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., NBLAST) can be used. In one embodiment, parameters
for sequence comparison can be set at score=100, wordlength=12, or
can be varied (e.g., W=5 or W=20).
[0094] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the Needleman et al.
(1970) (I Mol. Biol. 48:444-453) algorithm which has been
incorporated into the GAP program in the GCG software package using
either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6. In yet another preferred embodiment, the percent identity
between two nucleotide sequences is determined using the GAP
program in the GCG software package (Devereux et al. (1984) Nucleic
Acids Res. 12(1):387), using a NWSgapdna.CMP matrix and a gap
weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,
5, or 6.
[0095] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis et
al. (1994) Comput. Appi. Biosci. 10:3-5; and FASTA described in
Pearson et al. (1988) PNAS 85:2444-8.
[0096] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these. Variant
polypeptides can be fully functional or can lack function in one or
more activities. Thus, in the present case, variations can affect
the function, for example, of one or more of the regions
corresponding to the prodomain, the extracellular domain, the
extracellular loops, the transmembrane domain or the C-terminal
domain. Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids, which results in no change or
an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree. The activity of such functional variants can be determined
using assays that are standard in the art, such as those described
herein.
[0097] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0098] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for CaR2 polypeptide. This includes
preventing immunogenicity from pharmaceutical formulations by
preventing protein aggregation.
[0099] Useful variations further include alteration of catalytic
activity. For example, one embodiment involves a variation at the
binding site that results in binding but not calcium receptor
activity. A further useful variation at the same site can result in
altered affinity for substrate. Another useful variation provides a
fusion protein in which one or more domains or subregions are
operationally fused to one or more domains or subregions from
another CaR2 isoform or family.
[0100] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenisis or alanine scanning mutagenesis (Cunningham et al.
(1995) Science 244:1081-85). The later procedure introduces single
alanine mutations at every residue in the molecule. The relusting
mutant is then tested for biological activity. Sites that are
critical for activity for calcium binding or interaction with OC
can be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al.
(1992) J. Mol. Biol. 224:899-904; de Vos et al. (1992) Science
255:306-312).
[0101] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences. The
invention thus also includes polypeptide fragments of CaR2.
Fragments can be derived from the amino acid sequence shown in SEQ
ID NO:2. However, the invention also encompasses fragments of the
variants of CaR2 as described herein.
[0102] Accordingly, a fragment can comprise at least about 10, 15,
20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800,
900 or more contiguous amino acids. Fragments can retain one or
more of the biological activities of the protein.
[0103] The fragments to which the invention pertains, however, are
not to be construed as encompassing fragments that may be disclosed
prior to the present invention.
[0104] Fragments can also be used as an immunogen to generate CaR2
antibodies. Preferred antigenic regions of CaR2 protein are set
forth, for example, in FIG. 6.
[0105] Biologically active fragments (peptides which are, for
example, 5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900 or more amino acids in length) can comprise a domain or
motif, e.g., a catalytic site, calcium binding domain, OC binding
domain, G-protein binding domain, transmembrane domain,
extracellular domain, or the extracellular loops.
[0106] Such domains or motifs can be identified by means of routine
computerized homology searching procedures.
[0107] Accordingly useful fragments of CaR2, for example, can
extend in one or both directions from the functional sites or
regions of the protein described herein to encompass 5, 10, 15, 20,
30, 40, 50, or up to 100 amino acids. Further, fragments can
include sub-fragments of the specific domains mentioned above,
which sub-fragments retain the function of the domain from which
they are derived.
[0108] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of CaR2 and
variants. These epitope-bearing peptides are useful to raise
antibodies that bind specifically to a CaR2 polypeptide or region
or fragment. These peptides can contain at least 10, 12, at least
14, or between at least about 15 to about 30 amino acids, and can
be generated, for example, using the antigenic index profile of
CaR2 (FIG. 6).
[0109] Non-limiting examples of antigenic polypeptides that can be
used to generate antibodies include but are not limited to peptides
derived from an extracellular site. However, intracellularly-made
antibodies ("intrabodies") are also encompassed, which would
recognize intracellular peptide regions.
[0110] The epitope-bearing CaR2 polypeptides can be produced by any
conventional means (Houghten, R. A. (1985) Proc. Natl. Acad. Sci.
USA 82:5131-5135). Simultaneous multiple peptide synthesis is
described in U.S. Pat. No. 4,631,211.
[0111] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the CaR2 fragment and an additional
region fused to the carboxyl terminus of the fragment.
[0112] The invention thus provides chimeric or fusion proteins.
These comprise a CaR2 peptide sequence operatively linked to a
heterologous peptide having an amino acid sequence not
substantially homologous to the CaR2. "Operatively linked"
indicates that the CaR2 peptide and the heterologous peptide are
fused in-frame. The heterologous peptide can be fused to the
N-terminus or C-terminus of CaR2 or can be internally located.
[0113] In one embodiment the fusion protein does not affect CaR2
function per se. For example, the fusion protein can be a
GST-fusion protein in which the CaR2 sequences are fused to the N-
or C-terminus of the GST sequences. Other types of fusion proteins
include, but are not limited to, enzymatic fusion proteins, for
example beta-galactosidase fusions, yeast two-hybrid GAL-4 fusions,
poly-His fusions and Ig fusions. Such fusion proteins, particularly
poly-His fusions, can facilitate the purification of recombinant
CaR2. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a protein can be increased by using
a heterologous signal sequence. Therefore, in another embodiment,
the fusion protein contains a heterologous signal sequence at its
N-terminus.
[0114] EP-A-0 464533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(Bennett et al. (1995) J: Mol. Recog. 8:52-58 (1995) and Johanson
et al. J: Bio/. Chem. 270:9459-9471). Thus, this invention also
utilizes soluble fusion proteins containing a CaR2 polypeptide and
various portions of the constant regions of heavy or light chains
of immunoglobulins of various subclass (IgG, IgM, 1gA, 1gB).
Preferred as immunoglobulin is the constant part of the heavy chain
of human IgG, particularly IgGl, where fusion takes place at the
hinge region. For some uses it is desirable to remove the Fc after
the fusion protein has been used for its intended purpose, for
example when the fusion protein is to be used as antigen for
immunizations. In a particular embodiment, the Fc part can be
removed in a simple way by a cleavage sequence, which is also
incorporated and can be cleaved with factor Xa.
[0115] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. 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 which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A CaR2-encoding nucleic acid can be
cloned into such an expression vector such that the fusion moiety
is linked in-frame to CaR2.
[0116] Another form of fusion protein is one that directly affects
CaR2 functions. Accordingly, a CaR2 polypeptide is encompassed by
the present invention in which one or more of the CaR2 domains (or
parts thereof) has been replaced by homologous domains (or parts
thereof) from another calcium receptor family member. Accordingly,
various permutations are possible. For example, the aminoterminal
domain, or subregion thereof, can be replaced with the domain or
subregion from another isoform or calcium receptor family. As a
further example, the catalytic domain or parts thereof, can be
replaced; the carboxyterminal domain or subregion can be replaced.
Thus, chimeric CaR2s can be formed in which one or more of the
native domains or subregions has been replaced by another.
[0117] Additionally, chimeric CaR2 proteins can be produced in
which one or more functional sites is derived from a different
isoform, or from another calcium receptor family. It is understood,
however, that sites could be derived from calcium receptor families
that occur in the mammalian genome but which have not yet been
discovered or characterized.
[0118] The isolated CaR2 can be purified from cells that naturally
express it, purified from cells that naturally express it but have
been modified to overproduce CaR2, e.g., purified from cells that
have been altered to express it (recombinant), synthesized using
known protein synthesis methods, or by modifying cells that
naturally encode CaR2 to express it.
[0119] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
CaR2 polypeptide is cloned into an expression vector, the
expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques.
[0120] In other embodiments, the recombinant cell has been
manipulated to activate expression of the endogenous CaR2 gene. For
example, WO 99/15650, WO 00/49162, U.S. Pat. No. 6,361,972 and U.S.
Pat. No. 6,410,266 describe a method of expressing endogenous genes
termed, random activation of gene expression (RAGE), that can be
used to activate or increase expression of endogenous CaR2. The
RAGE methodology involves non-homologous recombination of a
regulatory sequence to activate expression of a downstream
endogenous gene. Alternatively, WO 94//12650, WO 95/31560, WO
96/29411, U.S. Pat. No. 5,733,761 and U.S. Pat. No. 6,270,985
describe a method of increasing expression of an endogenous gene
that involves homologous recombination of a DNA construct that
includes a targeting sequence, a regulatory sequence, an exon, and
a splice-donor site. Upon homologous recombination a downstream
endogenous gene is expressed. The methods of expressing endogenous
genes described in the forgoing patents are hereby expressly
incorporated by reference.
[0121] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally-occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in polypeptides are described in
basic texts, detailed monographs, and the research literature, and
they are well known to those of skill in the art.
[0122] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[0123] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0124] Such modifications are well-known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd ed., T. E.
Creighton, W.H. Freeman and Company, New York (1993). Many detailed
reviews are available on this subject, such as by Wold, F.,
Posttranslational Covalent Modification of proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990)
Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. NY:
Acad. Sci. 663:48-62).
[0125] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[0126] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
aminoterminal residue of polypeptides made in E. coli, prior to
proteolytic processing, almost invariably will be
N-formylmethionine.
[0127] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells, and, for this reason, insect
cell expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications. The same type of
modification may be present in the same or varying degree at
several sites in a given polypeptide. Also, a given polypeptide may
contain more than one type of modification.
[0128] B. CaR2 Antibodies
[0129] The methods for using antibodies described herein are based
on the generation of antibodies that specifically bind to CaR2 or
its variants or fragments.
[0130] To generate antibodies, an isolated CaR2 polypeptide is used
as an immunogen to generate antibodies using standard techniques
for polyclonal and monoclonal antibody preparation. Either the
full-length protein, or one or more antigenic peptide fragments can
be used.
[0131] Antibodies are preferably prepared from various regions of
CaR2 described herein, or from discrete fragments in these regions.
However, antibodies can be prepared from any region of the peptide
as described herein. A preferred fragment produces an antibody that
diminishes or completely prevents binding of, for example,
Ca.sup.++ or OC. Antibodies can be developed against the entire
CaR2 or domains of CaR2 as described herein. Antibodies can also be
developed against specific functional sites as disclosed
herein.
Antibody Uses
[0132] The antibodies can be used to isolate a CaR2 by standard
techniques, such as affinity chromatography or immunoprecipitation.
The antibodies can facilitate the purification of the natural CaR2
from cells and recombinantly produced CaR2 expressed in host
cells.
[0133] The antibodies are useful to detect the presence of CaR2 in
cells or tissues to determine the pattern of expression of the CaR2
among various tissues in an organism and over the course of normal
development.
[0134] The antibodies can be used to detect CaR2 in situ, in vitro,
or in a cell lysate or supernatant in order to evaluate the
abundance and pattern of expression.
[0135] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[0136] The antigenic peptide can comprise a contiguous sequence of
at least 8, 9, 10, 12, 14, 15, or 30 amino acid residues. In one
embodiment, fragments correspond to regions that are located on the
surface of the protein, e.g., hydrophilic regions (see FIG. 6).
These fragments are not to be construed, however, as encompassing
any fragments, which may be disclosed prior to the invention.
[0137] "Antibody" includes immunoglobulin molecules and
immunologically active determinants of immunoglobulin molecules,
i.e., molecules that contain an antigen binding site which
specifically binds (immunoreacts with) an antigen. Structurally,
the simplest naturally occurring antibody (e.g., IgG) comprises
four polypeptide chains, two copies of a heavy (H) chain and two of
a light (L) chain, all covalently linked by disulfide bonds.
Specificity of binding in the large and diverse set of antibodies
is found in the variable (V) determinant of the H and L chains;
regions of the molecules that are primarily structural are constant
(C) in this set. Antibody includes polyclonal antibodies,
monoclonal antibodies, whole immunoglobulins, and antigen binding
fragments of the immunoglobulins.
[0138] The binding sites of the proteins that comprise an antibody,
i.e., the antigen-binding functions of the antibody, are localized
by analysis of fragments of a naturally-occurring antibody. Thus,
antigen-binding fragments are also intended to be designated by the
term "antibody." Examples of binding fragments encompassed within
the term antibody include: a Fab fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; an F.sub.d fragment
consisting of the V.sub.H and C.sub.H1 domains; an F.sub.v fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546)
consisting of a V.sub.H domain; an isolated complementarity
determining region (CDR); and an F(ab').sub.2 fragment, a bivalent
fragment comprising two Fab' fragments linked by a disulfide bridge
at the hinge region. These antibody fragments are obtained using
conventional techniques well-known to those with skill in the art,
and the fragments are screened for utility in the same manner as
are intact antibodies. The term "antibody" is further intended to
include bispecific and chimeric molecules having at least one
antigen binding determinant derived from an antibody molecule.
[0139] In the diagnostic and prognostic assays of the invention,
the antibody can be a polyclonal antibody or a monoclonal antibody
and in a preferred embodiment is a labeled antibody.
[0140] Polyclonal antibodies are produced by immunizing animals,
usually a mammal, by multiple subcutaneous or intraperitoneal
injections of an immunogen (antigen) and an adjuvant as
appropriate. As an illustrative embodiment, animals are typically
immunized against a protein, peptide or derivative by combining
about 1 .mu.g to 1 mg of protein capable of eliciting an immune
response, along with an enhancing carrier preparation, such as
Freund's complete adjuvant, or an aggregating agent such as alum,
and injecting the composition intradermally at multiple sites.
Animals are later boosted with at least one subsequent
administration of a lower amount, as 1/5 to 1/10 the original
amount of immunogen in Freund's complete adjuvant (or other
suitable adjuvant) by subcutaneous injection at multiple sites.
Animals are subsequently bled, serum assayed to determine the
specific antibody titer, and the animals are again boosted and
assayed until the titer of antibody no longer increases (i.e.,
plateaus).
[0141] Such populations of antibody molecules are referred to as
"polyclonal" because the population comprises a large set of
antibodies each of which is specific for one of the many differing
epitopes found in the immunogen, and each of which is characterized
by a specific affinity for that epitope. An epitope is the smallest
determinant of antigenicity, which for a protein, comprises a
peptide of six to eight residues in length (Berzofsky, J. and I.
Berkower, (1993) in Paul, W., Ed., Fundamental Immunology, Raven
Press, N.Y., p. 246). Affinities range from low, e.g. 10.sup.-6 M,
to high, e.g., 10.sup.-11 M. The polyclonal antibody fraction
collected from mammalian serum is isolated by well known
techniques, e.g. by chromatography with an affinity matrix that
selectively binds immunoglobulin molecules such as protein A, to
obtain the IgG fraction. To enhance the purity and specificity of
the antibody, the specific antibodies may be further purified by
immunoaffinity chromatography using solid phase-affixed immunogen.
The antibody is contacted with the solid phase-affixed immunogen
for a period of time sufficient for the immunogen to immunoreact
with the antibody molecules to form a solid phase-affixed
immunocomplex. Bound antibodies are eluted from the solid phase by
standard techniques, such as by use of buffers of decreasing pH or
increasing ionic strength, the eluted fractions are assayed, and
those containing the specific antibodies are combined.
[0142] "Monoclonal antibody" or "monoclonal antibody composition"
as used herein refers to a preparation of antibody molecules of
single molecular composition. A monoclonal antibody composition
displays a single binding specificity and affinity for a particular
epitope. Monoclonal antibodies can be prepared using a technique
which provides for the production of antibody molecules by
continuous growth of cells in culture. These include but are not
limited to the hybridoma technique originally described by Kohler
and Milstein (1975, Nature 256:495-497; see also Brown et al. 1981
J. Immunol. 127:539-46; Brown et al., 1980, J Biol Chem
255:4980-83; Yeh et al., 1976, PNAS 76:2927-31; and Yeh et al.,
1982, Int. J. Cancer 29:269-75) and the more recent human B cell
hybridoma technique (Kozbor et al., 1983, Immunol Today 4:72),
EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), and trioma
techniques. The technology for producing hybridomas is well known
(see generally Current Protocols in Immunology, Coligan et al. ed.,
John Wiley & Sons, New York, 1994). Hybridoma cells producing a
monoclonal antibody of the invention are detected by screening the
hybridoma culture supernatants for antibodies that bind the
polypeptide of interest, e.g., using a standard ELISA assay.
