Note: Descriptions are shown in the official language in which they were submitted.
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Isoforms of Human Calcium Sensing Receptor
FIELD OF THE INVENTION
4
The present invention relates to isoforms of a human calcium sensing receptor,
and to the genes
encoding these isoforms. The invention further relates to methods of screening
for agonists or antagonists
of the isoforms, particularly with respect to calcium receptor activity, to
diagnostic uses of these isoforms
and to therapeutic uses of the agonists or antagonists. The invention also
relates to gene therapy using the
genes encoding the receptor isoforms or molecules capable of down-regulating
receptor activity, such as
antisense sequences.
BACKGROUND OF THE INVENTION
Calcium is an extracellular messenger (Brown et al. ( 1995) Cell 83:679-682}.
Serum calcium
levels are regulated by 1,25-dihydroxyvitamin D3, parathyroid hormone, and
calcitonin. A calcium
sensing receptor (CaSR) has been identified in bovine parathyroid (WO
94/18959; Brown et al. (1993)
Nature 366: 575-580). Cloning of the cDNA encoding this receptor (CaSRa)
revealed a G-protein-coupled
receptor featuring a large extracellular domain, coupled to a seven membrane
spanning domain similar to
those found in members of the G protein coupled receptor superfamily. This
receptor has been shown to
play a key role in Ca~" homeostatsis through regulation of parathyroid hormone
secretion and renal tubular
calcium reabsorption. This receptor recognizes calcium and other polyvalent
cations and is coupled by
changes in phosphoinositide turnover to the release of calcium from
intracellular stores. In addition to its
abundant expression in parathyroid gland and kidney, full length CaSRa
transcripts have also been found
in brain, thyroid, intestine, bone marrow and keratinocytes. The complete cDNA
sequence encoding the
corresponding human form of CaSRa has recently been reported (Freichel et al.
(1996) Endocrinology
137:3842-3848). This 3234 base pair nucleotide sequence {SEQ ID NO: 11)
encodes a protein having
1078 amino acids (SEQ ID NO: 12). Various forms of the CaSR, particularly from
bone marrow cells, are
also disclosed by House et al. ((1997) J. Bone Min. Res. 12:1959-1970) and in
US patents 5,688,938 and
5,763,569. The presence of calcium receptors in bone, suggests that they are
involved in bone remodeling
{Quarles (1997) J. Bone Min. Res. 12, 1971-1974).
An alternatively spliced form of CaSRa, has been identified in human medullary
thyroid
carcinoma and keratinocytes (see Freichel et al. supra). The medullary thyroid
carcinoma isoform,
designated CaSRb, contains a 307 base pair deletion between nucleotides 186
and 495, corresponding to
exon 2. This deletion results in a reading frame shift and premature
termination at nucleotide 766.
Translation of CaSRb transcript could yield an extracellular portion of the
receptor without the 7
transmembrane anchor and cytosolic tail. Therefore, CaSRb may be a secretory
protein, and, although its
exact function has yet to be determined, by analogy to other known souble
receptors it may play a role in
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modulating the interaction of native CaSR with its cationic ligands.
A second isoform of CaSRa has also been identified in kerotinocytes (Oda et
al. ( I 997) FAS'~B J.
11 (9): A925; Abstract #395). This form (CaSRc) lacks exon 4, encoding a
portion of the extracellular
domain of the receptor including a region of acidic amino acids which may
mediate calcium binding, and
is present in differentiated cells.
However, there is a need in the art to better understand calcium homeostasis.
In particular, there is
a need in the art to better understand calcium regulation through the CaSR.
Isoforms of the wild-type
CaSR could be expected to exhibit different pharmacological profiles and
signaling properties relative to
the wild-type. For example, different isoforms could couple differentially or
uniquely to known or
unknown signalling pathways, including phosphoinositide turnover, calcium
mobilization, protein kinase
C activation, cAMP production, and other ion channel activity. Alternatively,
variant isoforms may
exhibit different cell surface expression, metabolic half life, or
intracellular trafficking, when compared to
the wild-type, or behave in a dominant negative or positive fashion, thereby
over-riding the functionality
of the wild-type receptor expressed in the same tissue.
The present invention addresses the need in the art, as discussed below.
Specifically, Applicants
present evidence that CaSRb is also found in human kidney. Significantly,
Applicants have also identified
in human kidney two other alternatively spliced CaSR transcripts (CaSRc and d)
with deletions from
nucleotides 1378-1608 and 1075 to 1386, respectively.
The citation of any reference herein should not be construed as an admission
that such reference is
available as "Prior Art" to the instant application.
SUMMARY OF THE INVENTION
As noted above, the present invention concerns identification of isoforms of a
human calcium
sensing receptor (CaSR). The present invention reveals the presence of mutiple
alternatively spliced
transcripts of CaSR in human kidney. Two such isoforms identified, CaSRc and d
arise from partial
deletion of the wild type sequence. The nucleotide deletions in CaSRc and d
have no effect on the
reading frame. Thus, CaSRc and d will yield receptor proteins with about 90-
100 amino acid stretchs
deleted from the extracellular domain. These deletions cover a respective
extracellular sequence rich
in acidic residues (approximately 4%). The location and the charge
characteristics of the deleted
sequences in CaSRc and d suggest that these two CaSR splice variants may
exhibit different cation
sensing property from the wild type receptor.