[0143] A monoclonal antibody can be produced by the following
steps. In all procedures, an animal is immunized with an antigen
such as a protein (or peptide thereof) as described above for
preparation of a polyclonal antibody. The immunization is typically
accomplished by administering the immunogen to an immunologically
competent mammal in an immunologically effective amount, i.e., an
amount sufficient to produce an immune response. Preferably, the
mammal is a rodent such as a rabbit, rat or mouse. The mammal is
then maintained on a booster schedule for a time period sufficient
for the mammal to generate high affinity antibody molecules as
described. A suspension of antibody-producing cells is removed from
each immunized mammal secreting the desired antibody. After a
sufficient time to generate high affinity antibodies, the animal
(e.g., mouse) is sacrificed and antibody-producing lymphocytes are
obtained from one or more of the lymph nodes, spleens and
peripheral blood. Spleen cells are preferred, and can be
mechanically separated into individual cells in a physiological
medium using methods well known to one of skill in the art. The
antibody-producing cells are immortalized by fusion to cells of a
mouse myeloma line. Mouse lymphocytes give a high percentage of
stable fusions with mouse homologous myelomas, however rat, rabbit
and frog somatic cells can also be used. Spleen cells of the
desired antibody-producing animals are immortalized by fusing with
myeloma cells, generally in the presence of a fusing agent such as
polyethylene glycol. Any of a number of myeloma cell lines suitable
as a fusion partner are used with to standard techniques, for
example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma
lines, available from the American Type Culture Collection (ATCC),
Rockville, Md.
[0144] The fusion-product cells, which include the desired
hybridomas, are cultured in selective medium such as HAT medium,
designed to eliminate unfused parental myeloma or lymphocyte or
spleen cells. Hybridoma cells are selected and are grown under
limiting dilution conditions to obtain isolated clones. The
supernatants of each clonal hybridoma is screened for production of
antibody of desired specificity and affinity, e.g., by immunoassay
techniques to determine the desired antigen such as that used for
immunization. Monoclonal antibody is isolated from cultures of
producing cells by conventional methods, such as ammonium sulfate
precipitation, ion exchange chromatography, and affinity
chromatography (Zola et al., Monoclonal Hybridoma Antibodies:
Techniques And Applications, Hurell (ed.), pp. 51-52, CRC Press,
1982). Hybridomas produced according to these methods can be
propagated in culture in vitro or in vivo (in ascites fluid) using
techniques well known to those with skill in the art.
[0145] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody directed against a polypeptide of
the invention can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (e.g., an antibody
phage display library) with the polypeptide of interest. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display
Kit, Catalog No. 240612). Additionally, examples of methods and
reagents particularly amenable for use in generating and screening
an antibody display library can be found in, for example, U.S. Pat.
No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.
WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No.
WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No.
WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No.
WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et
al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; Griffiths et al. (1993) EMBO J.
12:725-734.
[0146] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are within the scope of the invention. Such
chimeric and humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art, for example using
methods described in PCT Publication No. WO 87/02671; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science
229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0147] Fully human antibodies can also be generated using
transgenic mice in which the endogenous immunoglobulin genes have
been inactivated and replaced with genes encoding the human light
and heavy chain immunoglobulins. Such mice, and methods for using
these mice to generate human polyclonal and monoclonal antibodies
to an antigen are described for example in U.S. Pat. Nos.
6,075,181, 6,091,001 and 6,300,129, and in Tomizuka et al. (2000)
Proc. Natl. Acad. Sci. 97:722-727.
[0148] Human antibodies are particularly desirable for therapeutic
treatment of human patients. For an overview of this technology for
producing human antibodies, see Lonberg et al. (1995) Int. Rev.
Immunol. 13:65-93. For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, e.g., U.S. Pat. No.
5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S.
Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806.
[0149] The invention also encompasses kits for using antibodies to
detect the presence of a CaR2 protein in a biological sample. The
kit can comprise antibodies such as a labeled or labelable antibody
and a compound or agent for detecting phosphodiesterase in a
biological sample; means for determining the amount of CaR2 in the
sample; and means for comparing the amount of CaR2 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 CaR2
[0150] "Labeled antibody" as used herein includes antibodies that
are labeled by a detectable means and includes enzymatically,
radioactively, fluorescently, chemiluminescently, and/or
bioluminescently labeled antibodies.
[0151] One of the ways in which an antibody can be detectably
labeled is by linking the same to an enzyme. This enzyme, in turn,
when later exposed to its substrate, will react with the substrate
in such a manner as to produce a chemical moiety which can be
detected, for example, by spectrophotometric, fluorometric or by
visual means. Enzymes which can be used to detectably label the
CaR2 specific antibody include, but are not limited to, malate
dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase,
triose phosphate isomerase, horseradish peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,
glucoamylase and acetylcholinesterase.
[0152] Detection may be accomplished using any of a variety of
immunoassays. For example, by radioactively labeling an antibody,
it is possible to detect the antibody through the use of
radioimmune assays. A description of a radioimmune assay (RIA) may
be found in Laboratory Techniques and Biochemistry in Molecular
Biology, by Work, T. S., et al., North Holland Publishing Company,
NY (1978), with particular reference to the chapter entitled "An
Introduction to Radioimmune Assay and Related Techniques" by Chard,
T.
[0153] The radioactive isotope can be detected by such means as the
use of a gamma counter or a scintillation counter or by
audioradiography. Isotopes which are particularly useful for the
purpose of the present invention are: .sup.3H, .sup.131I, .sup.35S,
.sup.14C, and preferably .sup.125I.
[0154] It is also possible to label an antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0155] An antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentaacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0156] An antibody also can be detectably labeled by coupling it to
a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, luciferin, isoluminol, theromatic
acridinium ester, imidazole, acridinium salt and oxalate ester.
[0157] Likewise, a bioluminescent compound may be used to label an
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0158] In the diagnostic and prognostic assays of the invention,
the amount of binding of the antibody to the biological sample can
be determined by the intensity of the signal emitted by the labeled
antibody and/or by the number cells in the biological sample bound
to the labeled antibody.
[0159] Antibodies directed toward a protein of interest can also be
connected to magnetic beads and used to enrich a cell population.
Immunomagnetic selection has been used previously for this purpose
and examples of this method can be found, for example, at U.S. Pat.
No. 5,646,001; Ree et al. (2002) Int. J. Cancer 97:28-33; Molnar et
al. (2001) Clin. Cancer Research 7:4080-4085; and Kasimir-Bauer et
al. (2001) Breast Cancer Res. Treat. 69:123-32. An antibody, either
polyclonal or monoclonal, that is specific for a cell surface
protein on a cell of interest is attached to a magnetic substrate
thereby allowing selection of only those cells that express the
surface protein of interest. Once a population of cells is
selected, the following assays, can be performed to test for the
presence of CaR2.
[0160] Antibodies of the present invention can be used as for
therapeutic administration. Antibodies can be administered to a
subject alone or in combination with one or more other substances,
i.e., in a pharmaceutical composition.
[0161] C. CaR2 Nucleic Acids
[0162] The invention further provides methods and uses for the
nucleotide sequence in SEQ ID NO:1. The specifically disclosed cDNA
comprises the coding region and 5' and 3' untranslated sequences in
SEQ ID NO:1. The expression vector comprising the nucleotide
sequence of SEQ ID NO:1 has been deposited with the ATCC and given
deposit number: ATCC ______.
[0163] The deposits will be maintained under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms. The deposits are provided as a convenience to those
of skill in the art and is not an admission that a deposit is
required under 35 U.S.C. .sctn. 112. The deposited sequence, as
well as the polypeptides encoded by the sequence, are incorporated
herein by reference and controls in the event of conflict, such as
a sequencing error, with description in this invention.
[0164] The invention provides isolated polynucleotides encoding
CaR2. The term "CaR2 polynucleotide" or "CaR2 nucleic acid" refers
to the sequences shown in SEQ ID NO:1. The term "CaR2
polynucleotide" or "CaR2 nucleic acid" further includes variants
and fragments of the CaR2 polynucleotides.
[0165] An "isolated" CaR2 nucleic acid is one that is separated
from other nucleic acid present in the natural source of the CaR2
nucleic acid. Preferably, an "isolated" nucleic acid is free of
sequences which naturally flank the CaR2 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.
However, there can be some flanking nucleotide sequences, for
example up to about 5 KB. The important point is that the CaR2
nucleic acid is isolated from flanking sequences such that it can
be subjected to the specific manipulations described herein, such
as recombinant expression, preparation of probes and primers, and
other uses specific to the CaR2 nucleic acid sequences.
[0166] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0167] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0168] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0169] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0170] The CaR2 polynucleotides can encode the mature protein plus
additional amino or carboxyterminal amino acids, or amino acids
interior to the mature polypeptide (when the mature form has more
than one polypeptide chain, for instance). Such sequences may play
a role in processing of a protein from precursor to a mature form,
facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0171] The CaR2 polynucleotides include, but are not limited to,
the sequence encoding the mature polypeptide alone, the sequence
encoding the mature polypeptide and additional coding sequences,
such as a leader or secretory sequence (e.g., a pre-pro or
pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[0172] CaR2 polynucleotides can be in the form of RNA, such as
mRNA, or in the form DNA, including cDNA and genomic DNA obtained
by cloning or produced by chemical synthetic techniques or by a
combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0173] In one embodiment, the CaR2 nucleic acid comprises only the
coding region.
[0174] The invention further provides variant CaR2 polynucleotides,
and fragments thereof, that differ from the nucleotide sequence
shown in SEQ ID NO:1 due to degeneracy of the genetic code and thus
encode the same protein as that encoded by the nucleotide sequence
shown in SEQ ID NO:1.
[0175] The invention also provides CaR2 nucleic acid molecules
encoding the variant polypeptides described herein. Such
polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[0176] Typically, variants have a substantial identity with a
nucleic acid molecule of SEQ ID NO:1, and the complements thereof.
Variation can occur in either or both the coding and non-coding
regions. The variations can produce both conservative and
non-conservative amino acid substitutions.
[0177] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding a CaR2 that is at least about 60-65%,
65-70%, typically at least about 70-75%, more typically at least
about 80-85%, and most typically at least about 90-95%, 96%, 97%,
98%, 99% or more homologous to the nucleotide sequence shown in SEQ
ID NO:1 or a fragment of this sequence. Such nucleic acid molecules
can readily be identified as being able to hybridize, under
stringent conditions, to the nucleotide sequence shown in SEQ ID
NO:1 or a fragment of the sequence. It is understood that stringent
hybridization does not indicate substantial homology where it is
due to general homology, such as poly A sequences, or sequences
common to all or most proteins, or all cyclic nucleotide CaR2.
[0178] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a polypeptide
at least about 60-65% homologous to each other typically remain
hybridized to each other. The conditions can be such that sequences
at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 90%, at least about 95%, 96%, 97%,
98%, 99% or more identical to each other remain hybridized to one
another. Such stringent conditions 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, incorporated by
reference. One example of stringent hybridization conditions are
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50.degree., 55.degree., 60.degree.,
62.degree. or 65.degree. C. In another non-limiting example,
nucleic acid molecules are allowed to hybridize in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more low stringency washes in 0.2.times.SSC/0.1% SDS at room
temperature, or by one or more moderate stringency washes in
0.2.times.SSC/0.1% SDS at 42.degree. C., or washed in
0.2.times.SSC/0.1% SDS at 65.degree. C. for high stringency. In one
embodiment, an isolated nucleic acid molecule that hybridizes under
stringent conditions to the sequence of SEQ ID NO:1.
[0179] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural
protein).
[0180] As understood by those of ordinary skill, the exact
conditions can be determined empirically and depend on ionic
strength, temperature and the concentration of destabilizing agents
such as formamide or denaturing agents such as SDS. Other factors
considered in determining the desired hybridization conditions
include the length of the nucleic acid sequences, base composition,
percent mismatch between the hybridizing sequences and the
frequency of occurrence of subsets of the sequences within other
non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while
maintaining a similar degree of identity or similarity between the
two nucleic acid molecules.
[0181] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to the nucleotide sequence of
SEQ ID NO:1 or the complement of SEQ ID NO:1. In one embodiment,
the nucleic acid consists of a portion of the nucleotide sequence
of SEQ ID NO 2 and the complement of SEQ ID NO:1. The nucleic acid
fragments of the invention are at least about 15, preferably at
least about 18, 20, 23 or 25 nucleotides, and can be 50, 100, 150,
200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500,
2600, 2700, 2800, or 2900 or more nucleotides in length. Longer
fragments, for example, 1000 or more nucleotides in length, which
encode antigenic proteins or polypeptides described herein are
useful.
[0182] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length CaR2 polynucleotide. The
fragment can be single or double-stranded and can comprise DNA or
RNA. The fragment can be derived from either the coding or the
non-coding sequence.
[0183] In another embodiment an isolated CaR2 nucleic acid encodes
the entire coding region. In another embodiment the isolated CaR2
nucleic acid encodes a sequence corresponding to the mature
protein. Other fragments include nucleotide sequences encoding the
amino acid fragments described herein.
[0184] Thus, CaR2 nucleic acid fragments further include sequences
corresponding to the domains described herein, subregions also
described, and specific functional sites. CaR2 nucleic acid
fragments also include combinations of the domains, segments, and
other functional sites described above. A person of ordinary skill
in the art would be aware of the many permutations that are
possible.
[0185] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary skill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[0186] However, it is understood that a CaR2 fragment includes any
nucleic acid sequence that does not include the entire gene.
[0187] The invention also provides CaR2 nucleic acid fragments that
encode epitope bearing regions of the CaR2 proteins described
herein.
[0188] The nucleic acid fragments useful to practice the invention
provide probes or primers in assays, such as those described
herein. "Probes" are oligonucleotides that hybridize in a
base-specific manner to a complementary strand of nucleic acid.
Such probes include polypeptide nucleic acids, as described in
Nielsen et al. (1991) Science 254: 1497-1500. Typically, a probe
comprises a region of nucleotide sequence that hybridizes under
highly stringent conditions to at least about 15, typically about
20-25, and more typically about 40, 50, 75 or 100 or more
consecutive nucleotides of the nucleic acid sequence shown in SEQ
ID NO:1 and the complements thereof. More typically, the probe
further comprises a label, e.g., radioisotope, fluorescent
compound, enzyme, or enzyme co-factor.
[0189] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[0190] The CaR2 polynucleotides are thus useful for probes, primers
and biological assays.
[0191] Where the polynucleotides are used to assess CaR2 properties
or functions, such as in the assays described herein, all or less
than all of the entire cDNA can be useful. Assays specifically
directed to CaR2 functions, such as assessing agonist or antagonist
activity, encompass the use of known fragments. Further, diagnostic
methods for assessing CaR2 function can also be practiced with any
fragment, including those fragments that may have been known prior
to the invention. Similarly, in methods involving treatment of CaR2
dysfunction, all fragments are encompassed including those, which
may have been known in the art.
[0192] The invention utilizes the CaR2 polynucleotides as a
hybridization probe for cDNA and genomic DNA to isolate a
full-length cDNA and genomic clones encoding variant polypeptides
and to isolate cDNA and genomic clones that correspond to variants
producing the same polypeptides shown in SEQ ID NO:2 or the other
variants described herein. This method is useful for isolating
variant genes and cDNA that are expressed in the cells, tissues,
and conditions disclosed herein.
[0193] The probe can correspond to any sequence along the entire
length of the gene encoding CaR2. Accordingly, it could be derived
from 5' noncoding regions, the coding region, and 3' noncoding
regions.
[0194] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NO:1, or a fragment thereof, such as an
oligonucleotide of at least 12, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to mRNA or DNA.
[0195] Fragments of the polynucleotides can also be used to
synthesize larger fragments or full-length polynucleotides
described herein. For example, a fragment can be hybridized to any
portion of an mRNA and a larger or full-length cDNA can be
produced.
[0196] Fragments can also be used to synthesize antisense molecules
of desired length and sequence.
[0197] Antisense nucleic acids, useful in treatment and diagnosis,
can be designed using the nucleotide sequences of SEQ ID NO:1, and
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense 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 which can
be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl)uracil, 5-carboxymethy
laminomethyl-2-thiouridine, 5-carboxymethy laminomethyl uracil,
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-methyl aminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-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-N2-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).
[0198] Additionally, the nucleic acid molecules useful to practice
the invention 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 deoxyribose
phosphate backbone of the nucleic acids can be modified to generate
peptide nucleic acids (see Hyrup et al. (1996) Bioorganic &
Medicinal Chemistry 4:5). As used herein, the terms "peptide
nucleic acids" or "PNAS" refer to nucleic acid mimics, e.g., DNA
mimics, in which the deoxyribose phosphate 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), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93: 14670. PNAs can be further modified, e.g., to
enhance their stability, specificity 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. The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5: 1119.
[0199] The nucleic acid molecules and fragments useful to practice
the invention can also include other appended groups such as
peptides (e.g., for targeting host cell CaR2 in vivo), or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;
Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No. WO 88/0918) or the blood brain barrier (see, e.g.,
PCT Publication No. WO 89/10134). In addition, oligonucleotides can
be modified with hybridization-triggered cleavage agents (see,
e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating
agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).
[0200] D. Vectors and Host Cells
[0201] The invention also provides methods using vectors containing
the CaR2 polynucleotides. The term "vector" refers to a vehicle,
preferably a nucleic acid molecule that can transport the CaR2
polynucleotides. When the vector is a nucleic acid molecule, the
CaR2 polynucleotides are covalently linked to the vector nucleic
acid. With this aspect of the invention, the vector includes a
plasmid, single or double stranded phage, a single or double
stranded RNA or DNA viral vector, or artificial chromosome, such as
a BAC, PAC, YAC, OR MAC.