Thus, in a first aspect, the present invention provides isolated nucleic acids
encoding an
isoform of a human calcium sensing receptor, wherein the nucleic acid
comprises about 2922 to about
3003 nucleotides and has a deletion of at least about 231 nucleotides when
compared to the wild-type
form of the receptor as depicted in SEQ ID NO:11. The term "about" or
"approximately" means
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within 20%, preferably within 10%, and more preferably within 5% of a given
value or range.
Preferably, the deletion is in the region encoding the extracellular domain of
the receptor.
In a preferred embodiment of the invention, the deletion is from about
nucleotides 1075-1386
of SEQ ID NO:11. In another preferred embodiment, the deletion is from about
nucleotides 1378-
S 1608 of SEQ ID NO:11. Alternatively, the deletion is from about nucleotides
1075-1608 of SEQ ID
NO:11.
In another embodiment, the isolated nucleic acid of the present invention has
at least one
property selected from:
it can be amplified by polymerase chain reaction (PCR) using an
oligonucleotide
primer derived from SEQ ID N0:7, SEQ ID N0:9 or SEQ ID NO:11;
it hybridizes under stringent conditions with a nucleic acid having a
nucleotide
sequence as depicted in SEQ ID N0:7 or SEQ ID N0:9; and
it encodes a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID N0:8, SEQ ID NO:10, and allelic variants thereof.
Preferably, 'the nucleic acid the invention can be amplified with at least one
oligonucleotide primer
selected from the group consisting of SEQ ID NO:1 and SEQ ID N0:6.
In still another preferred embodiment of the invention, the isolated nucleic
acid encodes a
CaSR isoform comprising an amino acid sequence as depicted in SEQ ID N0:8
(CaSRc) or SEQ ID
NO:10 (CaSRd), or allelic variants thereof. Preferably, the isolated nucleic
acid comprises a
nucleotide sequence as depicted in SEQ ID N0:7 or SEQ ID N0:9, or allelic
variants thereof.
As can be readily appreciated by one of ordinary skill in the art, one
effective way to prepare a
nucleic acid of the invention, particularly a cDNA, is to amplify the nucleic
acid from a cDNA library
comprising a coding sequence for a CasR isofom using PCR. Various PCR primers,
corresponding to any
desired segment from SEQ ID N0:7, SEQ ID N0:9 or SEQ ID NO:11 can be used in
accordance with the
invention. In specific embodiments, infra, PCR primers having the sequences
depicted in SEQ ID NOS:
1-6 were used to amplify and isolate nucleic acids of the invention.
Alternatively, a nucleic acid of the
invention can be isolated or identified with an oligonucleotide probe, e.g.,
of at least 10 bases, which
hybridizes under stringent conditions to a nucleotide having the sequence or
the complementary sequence
depicted in SEQ ID N0:7, SEQ ID N0:9 or SEQ ID NO:11. In a specific aspect,
the oligonucleotide can
be used in a method for detecting genomic DNA (Southern analysis) or
expression of mRNA (Northern
analysis) encoding a CaSR isoform in a cell. In either case, the method
comprises contacting a sample
from the cell with the oligonucleotide which is detectable, e.g., by labeling
with a radioisotope or a
chromophore or fluorophore, and detecting hybridization of the oligonucleotide
with genomic DNA or
mRNA in the sample, wherein detection of hybridization of the oligonucleotide
with genomic DNA
indicates the presence of a gene encoding a CaSR isoform in the genome, and
detection of hybridization
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with mRNA indicates expression of mRNA encoding a CaSR isoform. It is also
possible to use
quantitative methods; e.g., to detect the number of CaSR genes in the genome,
or to detect an increa3e or
decrease in the level of expression of mRNA.
An oligonucleotide of the invention can also be an antisense oligonucleotide,
i.e., one that
binds to mRNA encoding a CaSR isoform and prevents its translation in the
cell. Such an antisense
molecule can be encoded by a vector expressed in the cell, or can be a
synthetic oligonucleotide,
preferably one that includes non-phosphoester bonds so that it is resistant to
intracellular nucleases.
In another aspect, the invention provides a vector comprising nucleic acids
encoding an
isoform of a human calcium sensing receptor. Preferably, the nucleic acid is
operatively associated
with an expression control sequence permitting expression of the receptor in
an expression competent
host cell.
The vector may be an RNA molecule, a plasmid DNA molecule, or a viral vector.
The viral vector
may be a retrovirus, adenovirus, adeno-associated virus, herpes virus, or
vaccinia virus.
In still another aspect, the invention is directed to host cells transfected
with a vector
comprising nucleic acids encoding an isoform of a human calcium sensing
receptor. The host cell
may be a bacterial cell, a yeast cell, or a mammalian cell. The host cells of
the invention can be used
to produce a CaSR isofonm recombinantly. This method comprises culturing the
host cell in culture
medium under conditions permitting expression of the isoform. Therefore,
another aspect of this
invention is a method for expressing an isoform of human calcium sensing
receptor comprising:
culturing the host cell in culture medium under conditions permitting
expression
of the recombinant receptor; and
identifying cells expressing the receptor on their surface.