[0202] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the CaR2 polynucleotides.
[0203] Alternatively, the vector may integrate into the host cell
genome to produce additional copies of the CaR2 polynucleotides
when the host cell replicates, or to increase or activate
expression of the endogenous CaR2 coding sequences.
[0204] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the CaR2
polynucleotides. The vectors can function in procaryotic or
eukaryotic cells or in both (shuttle vectors).
[0205] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the CaR2 polynucleotides
such that transcription of the polynucleotides is allowed in a host
cell. The polynucleotides can be introduced into the host cell with
a separate polynucleotide capable of affecting transcription. Thus,
the second polynucleotide may provide a trans-acting factor
interacting with the cis-regulatory control region to allow
transcription of the CaR2 polynucleotides from the vector.
Alternatively, a trans-acting factor may be supplied by the host
cell. Finally, a trans-acting factor can be produced from the
vector itself.
[0206] It is understood, however, that in some embodiments,
transcription and/or translation of the CaR2 polynucleotides can
occur in a cell-free system.
[0207] The regulatory sequence to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lamda., the lac, TRP, and
TAC promoters from E. coli, the early and late promoters from SV40,
the CMV immediate early promoter, the adenovirus early and late
promoters, and retrovirus long-terminal repeats.
[0208] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV 40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0209] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[0210] A variety of expression vectors can be used to express a
CaR2 polynucleotide. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV 40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[0211] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e., tissue specific) or may provide
for inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0212] The CaR2 polynucleotides can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0213] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as HTI
080, HEK293, COS and CHO cells, and plant cells.
[0214] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the CaR2
polypeptides. Fusion vectors can increase the expression of a
recombinant protein, increase the solubility of the recombinant
protein, and aid in the purification of the protein by acting for
example as a ligand for affinity purification. A proteolytic
cleavage site may be introduced at the junction of the fusion
moiety so that the desired polypeptide can ultimately be separated
from the fusion moiety. Proteolytic enzymes include, but are not
limited to, factor Xa, TEY, thrombin, and enterokinase. Typical
fusion expression vectors include pGEX (Smith et al. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which 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 (Amann et al.
(1988) Gene 69:301-315) and pET lid (Studier et al. (1990) Gene
Expression Technology: Methods in Enzymology 185:60-89).
[0215] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S. (1990) Gene Expression Technology. Methods
in Enzymology 185, Academic Press, San Diego, Calif. 119-128).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118).
[0216] The CaR2 polynucleotides can also be expressed by expression
vectors that are operative in yeast. Examples of vectors for
expression in yeast e.g., S. cerevisiae include pYepSecl (Baldari
et al. (1987) EMBO J: 6:229-234), pMFa (Kurjan et al. (1982) Cell
30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and
pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0217] The CaR2 polynucleotides can also be expressed in insect
cells using, for example, 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 et al. (1989) Virology 170:31-39).
[0218] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman
et al. (1987) EMBO J. 6:187-195).
[0219] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the CaR2
polynucleotides. The person of ordinary skill in the art would be
aware of other vectors suitable for maintenance propagation or
expression of the polynucleotides described herein. These are found
for example in Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual 2nd; ed, Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0220] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0221] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0222] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.). Host cells can contain more than one vector. Thus,
different nucleotide sequences can be introduced on different
vectors of the same cell. Similarly, the CaR2 polynucleotides can
be introduced either alone or with other polynucleotides that are
not related to the CaR2 polynucleotides such as those providing
trans-acting factors for expression vectors. When more than one
vector is introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the CaR2 polynucleotide
vector.
[0223] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0224] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0225] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0226] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the CaR2 polypeptides or heterologous
to these polypeptides.
[0227] Where the polypeptide is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. The polypeptide can
then be recovered and purified by well-known purification methods
including ammonium sulfate precipitation, acid extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0228] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
[0229] In another embodiment, the endogenous CaR2 is expressed. In
one embodiment the cells of the present invention are cells that do
not produce CaR2 naturally but which have been modified to over
produce the CaR2 polypeptide. This can be done, for example, by the
technology known as RAGE, described in WO 99/15650 and WO 00/49162.
RAGE involves randomly incorporating a transcriptional activator in
the genome by non-homologous recombination, leading to activation
or increased expression of genes down stream of the activator.
Unlike other cloning methods the artisan needs no knowledge about
the gene sequences. Further, the gene is expressed in cells that
normally produce it rather than a host cell, e.g., E. coli. Once a
RAGE modified cell has been selected, e.g., by activity, or
phenotype, that cell can be cultured and used as an expression
vector for the CaR2 polypeptide. This can also be done by a
technology that relies on homologous recombination to incorporate a
transcriptional activator into the genome, as described in WO
94//12650, WO 95/31560, and WO 96/29411, U.S. Pat. No. 5,733,761
and U.S. Pat. No. 6,270,985.
[0230] In addition to the vectors and host cells described above,
the invention is intended to include cells in which CaR2 is
naturally expressed, e.g., normal cells such as kidney, prostate
and osteoblasts, cancerous cells, or cells involved with
osteoporosis.
[0231] The cells can disclosed herein be used in assays to
determine the effectiveness of potential CaR2 modulators with
regard to the ability of modulators to inhibit or activate
CaR2.
II. Diagnostic and Prognostic Assays of the Invention
[0232] The diagnostic and prognostic methods of the present
invention can be used to identify various types of conditions, such
as the conditions disclosed herein, mediated by CaR2. For example,
CaR2 has been identified in bone (osteoblasts), kidney and prostate
cells. It has also been observed that OC increases the sensitivity
of CaR2 to calcium.
[0233] The CaR2 polypeptides also are useful to provide a target
for diagnosing a conditions or predisposition to conditions
mediated by the CaR2, including, but not limited to, conditions
involving tissues in which the CaR2 are expressed as disclosed
herein. Accordingly, methods are provided for detecting the
presence, or levels of, CaR2 in a cell, tissue, or organism.
[0234] CaR2 has been found to be expressed in environments where
there are high levels of calcium. Immunohistochemical analysis has
shown expression of CaR2 in bone, kidney, prostate, salivary,
glands, testis, thymus, brain, trachea and thyroid. The present
invention shows that OC synergistically activates CaR2.
[0235] Accordingly, OC is a novel drug target for conditions
associated with CaR2. Therefore, the methods disclosed herein are
useful for treatment of conditions associated with the
above-mentioned tissues, including, but not limited to,
extracellular calcium concentration, metabolic conditions
associated with CaR2 or OC, osteoporosis, sperm motility and
viability, regulation of calcium flux in the kidneys, kidney stone
formation, regulation of calcium flux in the prostate, promotion of
osteoblast proliferation, e.g., for the production of osteoblasts
for medical use, metastasis of cancers, cancers, e.g., breast,
renal, prostate and bone cancers, regulation of bone
mineralization, bone overgrowth modulation of bone healing, e.g,
dental caries, osteoporosis, and other bone formation diseases, and
detection of a subset of cells, e.g., for forensic analysis.
[0236] As used herein, the term "cancer" refers to conditions
characterized by deregulated or uncontrolled cell growth, for
example, carcinomas, sarcomas, lymphomas. The term "cancer"
includes benign tumors, primary malignant tumors (e.g., those whose
cells have not migrated to sites in the subject's body other than
the site of the original tumor) and secondary malignant tumors
(e.g., those arising from metastasis, the migration of tumor cells
to secondary sites that are different from the site of the original
tumor).
[0237] The term "metastasis" as used herein refers to the condition
of spread of cancer from the organ of origin to additional distal
sites in the patient. The process of tumor metastasis is a
multistage event involving local invasion and destruction of
intercellular and extracellular matrix, intravasation into blood
vessels, lymphatics or other channels of transport, survival in the
circulation, extravasation out of the vessels in the secondary site
and growth in the new location (Fidler, et al., Adv. Cancer Res.
28, 149-250 (1978), Liotta, et al., Cancer Treatment Res. 40,
223-238 (1988), Nicolson, Biochim. Biophy. Acta 948, 175-224 (1988)
and Zetter, N. Eng. J. Med. 322, 605-612 (1990)). Increased
malignant cell motility has been associated with enhanced
metastatic potential in animal as well as human tumors (Hosaka, et
al., Gann 69, 273-276 (1978) and Haemmerlin, et al., Int. J. Cancer
27, 603-610 (1981)).
[0238] As used herein, the term "subject" includes living
organisms, e.g., prokaryotes and eukaryotes. Examples of subjects
include mammals, e.g., humans, dogs, cows, horses, kangaroos, pigs,
sheep, goats, cats, mice, rabbits, rats, and transgenic non-human
animals. Most preferably the subject is a human.
[0239] "Biological samples" include solid and body fluid samples.
The biological samples of the present invention may include cells,
protein or membrane extracts of cells, blood or biological fluids
such as ascites fluid or brain fluid (e.g., cerebrospinal fluid).
Examples of solid biological samples include samples taken from
feces, the rectum, central nervous system, bone, breast tissue,
renal tissue, the uterine cervix, the endometrium, the head/neck,
the gallbladder, parotid tissue, the metastatic, the brain, the
pituitary gland, kidney tissue, muscle, the esophagus, the stomach,
the small to intestine, the colon, the liver, the spleen, the
pancreas, thyroid tissue, heart tissue, lung tissue, the bladder,
adipose tissue, lymph node tissue, the uterus, ovarian tissue,
adrenal tissue, testis tissue, the tonsils, and the thymus.
Examples of "body fluid samples" include samples taken from the
blood, serum, semen, metastatic fluid, seminal fluid, urine,
saliva, sputum, mucus, bone marrow, lymph, and tears. For
amplifying CaR2 RNA, the preferred samples include peripheral
venous blood samples and metastatic tissue samples. Samples for use
in the assays of the invention can be obtained by standard methods
including venous puncture and surgical biopsy. In one embodiment,
the biological sample is a metastatic tissue sample obtained by
needle biopsy.
[0240] "Pharmacogenomics", as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market. More specifically, the term
refers the study of how a patient's genes determine a subject's
response to a drug (e.g., a patient's "drug response phenotype", or
"drug response genotype."
[0241] A. Antibody-Based Immunoassays
[0242] Methods for using antibodies as disclosed herein are
particularly applicable to the cells, tissues and conditions that
differentially express CaR2, or that are involved in CaR2 mediated
conditions and as otherwise discussed herein.
[0243] The invention provides methods using antibodies that
selectively bind to CaR2 and its variants and fragments. An
antibody is considered to selectively bind, even if it also binds
to other proteins that are not substantially homologous with CaR2.
These other proteins share homology with a fragment or domain of
the CaR2. This conservation in specific regions gives rise to
antibodies that bind to both proteins by virtue of the homologous
sequence. In this case, it would be understood that antibody
binding to CaR2 is still selective.
[0244] Antibodies accordingly can be used diagnostically to monitor
protein levels or activity in tissue as part of a clinical testing
procedure, for example, to determine the efficacy of a given
treatment regimen.
[0245] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic CaR2 can be
used to identify individuals that require modified treatment
modalities.
[0246] Antibodies can also be used in diagnostic procedures as an
immunological marker for aberrant CaR2 analyzed by electrophoretic
mobility, isoelectric point, tryptic peptide digest, and other
physical assays known to those in the art.
[0247] The antibodies are also useful for tissue typing. Thus,
where a specific CaR2 has been correlated with expression in a
specific tissue, antibodies that are specific for this CaR2 can be
used to identify a tissue type.
[0248] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[0249] Antibody detection of circulating fragments of the full
length CaR2 can be used to identify CaR2 turnover.
[0250] Further, the antibodies can be used to assess CaR2
expression in disease states such as in active stages of the
condition or in an individual with a predisposition toward a
condition related to CaR2 function. When a disorder is caused by an
inappropriate tissue distribution, developmental expression, or
level of expression of CaR2 protein, the antibody can be prepared
against the normal CaR2 protein. If a disorder is characterized by
a specific mutation in CaR2, antibodies specific for this mutant
protein can be used to assay for the presence of the specific
mutant CaR2. However, intracellularly-made antibodies
("intrabodies") are also encompassed, which would recognize
intracellular CaR2 peptide regions.
[0251] The antibodies can also be used to assess normal and
aberrant subcellular localization in cells in the various tissues
in an organism. Antibodies can be developed against the whole CaR2
or portions of CaR2.
[0252] The amount of an antigen (i.e. CaR2) in a biological sample
may be determined by a radioimmunoassay, an immunoradiometric
assay, and/or an enzyme immunoassay.
[0253] "Radioimmunoassay" is a technique for detecting and
measuring the concentration of an antigen using a labeled (i.e.
radioactively labeled) form of the antigen. Examples of radioactive
labels for antigens include 3H, .sup.14C, and .sup.125I. The
concentration of antigen (i.e. CaR2) in a sample (i.e. biological
sample) is measured by having the antigen in the sample compete
with a labeled (i.e. radioactively) antigen for binding to an
antibody to the antigen. To ensure competitive binding between the
labeled antigen and the unlabeled antigen, the labeled antigen is
present in a concentration sufficient to saturate the binding sites
of the antibody. The higher the concentration of antigen in the
sample, the lower the concentration of labeled antigen that will
bind to the antibody.
[0254] In a radioimmunoassay, to determine the concentration of
labeled antigen bound to antibody, the antigen-antibody complex
must be separated from the free antigen. One method for separating
the antigen-antibody complex from the free antigen is by
precipitating the antigen-antibody complex with an anti-isotype
antiserum. Another method for separating the antigen-antibody
complex from the free antigen is by precipitating the
antigen-antibody complex with formalin-killed S. aureus. Yet
another method for separating the antigen-antibody complex from the
free antigen is by performing a "solid-phase radioimmunoassay"
where the antibody is linked (i.e. covalently) to Sepharose beads,
polystyrene wells, polyvinylchloride wells, or microtiter wells. By
comparing the concentration of labeled antigen bound to antibody to
a standard curve based on samples having a known concentration of
antigen, the concentration of antigen in the biological sample can
be determined.
[0255] A "Immunoradiometric assay" (IRMA) is an immunoassay in
which the antibody reagent is radioactively labeled. An IRMA
requires the production of a multivalent antigen conjugate, by
techniques such as conjugation to a protein e.g., rabbit serum
albumin (RSA). The multivalent antigen conjugate must have at least
2 antigen residues per molecule and the antigen residues must be of
sufficient distance apart to allow binding by at least two
antibodies to the antigen. For example, in an IRMA the multivalent
antigen conjugate can be attached to a solid surface such as a
plastic sphere. Unlabeled "sample" antigen and antibody to antigen
which is radioactively labeled are added to a test tube containing
the multivalent antigen conjugate coated sphere. The antigen in the
sample competes with the multivalent antigen conjugate for antigen
antibody binding sites. After an appropriate incubation period, the
unbound reactants are removed by washing and the amount of
radioactivity on the solid phase is determined. The amount of bound
radioactive antibody is inversely proportional to the concentration
of antigen in the sample.
[0256] The most common enzyme immunoassay is the "Enzyme-Linked
Immunosorbent Assay (ELISA)." The "Enzyme-Linked Immunosorbent
Assay (ELISA)" is a technique for detecting and measuring the
concentration of an antigen using a labeled (i.e. enzyme linked)
form of the antibody.
[0257] In a "sandwich ELISA", an antibody (i.e. to CaR2) is linked
to a solid phase (i.e. a microtiter plate) and exposed to a
biological sample containing antigen (i.e. CaR2). The solid phase
is then washed to remove unbound antigen. A labeled (i.e. enzyme
linked) is then bound to the bound-antigen (if present) forming an
antibody-antigen-antibody sandwich. Examples of enzymes that can be
linked to the antibody are alkaline phosphatase, horseradish
peroxidase, luciferase, urease, and .beta.-galactosidase. The
enzyme linked antibody reacts with a substrate to generate a
colored reaction product that can be assayed for.
[0258] In a "competitive ELISA", antibody is incubated with a
sample containing antigen (i.e. CaR2). The antigen-antibody mixture
is then contacted with an antigen-coated solid phase (i.e. a
microtiter plate). The more antigen present in the sample, the less
free antibody that will be available to bind to the solid phase. A
labeled (i.e. enzyme linked) secondary antibody is then added to
the solid phase to determine the amount of primary antibody bound
to the solid phase.
[0259] In a "immunohistochemistry assay" a section of tissue for is
tested for specific proteins by exposing the tissue to antibodies
that are specific for the protein that is being assayed. The
antibodies are then visualized by any of a number of methods to
determine the presence and amount of the protein present. Examples
of methods used to visualize antibodies are, for example, through
enzymes linked to the antibodies (e.g., luciferase, alkaline
phosphatase, horseradish peroxidase, or .beta.-galactosidase), or
chemical methods (e.g., DAB/Substrate chromagen).
[0260] Antibody based assays can be used to determine the levels of
OC a given subject produces. As the calcium-sensing receptor of the
present invention is activated by OC, aberrant levels of OC
manifest the same CaR2 mediated conditions. OC levels can be
measured using techniques that are standard in the art. For
example, a kit for measuring OC is available from Biomedical
Technologies Inc. (Stoughton, Mass.). OC assays are described in
U.S. Pat. Nos. 5,866,364 and 5,681,707 and by Delmas P. D., 1990,
Endocrinol. Clin. North Am. 19: 1-18; Koyama et al (1991) J.
Immunol. Meth. 139, 17-23; and Grunhaberg et al. (1984) Meth.
Enzymology, 207, 516.
[0261] B. CaR2 Nucleic Acid-Based Diagnostic and Prognostic
Methods
[0262] Also encompassed by this invention is a method of diagnosing
CaR2 related conditions in a subject, comprising: detecting a level
of CaR2 nucleic acid in a biological sample; and comparing the
level of CaR2 in the biological sample with a level of CaR2 in a
control sample, wherein an elevation or reduction in the level of
CaR2 in the biological sample compared to the control sample is
indicative of CaR2 disorder.
[0263] In addition, this invention pertains to a method of
diagnosing a CaR2 disorder in a subject, comprising the steps of:
detecting a level of CaR2 nucleic acid in a biological sample; and
comparing the level of CaR2 in the biological sample with a level
of CaR2 in a control sample, wherein an elevation in the level of
CaR2 in the biological sample compared to the control sample is
indicative of an CaR2 condition. In preferred embodiments the CaR2
disorder is the metastasis of cancer, or other CaR2 mediated
conditions such as the conditions disclosed herein.
[0264] In an embodiment of the above methods, the detecting a level
of CaR2 nucleic acid in a biological sample includes amplifying
CaR2 RNA. In another embodiment of the above methods, the detecting
a level of CaR2 nucleic acid in a biological sample includes
hybridizing the CaR2 RNA with a probe.
[0265] As an alternative to making determinations based on the
absolute expression level of the CaR2 marker, determinations may be
based on the normalized expression level of the marker. Expression
levels are normalized by correcting the absolute expression level
of a marker by comparing its expression to the expression of a gene
that is not a marker, e.g., a housekeeping gene that is
constitutively expressed. Suitable genes for normalization include
housekeeping genes such as the actin gene, or epithelial
cell-specific genes. This normalization allows the comparison of
the expression level in one sample, e.g., a patient sample, to
another sample, e.g., a non-metastatic cancer sample, or between
samples from different sources.
[0266] Alternatively, the expression level can be provided as a
relative expression level. To determine a relative expression level
of a marker, the level of expression of the marker is determined
for 10 or more samples of normal versus cancer cell isolates,
preferably 50 or more samples, prior to the determination of the
expression level for the sample in question. The mean expression
level of each of the genes assayed in the larger number of samples
is determined and this is used as a baseline expression level for
the marker. The expression level of the marker determined for the
biological sample (absolute level of expression) is then divided by
the mean expression value obtained for that marker. This provides a
relative expression level.
[0267] One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. Probes based on the sequence of a nucleic acid
molecule of the invention can be used to detect transcripts
corresponding to CaR2. The nucleic acid probe can be, for example,
a full-length cDNA, or a portion thereof, such as an
oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to a mRNA or genomic DNA encoding a
marker of the present invention. Hybridization of an mRNA with the
probe indicates that the marker in question is being expressed. In
an embodiment, the probe includes a label group attached thereto,
e.g., a radioisotope, a fluorescent compound, an enzyme, or an
enzyme co-factor.
[0268] In one format, the mRNA is immobilized on a solid surface
and contacted with a probe, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probe(s) are immobilized on a solid surface and the mRNA is
contacted with the probe(s), for example, in an Affymetrix gene
chip array. A skilled artisan can readily adapt known mRNA
detection methods for use in detecting the level of mRNA encoded by
the markers of the present invention.
[0269] "Amplifying" refers to template-dependent processes and
vector-mediated propagation which result in an increase in the
concentration of a specific nucleic acid molecule relative to its
initial concentration, or in an increase in the concentration of a
detectable signal. As used herein, the term template-dependent
process is intended to refer to a process that involves the
template-dependent extension of a primer molecule. The term
template dependent process refers to nucleic acid synthesis of an
RNA or a DNA molecule wherein the sequence of the newly synthesized
strand of nucleic acid is dictated by the well-known rules of
complementary base pairing (see, for example, Watson, J. D. et al.,
In: Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc.,
Menlo Park, Calif. (1987). Typically, vector mediated methodologies
involve the introduction of the nucleic acid fragment into a DNA or
RNA vector, the clonal amplification of the vector, and the
recovery of the amplified nucleic acid fragment. Examples of such
methodologies are provided by Cohen et al. (U.S. Pat. No.
4,237,224), Maniatis, T. et al., Molecular Cloning (A Laboratory
Manual), Cold Spring Harbor Laboratory, 1982.
[0270] A number of template dependent processes are available to
amplify the target sequences of interest present in a sample. One
of the best known amplification methods is the polymerase chain
reaction (PCR) which is described in detail in Mullis, et al., U.S.
Pat. No. 4,683,195, Mullis, et al., U.S. Pat. No. 4,683,202, and
Mullis, et al., U.S. Pat. No. 4,800,159, and in Innis et al., PCR
Protocols, Academic Press, Inc., San Diego Calif., 1990. Briefly,
in PCR, two primer sequences are prepared which are complementary
to regions on opposite complementary strands of the target
sequence. An excess of deoxynucleoside triphosphates are added to a
reaction mixture along with a DNA polymerase (e.g., Taq
polymerase). If the target sequence is present in a sample, the
primers will bind to the target and the polymerase will cause the
primers to be extended along the target sequence by adding on
nucleotides. By raising and lowering the temperature of the
reaction mixture, the extended primers will dissociate from the
target to form reaction products, excess primers will bind to the
target and to the reaction products and the process is repeated.
Preferably a reverse transcriptase PCR amplification procedure may
be performed in order to quantify the amount of mRNA amplified.
Polymerase chain reaction methodologies are well known in the
art.
[0271] Another method for amplification is the ligase chain
reaction (LCR), disclosed in European Patent No. 320,308B1. In LCR,
two complementary probe pairs are prepared, and in the presence of
the target sequence, each pair will bind to opposite complementary
strands of the target such that they abut. In the presence of a
ligase, the two probe pairs will link to form a single unit. By
temperature cycling, as in PCR, bound ligated units dissociate from
the target and then serve as "target sequences" for ligation of
excess probe pairs. Whiteley, et al., U.S. Pat. No. 4,883,750
describes an alternative method of amplification similar to LCR for
binding probe pairs to a target sequence.
[0272] Qbeta Replicase, described in PCT Application No.
PCT/US87/00880 may also be used as still another amplification
method in the present invention. In this method, a replicative
sequence of RNA which has a region complementary to that of a
target is added to a sample in the presence of an RNA polymerase.
The polymerase will copy the replicative sequence which can then be
detected.
[0273] Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis, i.e.
nick translation. A similar method, called Repair Chain Reaction
(RCR) is another method of amplification which may be useful in the
present invention and is involves annealing several probes
throughout a region targeted for amplification, followed by a
repair reaction in which only two of the four bases are present.
The other two bases can be added as biotinylated derivatives for
easy detection. A similar approach is used in SDA.
[0274] CaR2 specific sequences can also be detected using a cyclic
probe reaction (CPR). In CPR, a probe having a 3' and 5' sequences
of non-metastatic specific DNA and middle sequence of metastatic
specific RNA is hybridized to DNA which is present in a sample.
Upon hybridization, the reaction is treated with RNaseH, and the
products of the probe identified as distinctive products generating
a signal which are released after digestion. The original template
is annealed to another cycling probe and the reaction is repeated.
Thus, CPR involves amplifying a signal generated by hybridization
of a probe to a metastatic cancer specific expressed nucleic
acid.
[0275] Still other amplification methods described in GB
Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025 may be used in accordance with the present
invention. In the former application, "modified" primers are used
in a PCR like, template and enzyme dependent synthesis. The primers
may be modified by labeling with a capture moiety (e.g., biotin)
and/or a detector moiety (e.g., enzyme). In the latter application,
an excess of labeled probes are added to a sample. In the presence
of the target sequence, the probe binds and is cleaved
catalytically. After cleavage, the target sequence is released
intact to be bound by excess probe. Cleavage of the labeled probe
signals the presence of the target sequence.
[0276] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS) (Kwoh D., et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 1989, 86:1173, Gingeras T. R., et
al., PCT Application WO 88/1D315), including nucleic acid sequence
based amplification (NASBA) and 3SR. In NASBA, the nucleic acids
can be prepared for amplification by standard phenol/chloroform
extraction, heat denaturation of a clinical sample, treatment with
lysis buffer and minispin columns for isolation of DNA and RNA or
guanidinium chloride extraction of RNA. These amplification
techniques involve annealing a primer which has metastatic specific
sequences. Following polymerization, DNA/RNA hybrids are digested
with RNase H while double stranded DNA molecules are heat denatured
again. In either case the single stranded DNA is made fully double
stranded by addition of second metastatic specific primer, followed
by polymerization. The double stranded DNA molecules are then
multiply transcribed by a polymerase such as T7 or SP6. In an
isothermal cyclic reaction, the RNAs are reverse transcribed into
double stranded DNA, and transcribed once against with a polymerase
such as T7 or SP6. The resulting products, whether truncated or
complete, indicate metastatic cancer specific sequences.
[0277] Davey, C., et al., European Patent No. 329,822B1 disclose a
nucleic acid amplification process involving cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and
double-stranded DNA (dsDNA), which may be used in accordance with
the present invention. The ssRNA is a first template for a first
primer oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from
resulting DNA:RNA duplex by the action of ribonuclease H(RNase H,
an RNase specific for RNA in a duplex with either DNA or RNA). The
resultant ssDNA is a second template for a second primer, which
also includes the sequences of an RNA polymerase promoter
(exemplified by T7 RNA polymerase) 5' to its homology to its
template. This primer is then extended by DNA polymerase
(exemplified by the large "Klenow" fragment of E. coli DNA
polymerase 1), resulting as a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA
between the primers and having additionally, at one end, a promoter
sequence. This promoter sequence can be used by the appropriate RNA
polymerase to make many RNA copies of the DNA. These copies can
then re-enter the cycle leading to very swift amplification. With
proper choice of enzymes, this amplification can be done
isothermally without addition of enzymes at each cycle. Because of
the cyclical nature of this process, the starting sequence can be
chosen to be in the form of either DNA or RNA.
[0278] Miller, H. I., et al., PCT Application WO 89/06700 discloses
a nucleic acid sequence amplification scheme based on the
hybridization of a promoter/primer sequence to a target
single-stranded DNA ("ssDNA") followed by transcription of many RNA
copies of the sequence. This scheme is not cyclic; i.e. new
templates are not produced from the resultant RNA transcripts.
Other amplification methods include "race" disclosed by Frohman, M.
A., In: PCR Protocols: A Guide to Methods and Applications 1990,
Academic Press, New York) and "one-sided PCR" (Ohara, O., et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 1989, 86:5673-5677).
[0279] 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. (1989) Bio Technology 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.
[0280] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide (Wu, D. Y. et al., Genomics 1989, 4:560), may
also be used in the amplification step of the present
invention.
[0281] Following amplification, the presence or absence of the
amplification product may be detected. The amplified product may be
sequenced by any method known in the art, including and not limited
to the Maxam and Gilbert method. The sequenced amplified product is
then compared to a sequence known to be in a metastatic cancer
specific sequence. Alternatively, the nucleic acids may be
fragmented into varying sizes of discrete fragments. For example,
DNA fragments may be separated according to molecular weight by
methods such as and not limited to electrophoresis through an
agarose gel matrix. The gels are then analyzed by Southern
hybridization. Briefly, DNA in the gel is transferred to a
hybridization substrate or matrix such as and not limited to a
nitrocellulose sheet and a nylon membrane. A labeled probe is
applied to the matrix under selected hybridization conditions so as
to hybridize with complementary DNA localized on the matrix. The
probe may be of a length capable of forming a stable duplex. The
probe may have a size range of about 200 to about 10,000
nucleotides in length, preferably about 200 nucleotides in length.
Various labels for visualization or detection are known to those of
skill in the art, such as and not limited to fluorescent staining,
ethidium bromide staining for example, avidin/biotin, radioactive
labeling such as .sup.32P labeling, and the like. Preferably, the
product, such as the PCR product, may be run on an agarose gel and
visualized using a stain such as ethidium bromide. The matrix may
then be analyzed by autoradiography to locate particular fragments
which hybridize to the probe.
[0282] The CaR2 polynucleotides are also useful for monitoring the
effectiveness of modulating compounds on the expression or activity
of the CaR2 gene in clinical trials or in a treatment regimen.
Thus, the gene expression pattern can serve as a barometer for the
continuing effectiveness of treatment with the compound,
particularly with compounds to which a patient can develop
resistance. The gene expression pattern can also serve as a marker
indicative of a physiological response of the affected cells to the
compound. Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[0283] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the mRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[0284] The CaR2 polynucleotides can be used in diagnostic assays
for qualitative changes in CaR2 nucleic acid, and particularly in
qualitative changes that lead to pathology. The polynucleotides can
be used to detect mutations in CaR2 genes and gene expression
products such as mRNA. The polynucleotides can be used as
hybridization probes to detect naturally-occurring genetic
mutations in the CaR2 gene and thereby to determine whether a
subject with the mutation is at risk for a disorder caused by the
mutation. Mutations include deletion, addition, or substitution of
one or more nucleotides in the gene, chromosomal rearrangement,
such as inversion or transposition, modification of genomic DNA,
such as aberrant methylation patterns or changes in gene copy
number, such as amplification. Detection of a mutated form of the
CaR2 gene associated with a dysfunction provides a diagnostic tool
for an active condition or susceptibility to a condition when the
condition results from overexpression, underexpression, or altered
expression of CaR2.
[0285] Mutations in the CaR2 gene can be detected at the nucleic
acid level by a variety of techniques. Genomic DNA can be analyzed
directly or can be amplified by using PCR prior to analysis. RNA or
cDNA can be used in the same way.
[0286] In certain embodiments, detection of the mutation 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
gene (see Abravaya et al. (1995) Nucleic 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 which specifically hybridize to a
gene under conditions such that hybridization and amplification of
the 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.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[0287] Alternatively, mutations in a CaR2 gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0288] Further, sequence-specific ribozymes (U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
[0289] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[0290] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and SI protection or
the chemical cleavage method.
[0291] Furthermore, sequence differences between a mutant CaR2 gene
and a wild-type gene can be determined by direct DNA sequencing. A
variety of automated sequencing procedures can be utilized when
performing the diagnostic assays ((1995) Biotechniques 19:448),
including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen et al. (1996) Adv.
Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.
Biotechnol. 38:147-159).
[0292] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.
(1985) Science 230: 1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton et al. (1993) Mutat. Res.
285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al. (1985)
Nature 313:495). 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 (Keen et al. (1991) Trends Genet. 7: 5).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[0293] In other embodiments, genetic mutations 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
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two dimensional
arrays containing light-generated DNA probes as described in Cronin
et al. supra. 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.
[0294] The CaR2 polynucleotides can also be used for testing an
individual for a genotype that while not necessarily causing the
condition, nevertheless affects the treatment modality. Thus, the
polynucleotides can be used to study the relationship between an
individual's genotype and the individual's response to a compound
used for treatment (pharmacogenomic relationship). In the present
case, for example, a mutation in the CaR2 gene that results in
altered affinity for substrate could result in an excessive or
decreased drug effect with standard concentrations substrate.
Accordingly, the CaR2 polynucleotides described herein can be used
to assess the mutation content of the gene in an individual in
order to select an appropriate compound or dosage regimen for
treatment.
[0295] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells or animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[0296] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control sample with the presence of mRNA or genomic DNA
in the test sample.
[0297] The CaR2 polynucleotides are also useful for chromosome
identification when the sequence is identified with an individual
chromosome and to a particular location on the chromosome. First,
the DNA sequence is matched to the chromosome by in situ or other
chromosome-specific hybridization. Sequences can also be correlated
to specific chromosomes by preparing PCR primers that can be used
for PCR screening of somatic cell hybrids containing individual
chromosomes from the desired species. Only hybrids containing the
chromosome containing the gene homologous to the primer will yield
an amplified fragment. Sublocalization can be achieved using
chromosomal fragments. Other strategies include prescreening with
labeled flow-sorted chromosomes and preselection by hybridization
to chromosome-specific libraries. Further mapping strategies
include fluorescence in situ hybridization, which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the chromosome, or panels of
reagents can be used for marking multiple sites and/or multiple
chromosomes. Reagents corresponding to noncoding regions of the
genes actually are preferred for mapping purposes. Coding sequences
are more likely to be conserved within gene families, thus
increasing the chance of cross hybridizations during chromosomal
mapping.
[0298] The CaR2 polynucleotides can also be used to identify
individuals from small biological samples. This can be done for
example using restriction fragment-length polymorphism (RFLP) to
identify an individual. Thus, the polynucleotides described herein
are useful as DNA markers for RIJLP (See U.S. Pat. No.
5,272,057).