The invention also provides an isolated isofonm of a human calcium sensing
receptor, wherein
the isoform comprises about 974 to about 1001 amino acids and has a deletion
of at least about 77
amino acids when compared to the wild-type form of the receptor as depicted in
SEQ ID N0:12. The
deletion may be from the extracellular domain of the receptor, such as from
about amino acids 358-
462, about amino acids 460-536 or about amino acids 358-536 of SEQ ID N0:12.
In particular, a
CaSR isoform of the invention comprises an amino acid sequence as depicted in
SEQ ID N0:8 or
SEQ ID NO:10, or allelic variants thereof.
The present invention advantageously provides methods of screening for
molecules that modulate
the activity of CaSR isoforms, and thus calcium levels. In particular, the
invention provides a method of
screening for agonists or antagonists of a CaSR isoform activity, the method
comprising incubating a test
sample with a CaSR isoform, measuring CaSR isoform activity and comparing the
activity to that in the
absence of the test sample. Any of the screening methods in the art can be
used, particularly high
throughput screening. In a specific embodiment, the method comprises screening
compounds for their
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ability to influence intracellular calcium concentration. The compounds may be
tested alone, in
conjunction with an elevation in extracellular calcium concentration, or in
the presence of other agoflists
or antagonists of CaSR isoform activity. Intracellular calcium can be measured
with a fluorescent
indicator, such as fura-2. Screening methods of the invention permit the
identification of CaSR agonists
(calcimimetics} or antagonists (calcilytics).
In yet a further embodiment, the present invention provides pharmaceutical
compositions and
methods for the treatment of a patient suffering from a disease or disorder
associated with abnormal
calcium levels, such as in the plasma, by the administration of a
therapeutically effective amount of a
compound capable of modulating the activity of a CaSR isoform. The compound
may be specific for a
CaSR isoform. Such diseases include, for example, hyperparathyroidism (primary
and secondary) and
osteoporosis. Other diseases include Paget's disease, hypercalcemia
malignancy, and hypertension. The
compound may be a calcimimetic or calcilytic identified using the screening
assays of the invention.
Alternatively, the disease or disorder is treated using gene therapy.
In one embodiment, the cells of a patient have been transfected with a vector
encoding a CaSR
isoform under conditions permitting expression of the isoform.
Alternatively, where desired, the invention provides a method of inhibiting
CaSR activity in a
patient's cell comprising decreasing the level of CaSR in the cell. The level
of CaSR protein can be
decreased by introducing a CaSR antisense nucleic acid into the cell, which
antisense nucleic acid
hybridizes under intracellular conditions to a CaSR mRNA. Alternatively, the
level of CaSR protein can
be decreased by introducing a single chain Fv antibody (scFv) that
specifically binds a CaSR isoform, or
nucleic acid encoding and intracellular antibody against the isoform, into the
cell at a level sufficient to
bind to and inactivate the CaSR.
Yet another object of the invention is to provide for high level expression of
CaSR isoforms,
either by fermentation of transfected or transduced cells to recover purified
protein, or in vivo in cells for
further testing in vitro or for regulation of calcium homeostasis in vivo,
e.g., for gene therapy.
A particular object of the invention is to provide for screening of small
molecule modulators, e.g.,
agonists and antagonists, of CaSR activity, particularly of specific CaSR
isoforms.
These and other objects are addressed by this invention, which is explained in
greater detail in the
attached drawings and the following Detailed Description and Examples.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE l . Diagram of the structures of the wild-type calcium sensing receptor
(CaSRa), splice
variant CaSRd (lacking amino acids encoded by nucleotides 1075-1386), and
splice variant CaSRc
(lacking amino acids encoded by nucleotides 1378-1608).
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DETAILED DESCRIPTION OF THE INVENTION
This invention is based, in part, on the identification of isoforms of a human
calcium sensing~
receptor, termed herein CaSR.
The invention accordingly relates to nucleic acids encoding CaSR isoforms, to
the purified
protein, to cells which express nucleic acids encoding isoforms of CaSR, in
particular splicing variants,
and to their use in screening for small molecules or natural products, which
agonize or antagonize the
activity of the CaSR.
The invention can also be used for the treatment of diseases or disorders
associated with an
abnormal level of calcium, including gene therapy applications (both coding
and antisense molecules can
be of use).
In addition, anti-CaSR antibodies can be used in diagnostic and purification
applications.
These and other aspects of the invention, particularly isolation of CaSR
genes, expression of CaSR
protein, generation of anti-CaSR antibodies, screening assays for agonists or
antagonists of CaSR activity
and delivery of CaSR encoding vectors, in particular for gene therapy
applications, are discussed in detail
in the following sections. Section headers are provided merely for the
reader's convenience, and are not to
be deemed limiting in any respect.
Genes Encoding Calcium Sensor Receptor Isoforms
The present invention contemplates isolation of genes encoding isoforms of a
human calcium
sensing receptor. As used herein, the term "gene" refers to an assembly of
nucleotides that encode a
polypeptide, and includes cDNA and genomic DNA nucleic acids.
In accordance with the present invention there may be employed conventional
molecular biology,
microbiology, and recombinant DNA techniques within the skill of the art. Such
techniques are explained
fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular
Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York
(herein "Sambrook et al., 1989"); DNA Cloning.' A Practical Approach, Volumes
I and II (D.N. Glover
ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid
Hybridization [B.D. Hames &
S.J. Higgins eds. ( 1985)]; Transcription And Translation [B.D. Hames & S.J.