[0299] Furthermore, the CaR2 sequence can be used to provide an
alternative technique, which determines the actual DNA sequence of
selected fragments in the genome of an individual. Thus, the CaR2
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 DNA from an individual for subsequent
sequencing.
[0300] 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. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. The
CaR2 sequences can be used to obtain such identification sequences
from individuals and from tissue. The sequences represent unique
fragments of the human genome. 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.
[0301] If a panel of reagents from the sequences is used to
generate a unique identification database for an individual, those
same reagents can later be used to identify tissue from that
individual. Using the unique identification database, positive
identification of the individual, living or dead, can be made from
extremely small tissue samples.
[0302] The CaR2 polynucleotides can also be used in forensic
identification procedures. PCR technology can be used to amplify
DNA sequences taken from very small biological samples, such as a
single hair follicle, body fluids (e.g. blood, saliva, or semen).
The amplified sequence can then be compared to a standard allowing
identification of the origin of the sample.
[0303] The CaR2 polynucleotides can thus be used to provide
polynucleotide reagents, e.g., PCR primers, targeted to specific
loci in the human genome, which 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 described 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 the noncoding region are
particularly useful since greater polymorphism occurs in the
noncoding regions, making it easier to differentiate individuals
using this technique.
[0304] The CaR2 polynucleotides can further be used to provide
polynucleotide reagents, e.g., labeled or labelable probes which
can be used in, for example, an in situ hybridization technique, to
identify a specific tissue. This is useful in cases in which a
forensic pathologist is presented with a tissue of unknown origin.
Panels of CaR2 probes can be used to identify tissue by species
and/or by organ type.
[0305] In a similar fashion, these primers and 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).
[0306] Alternatively, the CaR2 polynucleotides can be used directly
to block transcription or translation of CaR2 gene sequences by
means of antisense or ribozyme constructs. Thus, in a disorder
characterized by abnormally high or undesirable CaR2 gene
expression, nucleic acids can be directly used for treatment.
[0307] The CaR2 polynucleotides are thus useful as antisense
constructs to control CaR2 gene expression in cells, tissues, and
organisms. A DNA antisense polynucleotide is designed to be
complementary to a region of the gene involved in transcription,
preventing transcription and hence production of CaR2 protein. An
antisense RNA or DNA polynucleotide would hybridize to the mRNA and
thus block translation of mRNA into CaR2 protein.
[0308] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of SEQ ID NO:2 or SEQ ID
NO:4 which also includes the start codon and antisense molecules
which are complementary to a fragment of the 3' untranslated region
of SEQ ID NO:2 or SEQ ID NO:4.
[0309] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of CaR2 nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired CaR2 nucleic acid
expression. This technique involves cleavage by means of ribozymes
containing nucleotide sequences complementary to one or more
regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the CaR2 protein.
[0310] The CaR2 polynucleotides also provide vectors for gene
therapy in patients containing cells that are aberrant in CaR2 gene
expression. Thus, recombinant cells, which include the patient's
cells that have been engineered ex vivo and returned to the
patient, are introduced into an individual where the cells produce
the desired CaR2 protein to treat the individual.
[0311] The invention also encompasses kits for detecting the
presence of a CaR2 nucleic acid in a biological sample. For
example, the kit can comprise reagents such as a labeled or
labelable nucleic acid or agent capable of detecting CaR2 nucleic
acid in a biological sample; means for determining the amount of
CaR2 nucleic acid in the sample; and means for comparing the amount
of CaR2 nucleic acid 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 phosphodicsterase
mRNA or DNA.
[0312] C. OC Nucleic Acid-Based Diagnostic and Prognostic
Methods
[0313] The methods described above for CaR2 nucleic acid based
diagnostic and prognostic methods can be used in similar fashion to
determine the presence and amount of OC in a biological sample.
[0314] The determination of OC abundance can be used to diagnose
conditions associated with aberrant OC expression.
III. Methods for Identifying Modulators
[0315] A. CaR2 Modulators
[0316] Determining the ability of the calcium receptor to bind to a
target molecule can be accomplished using a technology such as
real-time Bimolecular Interaction Analysis (BIA). Sjolander et al.
(1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin.
Struct. Biol. 5:699-705. As used herein, "BIA" is a technology for
studying biospecific interactions in real time, without labeling
any of the interactants (e.g., BIAcore.TM.). Changes in the optical
phenomenon surface plasmon resonance (SPR) can be used as an
indication of real-time reactions between biological molecules.
[0317] The test compounds of the present invention can be 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 polypeptide libraries, while the other four
approaches are applicable to polypeptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145).
[0318] 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. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckerman et al. (1994) J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. 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), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '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. 97:6378-6382); Felici (1991)
J. Mol. Biol. 222:301-310); (Ladner supra).
[0319] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al. (1991)
Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0320] One candidate compound is a soluble full-length calcium
receptor or fragment that competes for ligand. Other candidate
compounds include mutant calcium receptor or approaching fragments
containing mutations that affect calcium receptor function and thus
compete for ligand. Accordingly, a fragment that competes for
ligand, for example with a higher affinity, or a fragment that
binds ligand, is encompassed by the invention.
[0321] Other candidate compounds are OC, OC-like molecules or a
fragment of OC that competes with CaR2 for binding. Other candidate
compounds include mutant OC or mutant OC fragments that affect the
ability of OC to bind to CaR2.
[0322] Protein inhibitors of the present invention can be selected
using the RNA-protein fusion method that was developed by Szostak,
J. W., et al. This method relies on a covalent fusion between an
mRNA and a protein or peptide that it encodes through a puromycin
at the 3' end of the RNA molecule. Fusion of the polypeptide to the
RNA that encodes it allows for the skilled artisan to isolate a
protein of interest while also isolating the nucleic acid that
encodes the protein. The technology is described in Roberts, R. W.
and Szostak, J. W. (1997) Prot. Natl. Acad. Sci. USA 11:12297-302
and Liu, R. et al. (2000) Methods Enzymol. 318:268-93, and in U.S.
Pat. Nos. 6,207,446, 6,214,553, 6,261,804, 6,258,558, and
6,281,344.
[0323] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) CaR2 activity. The
assays typically involve an assays that indicate CaR2 activity.
Thus, the expression of genes that are up- or down-regulated in
response to the calcium receptor dependent signal cascade can be
assayed. In one embodiment, the regulatory region of such genes can
be operably linked to a marker that is easily detectable, such as
luciferase. Alternatively, cleavage of the calcium receptor target
could also be measured.
[0324] Any of the biological or biochemical functions mediated by
the calcium receptor can be used as an endpoint assay. These
include all of the biochemical or biochemical/biological events
described herein, in the references cited herein, incorporated by
reference for these endpoint assay targets, and other functions
known to those of ordinary skill in the art.
[0325] Assays for calcium receptors are common in the field and
have been previously described. For example, the activity of a
calcium receptor can be conveniently measured using a Xenopus
expression assay to detect increases in intracellular Ca++ due to
receptor activation. Increases in intracellular Ca++ can be
measured by different techniques such as by measuring current
through the endogenous Ca++-activated Cl-channel; loading oocytes
with .sup.45Ca++ and measuring mobilization of .sup.45Ca++ from
intracellular stores; and using fluorescent Ca++ indicators.
[0326] The invention provides competition binding assays designed
to discover compounds that interact with the calcium receptor.
Thus, a compound is exposed to a calcium receptor polypeptide under
conditions that allow the compound to bind or to otherwise interact
with the polypeptide. Soluble CaR2 polypeptide is also added to the
mixture. If the test compound interacts with the soluble CaR2
polypeptide, it decreases the amount of complex formed or activity
from the CaR2 target. This type of assay is particularly useful in
cases in which compounds are sought that interact with specific
regions of the calcium receptor. Thus, the soluble polypeptide that
competes with the target calcium receptor region is designed to
contain peptide sequences corresponding to the region of
interest.
[0327] Another type of competition-binding assay can be used to
discover compounds that interact with specific functional sites.
Accordingly, compounds can be discovered that directly interact
with CaR2 and compete with substrate. Such assays can involve any
other component that interacts with CaR2.
[0328] To perform cell-free drug screening assays, it is desirable
to immobilize either the CaR2, or fragment, or its target molecule
to facilitate separation of complexes from uncomplexed forms of one
or both of the proteins, as well as to accommodate automation of
the assay.
[0329] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase/CaR2
fusion proteins can be absorbed onto glutathione sepharose beads
(Sigma Chemical, St. Louis, Mo.) or glutathione derivatized
microtitre plates, which are then combined with the cell lysates
(e.g., .sup.35S-labeled) and the candidate compound, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads are washed to remove any unbound label, and
the matrix immobilized and radiolabel determined directly, or in
the supernatant after the complexes is dissociated. Alternatively,
the complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of CaR2-binding protein found in the bead
fraction quantitated from the gel using standard electrophoretic
techniques. For example, either the polypeptide or its target
molecule can be immobilized utilizing conjugation of biotin and
streptavidin using techniques well known in the art. Alternatively,
antibodies reactive with the protein but which do not interfere
with binding of the protein to its target molecule can be
derivatized to the wells of the plate, and the protein trapped in
the wells by antibody conjugation. Preparations of a CaR2-binding
target component, and a candidate compound are incubated in the
CaR2-presenting wells and the amount of complex trapped in the well
can be quantitated. 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 CaR2 target molecule, or which are reactive with
CaR2 and compete with the target molecule; as well as enzyme-linked
assays which rely on detecting an enzymatic activity associated
with the target molecule.
[0330] Nucleic acid expression assays are also useful for drug
screening to identify compounds that modulate CaR2 nucleic acid
expression (e.g., antisense, polypeptides, peptidomimetics, small
molecules or other drugs). A cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of the mRNA in the presence of the candidate compound is
compared to the level of expression of the mRNA in the absence of
the candidate compound. The candidate compound can then be
identified as a modulator of nucleic acid expression based on this
comparison and be used, for example to treat a disorder
characterized by aberrant nucleic acid expression. The modulator
can bind to the nucleic acid or indirectly modulate expression,
such as by interacting with other cellular components that affect
nucleic acid expression.
[0331] 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 gene to a subject) in patients or in
transgenic animals.
[0332] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
expression of the CaR2 gene. The method typically includes assaying
the ability of the compound to modulate the expression of the CaR2
nucleic acid and thus identifying a compound that can be used to
treat a disorder characterized by excessive or deficient CaR2
nucleic acid expression.
[0333] The assays can be performed in cell-based and cell-free
systems, such as systems using the tissues described herein, in
which the gene is expressed or in model systems for the conditions
to which the invention pertains. Cell-based assays include cells
naturally expressing the CaR2 nucleic acid or recombinant cells
genetically engineered to express specific nucleic acid
sequences.
[0334] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals. The assay for CaR2 nucleic acid
expression can involve direct assay of nucleic acid levels, such as
mRNA levels.
[0335] Thus, modulators of CaR2 gene expression can be identified
in a method wherein a cell is contacted with a candidate compound
and the expression of mRNA determined. The level of expression of
CaR2 mRNA in the presence of the candidate compound is compared to
the level of expression of CaR2 mRNA in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of nucleic acid expression based on this comparison
and be used, for example, to treat a disorder characterized by
aberrant nucleic acid expression. When expression of mRNA is
statistically significantly greater in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of nucleic acid expression. When nucleic
acid expression is statistically significantly less in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of nucleic acid
expression.
[0336] B. Osteocalcin Modulators
[0337] The present invention is based on, in part, that OC
activation of CaR2 when pre-incubated with Ca.sup.++ in a
dose-dependent manner. This study, for the first time, identifies a
regulatory role for OC. Accordingly, modulators that effect the
ability of OC to activate CaR2, e.g., agonists or antagonists, are
useful therapeutically.
[0338] The methods disclosed above to screen compounds for the
ability to bind and/or modulate CaR2 are also useful for screening
compounds for the ability to bind and/or modulate OC activation of
CaR2. Compounds can be tested according to the methods disclosed
herein for the ability to bind to OC, or the DNA that encodes OC
and either increase or decrease the ability of OC to activate
CaR2.
IV. CaR2 Cell Assays and Transgenic Animal Models
[0339] The methods using vectors and cells described herein are
useful where the cells are those that naturally express the gene or
a recombinant cell expressing the gene. The cells of the present
invention are useful for identifying compounds that modulate CaR2
activity, as well as for testing the toxicity of compounds
identified to modulate CaR2.
[0340] It is understood that "cells", "host cells" and "recombinant
host cells" refer not only to the particular subject cell but also
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.
[0341] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing CaR2 proteins or
polypeptides that can be further purified to produce desired
amounts of CaR2 protein or fragments. Thus, host cells containing
expression vectors or cells expressing the endogenous CaR2 are
useful for polypeptide production, as well as cells producing
significant amounts of the polypeptide.
[0342] In one embodiment, host cells of the invention include cells
that naturally produce CaR2, e.g., a kidney, prostate or bone cell.
CaR2 has been shown to be produced in kidney, skeletal muscle,
spleen, fetal liver, heart, stomach, uterus, salivary gland,
adipose and prostate. CaR2 has been shown to be overproduced in
gastrointestinal tumors and may be overexpressed in other cancer
cells. Experiments have shown (see the Examples) that expression of
CaR2 results in cells that are more invasive, e.g., more likely to
be metastatic. Accordingly, assays can be performed using cancerous
cells, e.g., biopsied cells, to determine the aggressiveness of the
cancer. Invasiveness assays are common in the field and examples
are presented below.
[0343] Host cells can be natural cells which naturally contain the
CaR2 gene and have been modified using the Random Activation of
Gene Expression (RAGE) technology to over express CaR2 (for details
on the RAGE technology see WO 00/49162 and WO 99/15650,
incorporated herein by reference). The RAGE technology provides
methods of expressing an endogenous gene at levels higher than
normally found in the cell without having to clone the gene. RAGE
is based on the introduction of a transcriptional activator in to a
genome by non-homologous recombination. Host cells can be modified
by the introduction of a transcriptional activator by homologous
recombination as described in WO 94//12650, WO 95/31560, WO
96/29411, U.S. Pat. No. 5,733,761 and U.S. Pat. No. 6,270,985.
[0344] Host cells are also useful for conducting cell-based assays
involving the CaR2 or CaR2 fragments. Thus, a recombinant host cell
expressing CaR2 is useful to assay for compounds that stimulate or
inhibit CaR2 function.
[0345] Host cells are also useful for identifying CaR2 mutants in
which these functions are affected. If the mutants naturally occur
and give rise to a pathology, host cells containing the mutations
are useful to assay compounds that have a desired effect on the
mutant CaR2 (for example, stimulating or inhibiting function) which
may not be indicated by their effect on the native CaR2.
[0346] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation by means of a heterologous domain,
segment, site, and the like, as disclosed herein.
[0347] Further, mutant CaR2 can be designed in which one or more of
the various functions is engineered to be increased or decreased
and used to augment or replace CaR2 proteins in an individual.
Thus, host cells can provide a therapeutic benefit by replacing an
aberrant CaR2 or providing an aberrant CaR2 that provides a
therapeutic result. In one embodiment, the cells provide CaR2 that
are abnormally active.
[0348] In another embodiment, the cells provide CaR2 that is
abnormally inactive. This CaR2 can compete with endogenous CaR2 in
the individual.
[0349] In a related embodiment, the cell of the invention can
produce abnormally low levels of CaR2. This can be done, for
example, by a method called RNA interference (RNAi). The best
developed RNAi method is one that employs the siRNA technology
developed by Tuschl, et al. The siRNA technique is a method of post
translational gene silencing that is initiated by double stranded
RNA that is homologous to the sequence of the gene to be silenced.
The siRNA methodology is described in Elbashir, S. M., et al.
(2001) Nature 411:494-8 and Elbashir, S. M., et al. (2001) EMBO J.
3:6877-88. siRNAs have been used, for example, to silence genes in
Xenopus embryos (Zhou, Y. et al. (2002) Nucleic Acids Res.
30:1664-9) and to silence human tissue factor expression (Holen, T,
et al. (2002) Nucleic Acids Res. 30:1757-66).
[0350] In another embodiment, cells expressing CaR2 that cannot be
activated are introduced into an individual in order to compete
with endogenous CaR2.
[0351] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous CaR2 polynucleotide
sequences in a host cell genome. The host cell includes, but is not
limited to, a stable cell line, cell in vivo, or cloned
microorganism. This technology is more fully described in WO
93/09222, WO 91/12650, WO 91/06667, U.S. Pat. No. 5,272,071, and
U.S. Pat. No. 5,641,670. Briefly, specific polynucleotide sequences
corresponding to the CaR2 polynucleotides or sequences proximal or
distal to a CaR2 gene are allowed to integrate into a host cell
genome by homologous recombination where expression of the gene can
be affected. In one embodiment, regulatory sequences are introduced
that either increase or decrease expression of an endogenous
sequence. Accordingly, a CaR2 protein can be produced in a cell not
normally producing it. Alternatively, increased expression of CaR2
protein can be effected in a cell normally producing the protein at
a specific level. Further, expression can be decreased or
eliminated by introducing a specific regulatory sequence. The
regulatory sequence can be heterologous to the CaR2 protein
sequence or can be a homologous sequence with a desired mutation
that affects expression. Alternatively, the entire gene can be
deleted. The regulatory sequence can be specific to the host cell
or capable of functioning in more than one cell type. Still
further, specific mutations can be introduced into any desired
region of the gene to produce mutant CaR2 proteins. Such mutations
could be introduced, for example, into the specific functional
regions such as the OC, calcium or G-protein binding site.
[0352] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered CaR2 gene. Alternatively, the host
cell can be a stem cell or other early tissue precursor that gives
rise to a specific subset of cells and can be used to produce
transgenic tissues in an animal. See also Thomas et al., Cell
51:503 (1987) 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 gene
has homologously recombined with the endogenous CaR2e gene is
selected (see e.g., Li, E. 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, A. in
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed. (IRL, Oxford, 1987) 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, A. (1991) Current Opinion in Biotechnology 2:823-829 and
in PCT International Publication Nos. WO 90/11354; WO 91/01140; and
WO 93/04169.
[0353] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. A transgene
is exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of a CaR2 protein and identifying and evaluating
modulators of CaR2 protein activity.
[0354] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0355] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which CaR2 polynucleotide sequences have
been introduced.
[0356] A transgenic animal can be produced by introducing 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. Any of the CaR2
nucleotide sequences can be introduced as a transgene into the
genome of a non-human animal, such as a mouse.
[0357] Animal based models for studying cancer in vivo are well
known in the art (reviewed in Animal Models of Cancer
Predisposition Syndromes, Hiai, H. and Hino, O. (eds.) 1999,
Progress in Experimental Tumor Research, Vol. 35; Clarke, A. R.
(2000) Carcinogenesis 21:435-41) and include, for example,
carcinogen-induced tumors (Rithidech, K. et al. (1999) Mutat. Res.
428:33-39; Miller, M. L. et al. (2000) Environ. Mol. Mutagen.
35:319-327), injection and/or transplantation of tumor cells into
an animal, as well as animals bearing mutations in growth
regulatory genes, for example, oncogenes (e.g., ras) (Arbeit, J. M.
et al. (1993) Am. J. Pathol. 142:1187-1197; Sinn, E. et al. (1987)
Cell 49:465-475; Thorgeirsson, S S et al. (2000) Toxicol. Lett.
112-113:553-555) and tumor suppressor genes (e.g., p 53) (Vooijs,
M. et al. (1999) Oncogene 18:5293-5303; Clark A. R. (1995) Cancer
Metast. Rev. 14:125-148; Kumar, T. R. et al. (1995) J. Intern. Med.
238:233-238; Donehower, L. A. et al. (1992) Nature 356215-221).
Furthermore, experimental model systems are available for the study
of, for example, ovarian cancer (Hamilton, T. C. et al. (1984)
Semin. Oncol. 11:285-298; Rahman, N. A. et al. (1998) Mol. Cell.
Endocrinol. 145:167-174; Beamer, W. G. et al. (1998) Toxicol.
Pathol. 26:704-710), gastric cancer (Thompson, J. et al. (2000)
Int. J. Cancer 86:863-869; Fodde, R. et al. (1999) Cytogenet. Cell
Genet. 86:105-111), breast cancer (Li, M. et al. (2000) Oncogene
19:1010-1019; Green, J. E. et al. (2000) Oncogene 19:1020-1027),
melanoma (Satyamoorthy, K. et al. (1999) Cancer Metast. Rev.
18:401-405); lung cancer (Malkinson, A. M. (2001) Lung Cancer
32(3):265-79; Zhao, B. et al. (2001) Exp. Lung Res. 26(8):567-79);
colon cancer (Taketo, M. M. and Takaku (2000) Hum. Cell
13(3):85-95; Fodde, R. and Smits, R. (2001) Trends. Mol. Med.
7(8):369-73); and prostate cancer (Shirai, T. et al. (2000) Mutat.
Res. 462:219-226; Bostwick, D. G. et al. (2000) Prostate
43:286-294).
[0358] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the CaR2
protein to particular cells.
[0359] 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 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic 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 can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0360] In another embodiment, transgenic non-human animals can be
produced which contain selected systems, which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage PI. 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 S. cerevisiae (O'Gorman et al. (1991)
Science 251:1351-1355. 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 is
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.
[0361] 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 and PCT International Publication
Nos. WO 97/07668 and WO 97/07669. 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 G.sub.o 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 blastocyst and then
transferred to a pseudopregnant female foster animal. The offspring
born of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[0362] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, it is useful
to provide non-human transgenic animals to assay in vivo CaR2
function, including cAMP interaction, the effect of specific mutant
CaR2 on CaR2 function and cAMP interaction, and the effect of
chimeric CaR2. It is also possible to assess the effect of null
mutations, that is mutations that substantially or completely
eliminate one or more CaR2 functions.
[0363] In general, methods for producing transgenic animals include
introducing a nucleic acid sequence according to the present
invention, the nucleic acid sequence capable of expressing the
protein in a transgenic animal, into a cell in culture or in vivo.
When introduced in vivo, the nucleic acid is introduced into an
intact organism such that one or more cell types and, accordingly,
one or more tissue types, express the nucleic acid encoding the
protein. Alternatively, the nucleic acid can be introduced into
virtually all cells in an organism by transfecting a cell in
culture, such as an embryonic stem cell, as described herein for
the production of transgenic animals, and this cell can be used to
produce an entire transgenic organism. As described, in a further
embodiment, the host cell can be a fertilized oocyte. Such cells
are then allowed to develop in a female foster animal to produce
the transgenic organism.
V. Methods of Using CaR2 and OC Modulators
[0364] The identification of CaR2 as a high concentration calcium
sensor that is expressed in the epithelial ducts in kidney and
prostate, as well as in bone tissue, allows for predictions of the
physiological consequences expected to result from CaR2 hypo- and
hyper-activity. Normocalcemia in mammals and other tetrapods is
believed to be maintained primarily through actions of CaR.
Specific activating and inactivating mutations in CaR lead to
hyper- and hypocalcaemic disease states. In analogy with CaR, such
mutations in CaR2 would be expected to strongly influence its
specific microenvironments, such as kidney and bone. In kidney,
CaR2 is expected to respond to the Ca.sup.++ concentration in
fluids passing through the kidney tubules, resulting in either
reabsorbtion of Ca.sup.++ or its secretion in urine. Dysregulation
could lead to excessive calcium loss or the formation of kidney
stones. Similarly, osteoblast CaR2 activation might be expected to
affect bone formation and bone mass. Accordingly, using drugs to
modulate CaR2 activity may have therapeutic benefit for conditions
such as the conditions disclosed herein.
[0365] Modulators of CaR2 level or activity identified according to
these assays can be used to test the effects of modulation of
expression or activity of the receptor on the outcome of clinically
relevant conditions. This can be accomplished in vitro, in vivo,
such as in human clinical trials, and in test models derived from
other organisms, such as non-human transgenic subjects. Modulation
in such subjects includes, but is not limited to, modulation of the
cells, tissues, and conditions particularly disclosed herein.
Modulators of CaR2 activity identified according to these drug
screening assays can be used to treat a subject with a disorder
mediated by CaR2 or OC, by treating cells that express the calcium
receptor, such as those disclosed herein. In one embodiment, the
cells that are treated are derived from cancerous tissue or tumors.
Accordingly, conditions in which modulation is particularly
relevant can include the tissues disclosed herein. These methods of
treatment include the steps of administering the modulators of CaR2
activity or OC binding in a pharmaceutical composition as described
herein, to a subject in need of such treatment.
[0366] The invention thus provides methods for treating a disorder
characterized by aberrant expression or activity of a calcium
receptor, e.g., CaR2 or OC. 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) expression or activity of the
proteins. In another embodiment, the method involves administering
the calcium receptor or OC as therapy to compensate for reduced or
aberrant expression or activity of the proteins.
[0367] Methods for treatment include but are not limited to the use
of soluble CaR2 or OC or fragments of CaR2 or OC that compete for
ligand. CaR2, OC, or fragments thereof, can have a higher affinity
for the target so as to provide effective competition.
[0368] Stimulation of activity is desirable in situations in which
the protein is abnormally downregulated and/or in which increased
activity is likely to have a beneficial effect. Likewise,
inhibition of activity is desirable in situations in which the
protein is abnormally upregulated and/or in which decreased
activity is likely to have a beneficial effect. In one example of
such a situation, a subject has a disorder characterized by
metastasis of cancer to a bone, or the formation of kidney stones.
In another example, the subject has osteoporosis.
[0369] In another aspect, the invention pertains to using CaR2
modulators to treat subjects that have aberrant expression of OC.
Individuals who express aberrant levels, or mutated, OC can be
treated using modulators of CaR2 disclosed herein.
[0370] Pharmaceutical Compositions
[0371] The invention encompasses use of the polypeptides, nucleic
acids, and other agents in pharmaceutical compositions to
administer to the cells in which expression of the calcium receptor
(CaR2) or OC is relevant and in conditions as disclosed herein.
Uses are both diagnostic and therapeutic. The CaR2 and OC nucleic
acid molecules, protein, modulators of the protein, and antibodies
(also referred to herein as "active compounds") can be incorporated
into pharmaceutical compositions suitable for administration to a
subject, e.g., a human. Such compositions typically comprise the
nucleic acid molecule, protein, modulator, or antibody and a
pharmaceutically acceptable carrier. It is understood however, that
administration can also be to cells in vitro as well as to in vivo
model systems such as non-human transgenic animals.
[0372] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo as by transcription or translation, in
vivo, of polynucleotides that have been exogenously introduced into
a subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[0373] As used herein the language "pharmaceutically acceptable
carrier" 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. 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, such media can be used in the compositions of the
invention. 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 bisulfite; 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. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampules, disposable
syringes or multiple dose vials made of glass or plastic.
[0374] 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 carriers 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
syringability 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, 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.
[0375] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a CaR2 protein or
anti-CaR2 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 compound into
a sterile vehicle which 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, the preferred methods of preparation are vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0376] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tab lets, troches, or capsules. Oral compositions can
also be prepared using a fluid carrier for use as a mouthwash,
wherein the compound in the fluid carrier 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.
[0377] 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.
[0378] Systemic administration can also be by transmucosal or
transdermal means. For 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.
[0379] 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.
[0380] In one embodiment, the active compounds are prepared with
carriers that will protect 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.
[0381] 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 carrier. 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, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0382] 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, for example, intravenous
injection, local administration (U.S. Pat. No. 5,328,470) or by
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 which produce the gene delivery system. The
pharmaceutical compositions can be included in a container, pack,
or dispenser together with instructions for administration.
[0383] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
[0384] The skilled artisan will appreciate that certain factors may
influence the dosage required to effectively treat a subject,
including but not limited to the severity of the condition,
previous treatments, the general health and/or age of the subject,
and other conditions present. Moreover, treatment of a subject with
a therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments. In a preferred example, a subject is
treated with antibody, protein, or polypeptide in the range of
between about 0.1 to 20 mg/kg body weight, one time per week for
between about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. It will also be appreciated that the
effective dosage of antibody, protein, or polypeptide used for
treatment may increase or decrease over the course of a particular
treatment. Changes in dosage may result and become apparent from
the results of diagnostic assays as described herein.
[0385] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds.
[0386] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the ken of the
ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention. Exemplary doses
include milligram or microgram amounts of the small molecule per
kilogram of subject or sample weight (e.g., about 1 microgram per
kilogram to about 500 milligrams per kilogram, about 100 micrograms
per kilogram to about 5 milligrams per kilogram, or about 1
microgram per kilogram to about 50 micrograms per kilogram. It is
furthermore understood that appropriate doses of a small molecule
depend upon the potency of the small molecule with respect to the
expression or activity to be modulated. Such appropriate doses may
be determined using the assays described herein. When one or more
of these small molecules is to be administered to an animal (e.g.,
a human) in order to modulate expression or activity of a
polypeptide or nucleic acid of the invention, a physician,
veterinarian, or researcher may, for example, prescribe a
relatively low dose at first, subsequently increasing the dose
until an appropriate response is obtained. In addition, it is
understood that the specific dose level for any particular animal
subject will depend upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, gender, and diet of the subject, the time of
administration, the route of administration, the rate of excretion,
any drug combination, and the degree of expression or activity to
be modulated.
[0387] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate CaR2 nucleic
acid expression. Modulation includes both up-regulation (i.e.
activation or agonization) or down-regulation (suppression or
antagonization) or effects on nucleic acid activity (e.g. when
nucleic acid is mutated or improperly modified). Conditions
characterized by aberrant expression or activity of the nucleic
acid can be treated.
[0388] The gene is particularly relevant for the treatment of
conditions involving the cells and tissues that differentially
express CaR2, cells that are involved in cell proliferative
conditions, and metastisis of those cells, and cells and tissues
that are involved in conditions such as the conditions disclosed
herein.
[0389] Alternatively, a modulator for CaR2 nucleic acid expression
can be a small molecule or drug identified using the screening
assays described herein as long as the drug or small molecule
inhibits the CaR2 nucleic acid expression.
Computer Readable Means
[0390] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[0391] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[0392] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[0393] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0394] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0395] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0396] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0397] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software includes, but is not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0398] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
[0399] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference. Those skilled in the art will
understand that this invention may be embodied in many different
forms and should not be construed as limited to the embodiments set
forth herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
EXAMPLES
Example 1
Identification and Characterization of a Novel Family C Group II
G-Protein Coupled Receptor
[0400] RAGE (Random Activation of Gene Expression) libraries were
created and screened for novel sequences as previously described.
From the resulting gene sequence database, one RAGE sequence-tagged
product (167B12) was predicted to encode a partial protein that
showed similarity to the extracellular ligand-binding domain of the
metabotropic neurotransmitter receptor family C subfamily of GPCRs.
167B12, renamed CaR2, was found to be distributed over two exons of
a novel gene located on chromosome 6 and molecular acquisition of
the full-length cDNA led to the identification of the six coding
exons. This cDNA contains a deduced open reading frame encoding a
926 amino acid protein (SEQ ID NO:2).
Example 2
CaR2 Protein Structure
[0401] CaR2 has the typical protein structure of members of the
family C subfamily of GPCRs; a large N-terminal extracellular
domain (ECD) and a C-terminal seven transmembrane (7.TM.) domain
(for a review see, Hermans E. & Challiss R A (2001) Biochem J
359 465-484). Pfam analysis identifies two structural domains; an
ANF domain in the ECD that indicates the region of the protein that
is similar to the bacterial periplasmic binding proteins and the
7.TM. domain. Sequence comparisons indicate that CaR2 is a Group I
or Group II receptor (identity to human metabotropic glutamate
receptor is 27%, human calcium-sensing receptor is 32%, and human
GABA.sub.B receptor is less than 5%). The highest overall identity
(43%) was observed to a goldfish (Cassius auratus) receptor, termed
5.24. Receptor 5.24 functionally responds to positively charged
amino acids and is classified as a fish odorant receptor (Speca D J
et al. (1999) Neuron 23, 487-498). Recently, the CaR2/167B12 gene
was entered into the public protein database at NCBI and annotated
as the human homolog of the Goldfish receptor 5.24 gene
(XP.sub.--069224). This designation was based on bioinformatic and
not functional considerations. As discussed below, the data
indicate that receptor 5.24 is not the Goldfish ortholog of CaR2.
CaR2 is not activated by the amino acids that activate receptor
5.24 and secondarily, the expression pattern determined for
receptor 5.24 (olfactory epithelium but not kidney, liver, brain,
muscle, ovary, intestine and testis) is qualitatively different
from the CaR2 expression pattern.
Example 3
CaR2 mRNA Expression Profiling
[0402] To determine the tissue distribution of CaR2, an RT-PCR
based expression profiling experiment was performed with primer
pairs targeted to the 5'-end of the CaR2 mRNA, using first strand
cDNA corresponding to twenty major organ systems. The results of
this experiment indicated that CaR2 mRNA was produced in kidney,
prostate, adrenal gland, salivary gland, testis, thymus, thyroid
gland and trachea. RNA samples were treated with DNAse I (RNAse
free) before use in first strand cDNA synthesis. A negative control
(no Reverse Transcriptase) was included for all RNA samples.
Following first strand cDNA synthesis the reaction mix was diluted
from 20 .mu.l to 100 .mu.l. 1 .mu.l of this mix was used per
expression profiling PCR. In the first PCR reaction primers PP
315-PP 321 were utilized using Advantage 2 in a 50 .mu.l volume
with 36 cycles of PCR. 10 .mu.l of this sample was analyzed on an
agarose gel which invariably produced no visible fragments.
Subsequently, 1 .mu.l of this sample was diluted into 100 .mu.l of
water and a second round of PCR was performed using PP 315-PP 320.
This time 28 cycles of PCR were performed. 10 .mu.l of this sample
was analyzed on an agarose gel and tissues were scored as positive
or negative based on the pattern observed. A control PCR was
performed using .beta.-actin primers which produced a robust
positive signal in plus Reverse transcriptase sample in a single
round of 30 cycles.