Higgins, eds. ( 1984)];
Animal Cell Culture [R.I. Freshney, ed. ( 1986)]; Immobilized Cells And
Enzymes [IRL Press, ( 1986)];
B. Perbal, A Practical Guide To Molecular Cloning ( 1984); F.M. Ausubel et al.
(eds.), Current Protocols
in Molecular Biolog~, John Wiley & Sons, Inc. ( 1994).
Therefore, if appearing herein, the following terms shall have the definitions
set out below.
A "cloning vector" is a replicon, such as plasmid, phage or cosmid, to which
another DNA
segment may be attached so as to bring about the replication of the attached
segment. A "replicon" is any
genetic element (e.g., plasmid, chromosome, virus) that functions as an
autonomous unit of DNA
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replication in vivo, i.e., capable of replication under its own control.
Cloning vectors may be capable of
replication in one cell type, and expression in another ("shuttle vector").
A "cassette" refers to a segment of DNA that can be inserted into a vector at
specific restriction
sites. The segment of DNA encodes a polypeptide of interest, and the cassette
and restriction sites are
designed to ensure insertion of the cassette in the proper reading frame for
transcription and translation.
A cell has been "transfected" by exogenous or heterologous DNA when such DNA
has been
introduced inside the cell. A cell has been "transformed" by exogenous or
heterologous DNA when the
transfected DNA effects a phenotypic change. The transforming DNA can be
integrated (covalently
linked) into chromosomal DNA making up the genome of the cell.
A "nucleic acid molecule" refers to the phosphate ester polymeric form of
ribonucleosides
(adenosine, guanosine, uridine or cytidine; "RNA molecules") or
deoxyribonucleosides (deoxyadenosine,
deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any
phosphoester anoiogs
thereof, such as phosphorothioates and thioesters, in either single stranded
form, or a double-stranded
helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The
term nucleic
I S acid molecule, and in particular DNA or RNA molecule, refers only to the
primary and secondary
structure of the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes
double-stranded DNA found, inter alia, in linear or circular DNA molecules
(e.g., restriction fragments),
plasmids, and chromosomes. In.discussing the structure of particular double-
stranded DNA molecules,
sequences may be described herein according to the normal convention of giving
only the sequence in the
5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand
having a sequence homologous
to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has
undergone a molecular
biological manipulation.
A nucleic acid molecule is "hybridizable" to another nucleic acid molecule,
such as a cDNA,
genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule
can anneal to the other
nucleic acid molecule under the appropriate conditions of temperature and
solution ionic strength (see
Sambrook et al., supra). The conditions of temperature and ionic strength
determine the "stringency" of
the hybridization. For preliminary screening for homologous nucleic acids, low
stringency hybridization
conditions, corresponding to a Tm of 55°, can be used, e.g., Sx SSC,
0.1% SDS, 0.25% milk, and no
formamide; or 30% formamide, Sx SSC, 0.5% SDS). Moderate stringency
hybridization conditions
correspond to a higher Tm, e.g., 40% formamide, with Sx or 6x SCC. High
stringency hybridization
conditions correspond to the highest Tm, e.g., 50% formamide, Sx or 6x SCC.
Hybridization requires that
the two nucleic acids contain complementary sequences, although depending on
the stringency of the
hybridization, mismatches between bases are possible. The appropriate
stringency for hybridizing nucleic
acids depends on the length of the nucleic acids and the degree of
complementation, variables well known
in the art. The greater the degree of similarity or homology between two
nucleotide sequences, the greater
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the value of Tm for hybrids of nucleic acids having those sequences. The
relative stability (corresponding
to higher Tm) of nucleic acid hybridizations decreases in the following order:
RNA:RNA, DNA:RNf~,
DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for
calculating T,~ have been
derived (see Sambrook et al., supra, 9.50-0.51). For hybridization with
shorter nucleic acids, i.e.,
oligonucleotides, the position of mismatches becomes more important, and the
length of the
oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-
11.8). Preferably a minimum
length for a hybridizable nucleic acid is at least about 10 nucleotides;
preferably at least about 1 S
nucleotides; and more preferably the length is at least about 20 nucleotides.
In a specific embodiment, the term "standard hybridization conditions" refers
to a Tm of 55°C, and
utilizes conditions as set forth above. In a preferred embodiment, the Tm is
60°C; in a more preferred
embodiment, the Tm is 65°C.
As used herein, the term "oligonucleotide" refers to a nucleic acid, generally
of at least 18
nucleotides, that is specifically hybridizable to a genomic DNA molecule, a
cDNA molecule, or an mRNA
molecule encoding CaSR, or an isoform thereof. Oligonucleotides can be
labeled, e.g., with'ZP-
nucleotides or nucleotides to which a label, such as biotin, has been
covalently conjugated. In one
embodiment, a labeled oligonucleotide can be used as a probe to detect the
presence of a nucleic acid
encoding a CaSR, or an isoform thereof. In another embodiment,
oligonucleotides (one or both of which
may be labeled) can be used as PCR primers, either for cloning CaSR isoforms,
or to detect the presence
of nucleic acids encoding CaSR isoforms. In a further embodiment, an
oligonucleotide of the invention
can form a triple helix with a CaSR DNA molecule. Generally, oligonucleotides
are prepared
synthetically, preferably on a nucleic acid synthesizer. Accordingly,
oligonucleotides can be prepared
with non-naturally occurring phosphoester analog bonds, such as thioester
bonds, etc.