[0403] Immunohistochemical studies performed with antibodies
developed to this receptor indicated that CaR2 is most highly
expressed in kidney, prostate and osteoblasts.
Example 4
Immunohistochemical Profiling of CaR2
[0404] Antibodies were prepared to peptides corresponding to
20-amino acid regions within the amino-terminal extracellular
domain of CaR2, and these were used to study the expression of the
receptor. Anti-CaR2 antibodies were prepared using the
extracellular N-terminus domain of CaR2. Potential antigenic
sequences were compared with sequences from mouse to ensure 100%
homology. Two sequences were identified as potential antigenic
epitopes, and synthetic peptides were made for these sequences and
conjugated to KLH. The peptide sequences were: (1) [100]
SKFNCSRETVEFKCDYSSYM and (2) [473] MAEYDLQNDVFIIPDQETKN. The
peptides were injected in rabbits with complete Freund's adjuvant,
boosted twice in incomplete Freund's adjuvant and bled at 6 and 8
weeks. The rabbits were boosted again and bled at 10 and 12 weeks.
Antisera were titered by enzyme-linked immunosorbent assay
(ELISA).
[0405] The receptor was immunohistochemically localized in bone
(osteoblasts), kidney tubules, prostate, cerebellum, and
hippocampus.
Example 5
Identification of CaR2 Ligands
[0406] To facilitate pharmacological experimentation, CaR2 cDNA was
cloned into the mammalian expression vector pcDNA 3.1 (+) Neo with
or without a COOH-terminal FLAG epitope. The transgene was
introduced into the HEK293 cell line and stable transfectants were
identified. These clones were analyzed for CaR2 expression by qPCR
and Western blotting to identify those clones that were suitable
for ligand identification. The clone HEK-167B12-2.1 was chosen for
further experimentation.
[0407] Based on the homology to the characterized goldfish odorant
receptor 5.24, initial studies investigated the ability of 18
naturally occurring amino acids to trigger changes of intracellular
Ca.sup.++ levels in the transfected cells, as measured with a
fluorescent imaging plate reader (FLIPR).
[0408] FLIPR Assay
[0409] Intracellular Ca.sup.++ was measured using a fluorometric
imaging plate reader (FLIPR) [Molecular Devices]. Cells were seeded
at a density of 1.times.10.sup.5/well (96-well plate) or
1.times.10.sup.4/well (384-well plate) in collagen coated plates
and incubated overnight at 36.9.degree. C. with 5% CO.sub.2. Medium
was aspirated from the plates and replaced with equal volumes of a
no wash calcium dye (Molecular Devices) and modified Hank's
buffered saline solution [Ca.sup.2+-free, Mg.sup.2+-free] (140 mM
NaCl, 5.4 mM KCl, 0.64 mM KH.sub.2PO.sub.4, 3 mM NaHCO.sub.3, 5.5
mM C.sub.6H.sub.12O.sub.6, 20 mM HEPES, 2.5 mM Caprylic acid (or
2.5 mM probenecid)). The plates were incubated for 1 hour at
36.9.degree. C. with 5% CO.sub.2. FLIPR was used to measure changes
in intracellular calcium as relative fluorescence upon activation
by ligand.
[0410] None of the potential ligands caused a measurable effect,
suggesting either that these molecules were not binding to 167B 12
or that activation of the receptor did not elicit changes in
intracellular Ca.sup.++. Additional ligands that might activate a
family C group I/II receptor (NMDA, Ca.sup.++.sub.0 and GABA) were
tested next but also failed to elicit a positive FLIPR response.
Upon re-examination of the expression profiling results it was
realized that at least one 167B12 microenvironment, the residence
of osteoblasts in bone, had a demonstrated high Ca.sup.++.sub.0.
Also, the apical side of cells lining tubules in Henle's loop in
kidney and in epithelial cells lining prostate ducts are
microenvironments where a higher than standard extracellular
Ca.sup.++ can be expected. These deductions led to the further
testing of Ca.sup.++ as the activating ligand and showed that high
calcium levels (>20 mM) caused a marked increase in fluorescence
signal, and hence intracellular Ca.sup.++, in the clone expressing
CaR2 (FIG. 2). This effect was not observed in the parental cell
line, indicating that CaR2 acts as a low-affinity Ca.sup.++
receptor with pharmacology that is profoundly distinct from CaR as
evidenced by an EC.sub.50 of 85 mM for CaR2 as compared to an
EC.sub.50 of 4.1 mM for CaR in transfected HEK 293 cells.
[0411] Having shown that high calcium levels activate CaR2, the
effects of several agents known to modulate bone metabolism were
determined. In the presence of OC (OC), CaR2 was activated at lower
calcium levels and the activation by high mM calcium was
potentiated (FIG. 3). This synergistic effect is only witnessed
when calcium is pre-incubated with OC, but not when both ligands
are added separately. Moreover, the effect was not observed when OC
and Ca.sup.++ were added to the parental HEK293 cells that do not
express CaR2 or to HEK293 cells that express the previously
characterized CaR.
[0412] When the Ca.sup.++ concentration is held at a fixed value,
there is a dose-dependent effect of OC, with the apparent EC.sub.50
value for OC being between 1 nM and 10 nM (FIG. 4). The effect of
combined OC and Ca.sup.++ on receptor activation is blocked in the
presence of Pb.sup.++, which is known to prevent the formation of
OC/Ca.sup.++ complexes. These data suggest that CaR2 can bind free
Ca.sup.++ when the concentrations exceed .about.20 mM. Moreover,
the receptor can bind OC/Ca.sup.++ complexes.
[0413] In the absence of calcium, the combination of bone
morphogenic peptide (BMP-2) and calcitonin (CT) causes a modest
increase in intracellular Ca.sup.++ in the CaR2 clone, and not in
the parental cells (FIG. 5). This effect is not seen when either
ligand is added alone. The increased fluorescent signal is delayed
relative to that seen when CaR2 is triggered by Ca.sup.++,
suggesting that CaR2 is being activated by an unknown mechanism in
response to CT and BMP-2 binding to their cognate receptors.
Example 6
Osteoblast Survival in High mM Ca.sup.2++
[0414] Saos-2 osteoblasts (ATCC-HTB-85) express CaR2 as determined
by Western Blotting. Whereas these cells grew in buffer containing
2 mM Ca.sup.++, they did not proliferate in medium containing 10 mM
Ca.sup.++ concentration and survived only a couple of days in
culture before dying. In contrast, the Saos-2 cells survived and
proliferated normally in medium containing 40 mM Ca.sup.2+. These
data suggest that activation of CaR2 by high calcium concentrations
promotes osteoblast proliferation, and is consistent with the high
calcium concentration found in the bone matrix.
Example 7
Physiology of CaR2
[0415] The characterization of a novel GPCR that is functionally
activated by elevated concentrations of Ca.sup.++.sub.0 and
physically located in the cellular microenvironments where high
Ca.sup.++.sub.0 is expected (kidney epithelial cells lining tubules
in the thick ascending limb of Henle's loop and in the skeletal
osteoblasts) suggests that CaR2 is responsible for regulation of
Ca.sup.++.sub.0 in these extreme Ca.sup.++ environments.
[0416] This analysis suggests that OC may function as a signal to
help regulate aspects of bone remodeling and mineralization that
are mediated by CaR2. The fact that OC is an active participant in
stimulating CaR2 is shown by the ability of OC to potentiate the
activation of CaR2 in the presence of Ca.sup.++. The existence of
newly synthesized and catabolically generated soluble OC in
remodeling bone matrix suggests that OC could be the proximal
signal detected by CaR2 to integrate osteoblast function with the
process of bone remodeling and mineralization. This premise is
supported by data showing a significant linkage between
osteoporosis and both the OC and vitamin D receptor genes (Deng H
W, et al. (2002) J Bone Miner Res (4):678-86).
Example 8
CaR2 and Disease
[0417] CaR2 and OC expressing osteoblastic cells have a survival
and proliferative advantage when challenged with the high
concentration of free Ca++ that activates CaR2 and that is normally
present in mineralizing bone.
[0418] In support of the relevance of CaR2 activity to conditions
disclosed herein, some genetic predisposition to diseases of bone
and kidney, as well as to cancer, have been mapped to human
chromosome 6q22 in the vicinity of CaR2. Mapped diseases include
two distinct bone dysplasias, craniometaphyseal dysplasia and
oculodentodigital dysplasia, both of which map to this region of
chromosome 6. In addition, a genetic cause for IgA nephropathy, a
form of degenerative kidney disease, also maps to human chromosome
6q22-q23. A major factor in the progression of this disease is
thought to be defects in the function of uteroglobin, a gene that
is normally expressed in lung and that maps to chromosome 11.
Uteroglobin hypomorphic mutations have been shown to promote the
glomerular protein deposits that are a major part of IgA
nephropathy. However, the genetic predisposition to this disease
also maps to 6q, and an alteration of CaR2 function in the kidney
may modify the progression of IgA nephropathy by altering kidney
Ca.sup.++ homeostasis and the ability of specialized kidney cells
to survive or function.
[0419] Other human diseases have been mapped less precisely to the
general region of chromosome 6q. Relevant diseases fall into three
groups: diseases of bone, parathyroid and calcium homeostasis;
factors affecting the development and progression of prostate,
renal and bone cancer; and factors affecting the incidence of a
variety of leukemias. Association of one or more of these diseases
with CaR2 is plausible based upon overlap of disease pathology with
CaR2 tissue distribution, and the demonstration of a calcium link
to some of these diseases.
[0420] Genes for calcium receptors have been implicated in the
development of hyperparathyroidism and parathyroid tumors, and
factors increasing the propensity to develop primary
hyperparathyroidism and parathyroid tumors have been mapped to 6q.
Another genetic syndrome with a potential calcium link that has
also been mapped to 6q is the Shwachman-Diamond syndrome. This
disease is characterized by exocrine pancreatic insufficiency and
hematological and skeletal abnormalities. Factors that promote the
development and progression of prostate, renal and bone and blood
cancers have also been mapped to 6q, and the fact that CaR2 is
normally expressed in many of these tissues and might promote cell
proliferation in high calcium environments suggests that CaR2 might
play at least a supportive role in the development of these
cancers.
[0421] It is not expected that these mapped diseases will represent
a complete list of all 6q and 6q22 genes that are important in
determining bone, prostate and kidney biology, cancer metastatic
tissue tropism, and calcium homeostasis. Genes relevant to these
processes may have been missed because the current mapping of human
diseases has not yet been completed.
[0422] As mentioned, CaR2 may modify the progression of renal,
prostate and bone cancer. In addition, CaR2 may influence the
tissue tropism of metastases of these and other cancers. Breast
cancers also often undergo metastasis to bone. If CaR2 does indeed
play a role in promoting the colonization of bone by primary cancer
cells from the prostate and kidney, then CaR2 expression in breast
cancer cells may also promote the ability of these cells to
proliferate in bone. In light of the bone tropism of breast
cancers, it is interesting to consider that lactating mammary gland
epithelium is an additional high calcium environment that might
require a high threshold calcium receptor to regulate milk
Ca.sup.++ concentration. Although the induction of CaR2 expression
during lactation has not yet been tested in lactating mammary
gland, calcium concentration in human and other mammalian milks
ranges from 8-40 mM, a range that is within the physiological
detection limits of CaR2. Since Ca.sup.++ concentration in milk
seems to be regulated during lactation, CaR2 is an ideal candidate
to help maintain appropriate Ca.sup.++ levels, and the hypothesized
presence of CaR2 in lactating mammary cells could link the
mechanism driving the skeletal metastases of breast with those of
prostate and kidney.
Sequence CWU 1
1
4 1 3120 DNA Homo sapiens CDS (147)...(2927) 1 aatactgagt
gtttctggcc tttgacactg tcctatacct tataaggtgt ttacaggtga 60
aataggtgaa ataggaatct tgctggcact ccgtgcactt aatgattcct aagaactcac
120 atgaactgag caaatgagat agaaac atg gca ttc tta att ata cta att
acc 173 Met Ala Phe Leu Ile Ile Leu Ile Thr 1 5 tgc ttt gtg att att
ctt gct act tca cag cct tgc cag acc cct gat 221 Cys Phe Val Ile Ile
Leu Ala Thr Ser Gln Pro Cys Gln Thr Pro Asp 10 15 20 25 gac ttt gtg
gct gcc act tct ccg gga cat atc ata att gga ggt ttg 269 Asp Phe Val
Ala Ala Thr Ser Pro Gly His Ile Ile Ile Gly Gly Leu 30 35 40 ttt
gct att cat gaa aaa atg ttg tcc tca gaa gac tct ccc aga cga 317 Phe
Ala Ile His Glu Lys Met Leu Ser Ser Glu Asp Ser Pro Arg Arg 45 50