A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed
and translated
into a polypeptide in a cell in vitro or in vivo when placed under the control
of appropriate regulatory
sequences. The boundaries of the coding sequence are determined by a start
codon at the 5' (amino)
terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding
sequence can include, but is
not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA
sequences from
eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the
coding sequence is
intended for expression in a eukaryotic cell, a polyadenylation signal and
transcription termination
sequence will usually be located 3' to the coding sequence. In this case, the
nucleic acid is "operatively
associated" with an expression control sequence permitting expression of the
protein in an expression
competent host cell
Transcriptions) and translational control sequences are DNA regulatory
sequences, such as
promoters, enhancers, terminators, and the like, that provide for the
expression of a coding sequence in a
host cell. In eukaryotic cells, polyadenylation signals are control sequences.
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A "promoter sequence" is a DNA regulatory region capable of binding RNA
polymerise in a cell
and initiating transcription of a downstream (3' direction) coding sequence.
For purposes of defining the
present invention, the promoter sequence is bounded at its 3' terminus by the
transcription initiation site
and extends upstream (5' direction) to include the minimum number of bases or
elements necessary to
S initiate transcription at levels detectable above background. Within the
promoter sequence will be found a
transcription initiation site (conveniently defined for example, by mapping
with nuclease S 1 ), as well as
protein binding domains (consensus sequences) responsible for the binding of
RNA polymerise.
A coding sequence is "under the control" of transcriptionai and translational
control sequences in a
cell when RNA polymerise transcribes the coding sequence into mRNA, which is
then traps-RNA spliced
(if the coding sequence contains introns) and translated into the protein
encoded by the coding sequence.
As used herein, the term "homologous" in all its grammatical forms and
spelling variations refers
to the relationship between proteins that possess a "common evolutionary
origin," including proteins from
superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins
from different species
(e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell 50:667). Such
proteins (and their encoding genes)
have sequence homology, as reflected by their high degree of sequence
similarity.
Accordingly, the term "sequence similarity" in all its grammatical forms
refers to the degree of
identity or correspondence between nucleic acid or amino acid sequences of
proteins that may or may not
share a common evolutionary origin (see Reeck et al., supra). However, in
common usage and in the
instant application, the term "homologous," when modified with an adverb such
as "highly," may refer to
sequence similarity and not a common evolutionary origin.
In a specific embodiment, two DNA sequences are "substantially homologous" or
"substantially
similar" when at least about 50% (preferably at least about 75%, and most
preferably at least about 90 or
95%) of the nucleotides match over the defined length of the DNA sequences.
Sequences that are
substantially homologous can be identified by comparing the sequences using
standard software available
in sequence data banks, or in a Southern hybridization experiment under, for
example, stringent conditions
as defined for that particular system. Defining appropriate hybridization
conditions is within the skill of
the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & Il, supra;
Nucleic Acid Hybridization,
supra.
Similarly, in a particular embodiment, two amino acid sequences are
"substantially homologous"
or "substantially similar" when greater than 30% of the amino acids are
identical, or greater than about
60% are similar (functionally identical). Preferably, the similar or
homologous sequences are identified
by alignment using, for example, the GCG (Genetics Computer Group, Program
Manual for the GCG
Package, Version 7, Madison, Wisconsin) pileup program.
The term "corresponding to" is used herein to refer similar or homologous
sequences, whether the
exact position is identical or different from the molecule to which the
similarity or homology is measured.
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A nucleic acid or amino acid sequence alignment may include spaces. Thus, the
term "corresponding to"
refers to the sequence similarity, and not the numbering of the amino acid
residues or nucleotide bases.
A gene encoding a CaSR isoform, whether genomic DNA or cDNA, can be isolated
from a human
cDNA or genomic library. Methods for obtaining genes encoding CaSR isoforms
are well known in the
art, as described above (see, e.g., Sambrook et al., 1989, supra).
Accordingly, any human cell potentially can serve as the nucleic acid source
for the molecular
cloning of a CaSR isoform gene. The DNA may be obtained by standard procedures
known in the art
from cloned DNA (e.g., a DNA "library"), and preferably is obtained from a
cDNA library prepared from
tissues with high level expression of the protein (e.g., brain, thyroid and
kidney cDNA), by chemical
synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments
thereof, purified from the
desired cell (See, for example, Sambrook et al., 1989, supra; Glover, D.M.
(ed.), 1985, DNA Cloning: A
Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II). Clones derived
from genomic DNA may
contain regulatory and intron DNA regions in addition to coding regions;
clones derived from cDNA will
not contain intron sequences. In specific embodiments, isoforms CaSRc and
CaSRd were isolated from a
1 S human kidney cell library. Whatever the source, the gene should be
molecularly cloned into a suitable
vector for propagation of the gene.
Once the DNA fragments are generated, identification of the specific DNA
fragment containing
gene encoding a CaSR isoform may be accomplished in a number of ways. For
example, DNA fragments
may be screened by nucleic acid hybridization to a labeled probe (Benton and
Davis, 1977, Science
196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961 ).
Those DNA fragments
with substantial homology to the probe will hybridize. As noted above, the
greater the degree of
homology, the more stringent hybridization conditions can be used. In a
speciFc embodiment, Northern
hybridization conditions are used to identify mRNA splicing variants of a CaSR
gene.
Further selection can be carried out on the basis of the properties of the
gene, e.g., if the gene
encodes a protein product having the isoelectric, electrophoretic, amino acid
composition, or partial amino
acid sequence of a CaSR isoform as disclosed herein. Thus, the presence of the
gene may be detected by
assays based on the physical, chemical, or immunological properties of its
expressed product. For
example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can
be selected which
produce a protein that, e.g., has similar or identical electrophoretic
migration, isoelectric focusing or non-
equilibrium pH gel electrophoresis behavior, proteolytic digestion maps, or
antigenic properties as known
for CaSR. In a specific embodiment, the isoform is recognized by a polyclonal
antibody that does not
recognize wild-type CaSR.
The present invention relates to genes (e.g., cDNAs) encoding allelic
variants, splicing variants,
analogs, and derivatives of CaSR isoforms of the invention that have the same
or homologous functional
activity as the isoforms. The production and use of derivatives and analogs
related to CaSR isoforms are
CA 02336543 2001-O1-26
WO 00/06601 11 PCT/US99/17116 .
within the scope of the present invention. In a specific embodiment, the
derivative or analog is
functionally active, i.e., capable of exhibiting one or more functional
activities associated with an isbform
of the invention. In particular, such an analog can bind calcium.
Alternatively, an allelic variant can
comprise a mutation that results an inability to bind calcium.
Derivatives can be made by altering encoding nucleic acid sequences by
substitutions, additions or
deletions that provide for functionally equivalent molecules. Preferably,
derivatives are made that have
enhanced or increased functional activity relative to native CaSR isoforms.
Due to the degeneracy of nucleotide coding sequences, other DNA sequences
which encode
substantially the same amino acid sequence as a gene encoding a CaSR isoform,
including an amino acid
sequence that contains a single amino acid variant, may be used in the
practice of the present invention.
These include but are not limited to allelic genes, homologous genes from
other species, and nucleotide
sequences comprising all or portions of CaSR isoform genes which are altered
by the substitution of
different codons that encode the same amino acid residue within the sequence,
thus producing a silent
change. Likewise, the derivatives of the invention include, but are not
limited to, those containing, as a
primary amino acid sequence, all or part of the amino acid sequence of a CaSR
isoform including altered
sequences in which functionally equivalent amino acid residues are substituted
for residues within the
sequence resulting in a conservative amino acid substitution. For example, one
or more amino acid
residues within the sequence can be substituted by another amino acid of a
similar polarity, which acts as a
functional equivalent, resulting in a silent alteration. Substitutes for an
amino acid within the sequence
may be selected from other members of the class to which the amino acid
belongs. For example, the
nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine,
tryptophan and methionine. Amino acids containing aromatic ring structures are
phenylalanine,
tryptophan, and tyrosine. The polar neutral amino acids include glycine,
serine, threonine, cysteine,
tyrosine, asparagine, and glutamine. The positively charged (basic) amino
acids include arginine, lysine
and histidine. The negatively charged (acidic) amino acids include aspartic
acid and glutamic acid. Such
alterations will not be expected to affect apparent molecular weight as
determined by polyacrylamide gel
electrophoresis, or isoelectric point.
Particularly preferred substitutions are:
- Lys for Arg and vice versa such that a positive charge may be maintained;
- Glu for Asp and vice versa such that a negative charge may be maintained;
- Ser for Thr such that a free -OH can be maintained; and
- Gin for Asn such that a free CONHZ can be maintained.
Amino acid substitutions may also be introduced to substitute an amino acid
with a particularly
preferable property. For example, a Cys may be introduced a potential site for
disulfide bridges with
another Cys. A His may be introduced as a particularly "catalytic" site (i.e.,
His can act as an acid or base
CA 02336543 2001-O1-26
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WO 00/06601 PCT/US99/17116
and is the most common amino acid in biochemical catalysis). Pro may be
introduced because of its
particularly planar structure, which induces b-turns in the protein's
structure.
The genes encoding CaSR isoforms, derivatives and analogs of the invention can
be produced by
various methods known in the art. The manipulations which result in their
production can occur at the
gene or protein level. For example, the cloned gene sequence can be modified
by any of numerous
strategies known in the art (Sambrook et al., 1989, supra). The sequence can
be cleaved at appropriate
sites with restriction endonuclease(s), followed by further enzymatic
modification if desired, isolated, and
ligated in vitro. In the production of the gene encoding an CaSR isoform,
derivative or analog, care
should be taken to ensure that the modified gene remains within the same
translational reading frame as
the CaSR gene (SEQ ID NO: l I ), uninterrupted by translational stop signals,
in the gene region where the
desired activity is encoded.