55 cca caa atc cag gag tgt gtt ggc ttt gaa ata tca gtt ttt ctt caa
365 Pro Gln Ile Gln Glu Cys Val Gly Phe Glu Ile Ser Val Phe Leu Gln
60 65 70 act ctt gcc atg ata cac agc att gag atg atc aac aat tca
aca ctc 413 Thr Leu Ala Met Ile His Ser Ile Glu Met Ile Asn Asn Ser
Thr Leu 75 80 85 tta tct gga gtc aaa ctg ggg tat gaa atc tat gac
act tgt aca gaa 461 Leu Ser Gly Val Lys Leu Gly Tyr Glu Ile Tyr Asp
Thr Cys Thr Glu 90 95 100 105 gtc aca gtg gca atg gcg gcc act ctg
agg ttt ctt tct aaa ttc aac 509 Val Thr Val Ala Met Ala Ala Thr Leu
Arg Phe Leu Ser Lys Phe Asn 110 115 120 tgc tcc aga gaa act gtg gag
ttt aag tgt gac tat tcc agc tac atg 557 Cys Ser Arg Glu Thr Val Glu
Phe Lys Cys Asp Tyr Ser Ser Tyr Met 125 130 135 cca aga gtt aag gct
gtc ata ggt tct ggg tac tca gaa ata act atg 605 Pro Arg Val Lys Ala
Val Ile Gly Ser Gly Tyr Ser Glu Ile Thr Met 140 145 150 gct gtc tcc
agg atg ttg aat tta cag ctc atg cca cag gtg ggt tat 653 Ala Val Ser
Arg Met Leu Asn Leu Gln Leu Met Pro Gln Val Gly Tyr 155 160 165 gaa
tca act gca gaa atc ctg agt gac aaa att cgc ttt cct tca ttt 701 Glu
Ser Thr Ala Glu Ile Leu Ser Asp Lys Ile Arg Phe Pro Ser Phe 170 175
180 185 tta cgg act gtg ccc agt gac ttc cat caa att aaa gca atg gct
cac 749 Leu Arg Thr Val Pro Ser Asp Phe His Gln Ile Lys Ala Met Ala
His 190 195 200 ctg att cag aaa tct ggt tgg aac tgg att ggc atc ata
acc aca gat 797 Leu Ile Gln Lys Ser Gly Trp Asn Trp Ile Gly Ile Ile
Thr Thr Asp 205 210 215 gat gac tat gga cga ttg gct ctt aac act ttt
ata att cag gct gaa 845 Asp Asp Tyr Gly Arg Leu Ala Leu Asn Thr Phe
Ile Ile Gln Ala Glu 220 225 230 gca aat aac gtg tgc ata gcc ttc aaa
gag gtt ctt cca gcc ttt ctt 893 Ala Asn Asn Val Cys Ile Ala Phe Lys
Glu Val Leu Pro Ala Phe Leu 235 240 245 tca gat aat acc att gaa gtc
aga atc aat cgg aca ctg aag aaa atc 941 Ser Asp Asn Thr Ile Glu Val
Arg Ile Asn Arg Thr Leu Lys Lys Ile 250 255 260 265 att tta gaa gcc
cag gtt aat gtc att gtg gta ttt ctg agg caa ttc 989 Ile Leu Glu Ala
Gln Val Asn Val Ile Val Val Phe Leu Arg Gln Phe 270 275 280 cat gtt
ttt gat ctc ttc aat aaa gcc att gaa atg aat ata aat aag 1037 His
Val Phe Asp Leu Phe Asn Lys Ala Ile Glu Met Asn Ile Asn Lys 285 290
295 atg tgg att gct agt gat aat tgg tca act gcc acc aag att acc acc
1085 Met Trp Ile Ala Ser Asp Asn Trp Ser Thr Ala Thr Lys Ile Thr
Thr 300 305 310 att cct aat gtt aaa aag att ggc aaa gtt gta ggg ttt
gcc ttt aga 1133 Ile Pro Asn Val Lys Lys Ile Gly Lys Val Val Gly
Phe Ala Phe Arg 315 320 325 aga ggg aat ata tcc tct ttc cat tcc ttt
ctt caa aat ctg cac ttg 1181 Arg Gly Asn Ile Ser Ser Phe His Ser
Phe Leu Gln Asn Leu His Leu 330 335 340 345 ctt ccc agt gac agt cac
aaa ctc tta cat gaa tat gcc atg cat tta 1229 Leu Pro Ser Asp Ser
His Lys Leu Leu His Glu Tyr Ala Met His Leu 350 355 360 tct gcc tgc
gca tat gtc aag gac act gat ttg agt caa tgc ata ttc 1277 Ser Ala
Cys Ala Tyr Val Lys Asp Thr Asp Leu Ser Gln Cys Ile Phe 365 370 375
aat cat tct caa agg act ttg gcc tac aag gct aac aag gct ata gaa
1325 Asn His Ser Gln Arg Thr Leu Ala Tyr Lys Ala Asn Lys Ala Ile
Glu 380 385 390 agg aac ttc gtc atg aga aat gac ttc ctc tgg gac tat
gct gag cca 1373 Arg Asn Phe Val Met Arg Asn Asp Phe Leu Trp Asp
Tyr Ala Glu Pro 395 400 405 gga ctc att cat agt att cag ctt gca gtg
ttt gcc ctt ggt tat gcc 1421 Gly Leu Ile His Ser Ile Gln Leu Ala
Val Phe Ala Leu Gly Tyr Ala 410 415 420 425 att cgg gat ctg tgt caa
gct cgt gac tgt cag aac ccc aac gcc ttt 1469 Ile Arg Asp Leu Cys
Gln Ala Arg Asp Cys Gln Asn Pro Asn Ala Phe 430 435 440 caa cca tgg
gag tta ctt ggt gtg cta aaa aat gtg aca ttc act gat 1517 Gln Pro
Trp Glu Leu Leu Gly Val Leu Lys Asn Val Thr Phe Thr Asp 445 450 455
gga tgg aat tca ttt cat ttt gat gct cat ggg gat tta aat act gga
1565 Gly Trp Asn Ser Phe His Phe Asp Ala His Gly Asp Leu Asn Thr
Gly 460 465 470 tat gat gtt gtg ctc tgg aag gag atc aat gga cac atg
act gtc act 1613 Tyr Asp Val Val Leu Trp Lys Glu Ile Asn Gly His
Met Thr Val Thr 475 480 485 aag atg gca gaa tat gac cta cag aat gat
gtc ttc atc atc cca gat 1661 Lys Met Ala Glu Tyr Asp Leu Gln Asn
Asp Val Phe Ile Ile Pro Asp 490 495 500 505 cag gaa aca aaa aat gag
ttc agg aat ctt aag caa att caa tct aaa 1709 Gln Glu Thr Lys Asn
Glu Phe Arg Asn Leu Lys Gln Ile Gln Ser Lys 510 515 520 tgc tcc aag
gaa tgc agt cct ggg caa atg aag aaa act aca aga agt 1757 Cys Ser
Lys Glu Cys Ser Pro Gly Gln Met Lys Lys Thr Thr Arg Ser 525 530 535
caa cac atc tgt tgc tat gaa tgt cag aac tgt cct gaa aat cat tac
1805 Gln His Ile Cys Cys Tyr Glu Cys Gln Asn Cys Pro Glu Asn His
Tyr 540 545 550 act aat cag aca gat atg cct cat tgc ctt tta tgc aac
aac aaa act 1853 Thr Asn Gln Thr Asp Met Pro His Cys Leu Leu Cys
Asn Asn Lys Thr 555 560 565 cac tgg gcc cct gtt agg agc act atg tgc
ttt gaa aag gaa gtg gaa 1901 His Trp Ala Pro Val Arg Ser Thr Met
Cys Phe Glu Lys Glu Val Glu 570 575 580 585 tat ctc aac tgg aat gac
tcc ttg gcc atc cta ctc ctg att ctc tcc 1949 Tyr Leu Asn Trp Asn
Asp Ser Leu Ala Ile Leu Leu Leu Ile Leu Ser 590 595 600 cta ctg gga
atc ata ttt gtt ctg gtt gtt ggc ata ata ttt aca aga 1997 Leu Leu
Gly Ile Ile Phe Val Leu Val Val Gly Ile Ile Phe Thr Arg 605 610 615
aac ctg aac act ccc gtt gtg aaa tca tcc ggg gga tta aga gtc tgc
2045 Asn Leu Asn Thr Pro Val Val Lys Ser Ser Gly Gly Leu Arg Val
Cys 620 625 630 tat gtg atc ctt ctc tgt cat ttc ctc aat ttt gcc agc
acg agc ttt 2093 Tyr Val Ile Leu Leu Cys His Phe Leu Asn Phe Ala
Ser Thr Ser Phe 635 640 645 ttc att gga gaa cca caa gac ttc aca tgt
aaa acc agg cag aca atg 2141 Phe Ile Gly Glu Pro Gln Asp Phe Thr
Cys Lys Thr Arg Gln Thr Met 650 655 660 665 ttt gga gtg agc ttt act
ctt tgc atc tcc tgc att ttg acg aag tct 2189 Phe Gly Val Ser Phe
Thr Leu Cys Ile Ser Cys Ile Leu Thr Lys Ser 670 675 680 ctg aaa att
ttg cta gct ttc agc ttt gat ccc aaa tta cag aaa ttt 2237 Leu Lys
Ile Leu Leu Ala Phe Ser Phe Asp Pro Lys Leu Gln Lys Phe 685 690 695
ctg aag tgc ctc tat aga ccg atc ctt att atc ttc act tgc acg ggc
2285 Leu Lys Cys Leu Tyr Arg Pro Ile Leu Ile Ile Phe Thr Cys Thr
Gly 700 705 710 atc cag gtt gtc att tgc aca ctc tgg cta atc ttt gca
gca cct act 2333 Ile Gln Val Val Ile Cys Thr Leu Trp Leu Ile Phe
Ala Ala Pro Thr 715 720 725 gta gag gtg aat gtc tcc ttg ccc aga gtc
atc atc ctg gag tgt gag 2381 Val Glu Val Asn Val Ser Leu Pro Arg
Val Ile Ile Leu Glu Cys Glu 730 735 740 745 gag gga tcc ata ctt gca
ttt ggc acc atg ctg ggc tac att gcc atc 2429 Glu Gly Ser Ile Leu
Ala Phe Gly Thr Met Leu Gly Tyr Ile Ala Ile 750 755 760 ctg gcc ttc
att tgc ttc ata ttt gct ttc aaa ggc aaa tat gag aat 2477 Leu Ala
Phe Ile Cys Phe Ile Phe Ala Phe Lys Gly Lys Tyr Glu Asn 765 770 775
tac aat gaa gcc aaa ttc att aca ttt ggc atg ctc att tac ttc ata
2525 Tyr Asn Glu Ala Lys Phe Ile Thr Phe Gly Met Leu Ile Tyr Phe
Ile 780 785 790 gct tgg atc aca ttc atc cct atc tat gct acc aca ttt
ggc aaa tat 2573 Ala Trp Ile Thr Phe Ile Pro Ile Tyr Ala Thr Thr
Phe Gly Lys Tyr 795 800 805 gta ccg gct gtg gag att att gtc ata tta
ata tct aac tat gga atc 2621 Val Pro Ala Val Glu Ile Ile Val Ile
Leu Ile Ser Asn Tyr Gly Ile 810 815 820 825 ctg tat tgc aca ttc atc
ccc aaa tgc tat gtt att att tgt aag caa 2669 Leu Tyr Cys Thr Phe
Ile Pro Lys Cys Tyr Val Ile Ile Cys Lys Gln 830 835 840 gag att aac
aca aag tct gcc ttt ctc aag atg atc tac agt tat tct 2717 Glu Ile
Asn Thr Lys Ser Ala Phe Leu Lys Met Ile Tyr Ser Tyr Ser 845 850 855
tcc cat agt gtg agc agc att gcc ctg agt cct gct tca ctg gac tcc
2765 Ser His Ser Val Ser Ser Ile Ala Leu Ser Pro Ala Ser Leu Asp
Ser 860 865 870 atg agc ggc aat gtc aca atg acc aat ccc agc tct agt
ggc aag tca 2813 Met Ser Gly Asn Val Thr Met Thr Asn Pro Ser Ser
Ser Gly Lys Ser 875 880 885 gca acc tgg cag aaa agc aaa gat ctt cag
gca caa gca ttt gca cac 2861 Ala Thr Trp Gln Lys Ser Lys Asp Leu
Gln Ala Gln Ala Phe Ala His 890 895 900 905 ata tgc agg gaa aat gcc
aca agt gta tct aaa act ttg cct cga aaa 2909 Ile Cys Arg Glu Asn
Ala Thr Ser Val Ser Lys Thr Leu Pro Arg Lys 910 915 920 aga atg tca
agt ata tga ataagcctta ggagagatgc cacattccag 2957 Arg Met Ser Ser
Ile * 925 aataaaatgt ttccagggtc tttgcatcta agatataaat ttactttccc
agcaaatatg 3017 tcatatatat ttccttgcca ccatctttac caagttttag
ttgaacagtc actctgttca 3077 atcacctatt taacaaatag aattgagcct
tcagcctgaa gct 3120 2 926 PRT Homo sapiens 2 Met Ala Phe Leu Ile
Ile Leu Ile Thr Cys Phe Val Ile Ile Leu Ala 1 5 10 15 Thr Ser Gln
Pro Cys Gln Thr Pro Asp Asp Phe Val Ala Ala Thr Ser 20 25 30 Pro
Gly His Ile Ile Ile Gly Gly Leu Phe Ala Ile His Glu Lys Met 35 40
45 Leu Ser Ser Glu Asp Ser Pro Arg Arg Pro Gln Ile Gln Glu Cys Val
50 55 60 Gly Phe Glu Ile Ser Val Phe Leu Gln Thr Leu Ala Met Ile
His Ser 65 70 75 80 Ile Glu Met Ile Asn Asn Ser Thr Leu Leu Ser Gly
Val Lys Leu Gly 85 90 95 Tyr Glu Ile Tyr Asp Thr Cys Thr Glu Val
Thr Val Ala Met Ala Ala 100 105 110 Thr Leu Arg Phe Leu Ser Lys Phe
Asn Cys Ser Arg Glu Thr Val Glu 115 120 125 Phe Lys Cys Asp Tyr Ser
Ser Tyr Met Pro Arg Val Lys Ala Val Ile 130 135 140 Gly Ser Gly Tyr
Ser Glu Ile Thr Met Ala Val Ser Arg Met Leu Asn 145 150 155 160 Leu
Gln Leu Met Pro Gln Val Gly Tyr Glu Ser Thr Ala Glu Ile Leu 165 170
175 Ser Asp Lys Ile Arg Phe Pro Ser Phe Leu Arg Thr Val Pro Ser Asp
180 185 190 Phe His Gln Ile Lys Ala Met Ala His Leu Ile Gln Lys Ser
Gly Trp 195 200 205 Asn Trp Ile Gly Ile Ile Thr Thr Asp Asp Asp Tyr
Gly Arg Leu Ala 210 215 220 Leu Asn Thr Phe Ile Ile Gln Ala Glu Ala
Asn Asn Val Cys Ile Ala 225 230 235 240 Phe Lys Glu Val Leu Pro Ala
Phe Leu Ser Asp Asn Thr Ile Glu Val 245 250 255 Arg Ile Asn Arg Thr
Leu Lys Lys Ile Ile Leu Glu Ala Gln Val Asn 260 265 270 Val Ile Val
Val Phe Leu Arg Gln Phe His Val Phe Asp Leu Phe Asn 275 280 285 Lys
Ala Ile Glu Met Asn Ile Asn Lys Met Trp Ile Ala Ser Asp Asn 290 295
300 Trp Ser Thr Ala Thr Lys Ile Thr Thr Ile Pro Asn Val Lys Lys Ile
305 310 315 320 Gly Lys Val Val Gly Phe Ala Phe Arg Arg Gly Asn Ile
Ser Ser Phe 325 330 335 His Ser Phe Leu Gln Asn Leu His Leu Leu Pro
Ser Asp Ser His Lys 340 345 350 Leu Leu His Glu Tyr Ala Met His Leu
Ser Ala Cys Ala Tyr Val Lys 355 360 365 Asp Thr Asp Leu Ser Gln Cys
Ile Phe Asn His Ser Gln Arg Thr Leu 370 375 380 Ala Tyr Lys Ala Asn
Lys Ala Ile Glu Arg Asn Phe Val Met Arg Asn 385 390 395 400 Asp Phe
Leu Trp Asp Tyr Ala Glu Pro Gly Leu Ile His Ser Ile Gln 405 410 415
Leu Ala Val Phe Ala Leu Gly Tyr Ala Ile Arg Asp Leu Cys Gln Ala 420
425 430 Arg Asp Cys Gln Asn Pro Asn Ala Phe Gln Pro Trp Glu Leu Leu
Gly 435 440 445 Val Leu Lys Asn Val Thr Phe Thr Asp Gly Trp Asn Ser
Phe His Phe 450 455 460 Asp Ala His Gly Asp Leu Asn Thr Gly Tyr Asp
Val Val Leu Trp Lys 465 470 475 480 Glu Ile Asn Gly His Met Thr Val
Thr Lys Met Ala Glu Tyr Asp Leu 485 490 495 Gln Asn Asp Val Phe Ile
Ile Pro Asp Gln Glu Thr Lys Asn Glu Phe 500 505 510 Arg Asn Leu Lys
Gln Ile Gln Ser Lys Cys Ser Lys Glu Cys Ser Pro 515 520 525 Gly Gln
Met Lys Lys Thr Thr Arg Ser Gln His Ile Cys Cys Tyr Glu 530 535 540
Cys Gln Asn Cys Pro Glu Asn His Tyr Thr Asn Gln Thr Asp Met Pro 545
550 555 560 His Cys Leu Leu Cys Asn Asn Lys Thr His Trp Ala Pro Val
Arg Ser 565 570 575 Thr Met Cys Phe Glu Lys Glu Val Glu Tyr Leu Asn
Trp Asn Asp Ser 580 585 590 Leu Ala Ile Leu Leu Leu Ile Leu Ser Leu
Leu Gly Ile Ile Phe Val 595 600 605 Leu Val Val Gly Ile Ile Phe Thr
Arg Asn Leu Asn Thr Pro Val Val 610 615 620 Lys Ser Ser Gly Gly Leu
Arg Val Cys Tyr Val Ile Leu Leu Cys His 625 630 635 640 Phe Leu Asn
Phe Ala Ser Thr Ser Phe Phe Ile Gly Glu Pro Gln Asp 645 650 655 Phe
Thr Cys Lys Thr Arg Gln Thr Met Phe Gly Val Ser Phe Thr Leu 660 665
670 Cys Ile Ser Cys Ile Leu Thr Lys Ser Leu Lys Ile Leu Leu Ala Phe
675 680 685 Ser Phe Asp Pro Lys Leu Gln Lys Phe Leu Lys Cys Leu Tyr
Arg Pro 690 695 700 Ile Leu Ile Ile Phe Thr Cys Thr Gly Ile Gln Val
Val Ile Cys Thr 705 710 715 720 Leu Trp Leu Ile Phe Ala Ala Pro Thr
Val Glu Val Asn Val Ser Leu 725 730 735 Pro Arg Val Ile Ile Leu Glu
Cys Glu Glu Gly Ser Ile Leu Ala Phe 740 745 750 Gly Thr Met Leu Gly
Tyr Ile Ala Ile Leu Ala Phe Ile Cys Phe Ile 755 760 765 Phe Ala Phe
Lys Gly Lys Tyr Glu Asn Tyr Asn Glu Ala Lys Phe Ile 770 775 780 Thr
Phe Gly Met Leu Ile Tyr Phe Ile Ala Trp Ile Thr Phe Ile Pro 785 790
795 800 Ile Tyr Ala Thr Thr Phe Gly Lys Tyr Val Pro Ala Val Glu Ile
Ile 805 810 815 Val Ile Leu Ile Ser Asn Tyr Gly Ile Leu Tyr Cys Thr
Phe Ile Pro 820 825 830 Lys Cys Tyr Val Ile Ile Cys Lys Gln Glu Ile
Asn Thr Lys Ser Ala 835 840 845 Phe Leu Lys Met Ile Tyr Ser Tyr Ser
Ser His Ser Val Ser Ser Ile 850 855 860 Ala Leu Ser Pro Ala Ser Leu
Asp Ser Met Ser Gly Asn Val Thr Met 865 870 875 880 Thr Asn
Pro Ser Ser Ser Gly Lys Ser Ala Thr Trp Gln Lys Ser Lys 885 890 895
Asp Leu Gln Ala Gln Ala Phe Ala His Ile Cys Arg Glu Asn Ala Thr 900
905 910 Ser Val Ser Lys Thr Leu Pro Arg Lys Arg Met Ser Ser Ile 915
920 925 3 20 PRT Artificial Sequence synthetic construct 3 Ser Lys
Phe Asn Cys Ser Arg Glu Thr Val Glu Phe Lys Cys Asp Tyr 1 5 10 15
Ser Ser Tyr Met 20 4 20 PRT Artificial Sequence synthetic construct
4 Met Ala Glu Tyr Asp Leu Gln Asn Asp Val Phe Ile Ile Pro Asp Gln 1
5 10 15 Glu Thr Lys Asn 20
* * * * *