Additionally, the CaSR isoform-encoding nucleic acid sequence can be mutated
in vitro or in vivo,
to create and/or destroy translation, initiation, and/or termination
sequences, or to create variations in
coding regions and/or form new restriction endonuclease sites or destroy
preexisting ones, to facilitate
further in vitro modification. Preferably, such mutations enhance the
functional activity of the mutated
gene product. Any technique for mutagenesis known in the art can be used,
including but not limited to, in
vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem.
253:6551; Zoller and Smith,
1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al.,
1986, Proc. Natl. Acad. Sci.
U.S.A. 83:710), use of TAB~ linkers (Pharmacia), etc. PCR techniques are
preferred for site directed
mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR
Technology: Principles and
Applications for DNA Amplifrcation, H. Erlich, ed., Stockton Press, Chapter 6,
pp. 61-70).
The identified and isolated gene can then be inserted into an appropriate
cloning vector. A large
number of vector-host systems known in the art may be used. Possible vectors
include, but are not limited
to, plasmids or modified viruses, but the vector system must be compatible
with the host cell used.
Examples of vectors include, but are not limited to, E. coli, bacteriophages
such as lambda derivatives, or
plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX
vectors, pmal-c, pFLAG,
etc. The insertion into a cloning vector can, for example, be accomplished by
ligating the DNA fragment
into a cloning vector which has complementary cohesive termini. However, if
the complementary
restriction sites used to fragment the DNA are not present in the cloning
vector, the ends of the DNA
molecules may be enzymatically modified. Alternatively, any site desired may
be produced by ligating
nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may
comprise specific
chemically synthesized oligonucleotides encoding restriction endonuclease
recognition sequences.
Recombinant molecules can be introduced into host cells via transformation,
transfection, infection,
electroporation, etc., so that many copies of the gene sequence are generated.
Preferably, the cloned gene
is contained on a shuttle vector plasmid, which provides for expansion in a
cloning cell, e.g., E. coli, and
CA 02336543 2001-O1-26
WO 00/06601 13 PCT/US99/17116 .
facile purification for subsequent insertion into an appropriate expression
cell line, if such is desired. For
example, a shuttle vector, which is a vector that can replicate in more than
one type of organism, cattbe
prepared for replication in both E. coli and Saccharomyces cerevisiae by
linking sequences from an E. coli
plasmid with sequences form the yeast 2m plasmid.
Expression of CaSR Isoforms
The nucleotide sequence coding for CaSR isoforms, or derivatives or analogs
thereof, including a
chimeric protein, can be inserted into an appropriate expression vector, i.e.,
a vector which contains the
necessary elements for the transcription and translation of the inserted
protein-coding sequence. Such
elements are termed herein a "promoter." Thus, the nucleic acid encoding a
CaSR isoform of the
invention is operationally associated with a promoter in an expression vector
of the invention. Both
cDNA and genomic sequences can be cloned and expressed under control of such
regulatory sequences.
An expression vector also preferably includes a replication origin.
The necessary transcriptional and translational signals can be provided on a
recombinant
I S expression vector, or they may be supplied by the native gene encoding a
CaSR, a CaSR isoform and/or its
flanking regions.
Potential host-vector systems include but are not limited to mammalian cell
systems infected with
virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected
with virus (e.g., baculovirus);
microorganisms such as yeast containing yeast vectors; or bacteria transformed
with bacteriophage, DNA,
plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their
strengths and
specificities. Depending on the host-vector system utilized, any one of a
number of suitable transcription
and translation elements may be used.
A recombinant CaSR isoform of the invention, derivative, or analog thereof,
may be expressed
chromosomally, after integration of the coding sequence by recombination. In
this regard, any of a
number of amplification systems may be used to achieve high levels of stable
gene expression (See
Sambrook et al., 1989, supra).
The cell into which the recombinant vector comprising the nucleic acid
encoding a CaSR isoform
is cultured in an appropriate cell culture medium under conditions that
provide for expression of protein
by the cell.
Any of the methods previously described for the insertion of DNA fragments
into a cloning vector
may be used to construct expression vectors containing a gene consisting of
appropriate
transcriptional/translational control signals and the protein coding
sequences. These methods may include
in vitro recombinant DNA and synthetic techniques and in vivo recombination
(genetic recombination).
Expression of a gene may be controlled by any promoter/enhancer element known
in the art, but
these regulatory elements must be functional in the host selected for
expression. Promoters which may be
CA 02336543 2001-O1-26
WO 00/06601 14 PGT/US99/17116 -
used to control gene expression include, but are not limited to, the SV40
early promoter region (Benoist
and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long
terminal repeat of ltous
sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine
kinase promoter (Wagner
et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory
sequences of the metallothionein
gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors
such as the b-lactamase
promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-
3731 ), or the tac promoter
(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also
"Useful proteins from recombinant
bacteria" in Scientific American, 1980, 242:74-94; promoter elements from
yeast or other fungi such as
the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK
(phosphoglycerol kinase) promoter,
alkaline phosphatase promoter; and the animal transcriptiona) control regions,
which exhibit tissue
specificity and have been utilized in transgenic animals: elastase I gene
control region which is active in
pancreatic acinar cells (Swift et al., 1984, Cel138:639-646; Ornitz et al.,
1986, Cold Spring Harbor Symp.
Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene
control region which is
active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),
immunoglobulin gene control region
which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;
Adames et al., 1985, Nature
318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse
mammary tumor virus control
region which is active in testicular, breast, lymphoid and mast cells (Leder
et al., 1986, Cell 45:485-495),
albumin gene control region which is active in liver (Pinkert et al., 1987,
Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in liver (Krumlauf et
al., 1985, Mol. Cell. Biol.
5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha 1-antitrypsin gene
control region which is
active in the liver (Kelsey et al., 1987, Genes and, Devel. 1:161-171), beta-
globin gene control region
which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340;
Kollias et al., 1986, Cell
46:89-94); myelin basic protein gene control region which is active in
oligodendrocyte cells in the brain
(Readhead et al., 1987, Ceil 48:703-712), myosin light chain-2 gene control
region which is active in
skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing
hormone gene control
region which is active in the hypothalamus (Mason et al., 1986, Science
234:1372-1378).
Expression vectors containing a nucleic acid encoding a CaSR isoform of the
invention can be
identified by five general approaches: (a) PCR amplification of the desired
plasmid DNA or specific
mRNA, (b) nucleic acid hybridization, (c) presence or absence of selection
marker gene functions, (d)
analyses with appropriate restriction endonucleases, and (e) expression of
inserted sequences. In the first
approach, the nucleic acids can be amplified by PCR to provide for detection
of the amplified product. In
the second approach, the presence of a foreign gene inserted in an expression
vector can be detected by
nucleic acid hybridization using probes comprising sequences that are
homologous to an inserted marker
gene. In the third approach, the recombinant vector/host system can be
identified and selected based upon
the presence or absence of certain "selection marker" gene functions (e.g., b-
gaiactosidase activity,
CA 02336543 2001-O1-26
WO 00/06601 I 5 PCT/US99/17116
thymidine kinase activity, resistance to antibiotics, transformation
phenotype, occlusion body formation in
baculovirus, etc.) caused by the insertion of foreign genes in the vector. In
another example, if the nbcleic
acid encoding a CaSR isoform is inserted within the "selection marker" gene
sequence of the vector,
recombinants containing the nucleic acid insert can be identified by the
absence of the gene function. In
S the fourth approach, recombinant expression vectors are identified by
digestion with appropriate
restriction enrymes. In the fifth approach, recombinant expression vectors can
be identified by assaying
for the activity, biochemical, or immunological characteristics of the gene
product expressed by the
recombinant, provided that the expressed protein assumes a functionally active
conformation.
A wide variety of host/expression vector combinations may be employed in
expressing the DNA
sequences of this invention. Useful expression vectors, for example, may
consist of segments of
chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors
include derivatives of
SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRI,
pBR322, pMal-C2, pET, pGEX
(Smith et al., 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such
as RP4; phage DNAS,
e.g., the numerous derivatives of phage l, e.g., NM989, and other phage DNA,
e.g., M13 and filamentous
single stranded phage DNA; yeast plasmids such as the 2m plasmid or
derivatives thereof; vectors useful
in eukaryotic cells, such as vectors useful in insect or mammalian cells;
vectors derived from
combinations of plasmids and phage DNAs, such as plasmids that have been
modified to employ phage
DNA or other expression control sequences; and the like.
For example, in a baculovirus expression systems, both non-fusion transfer
vectors, such as but
not limited to pVL941 (BamHl cloning site; Summers), pVL1393 (BamHl, SmaI,
XbaI, EcoRl, NotI,
XmaIII, BglIl, and PstI cloning site; Invitrogen), pVL 1392 (BgIII, Pstl,
NotI, XmaIII, EcoRI, XbaI, Smal,
and BamHl cloning site; Summers and Invitrogen), and pBlueBacIII (BamHl,
BgIII, PstI, NcoI, and
HindIII cloning site, with blue/white recombinant screening possible;
Invitrogen), and fusion transfer
vectors, such as but not limited to pAc700 (BamHl and KpnI cloning site, in
which the BamHl
recognition site begins with the initiation codon; Summers), pAc701 and pAc702
(same as pAc700, with
different reading frames), pAc360 (BamHl cloning site 36 base pairs downstream
of a polyhedrin
initiation codon; lnvitrogen(195)), and pBlueBacHisA, B, C (three different
reading frames, with BamHl,
BgIII, PstI, NcoI, and HindIII cloning site, an N-terminal peptide for ProBond
purification, and blue/white
recombinant screening of plaques; lnvitrogen (220)) can be used.
Mammalian expression vectors contemplated for use in the invention include
vectors with
inducible promoters, such as the dihydrofolate reductase (DHFR) promoter,
e.g., any expression vector
with a DHFR expression vector, or a DHFR/methotrexate co-amplification vector,
such as pED (Pstl, Sall,
SbaI, SmaI, and EcoRl cloning site, with the vector expressing both the cloned
gene and DHFR; see
Kaufman, Current Protocols in Molecular Biology, 16.12 ( 1991 ).
Alternatively, a glutamine
synthetase/methionine sulfoximine co-amplification vector, such as pEEl4
(HindIII, XbaI, SmaI, SbaI,
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WO 00/06601 16 PCT/US99/17116
EcoRI, and BcII cloning site, in which the vector expresses glutamine synthase
and the cloned gene;
Celltech). In another embodiment, a vector that directs episomal expression
under control of EpstelTt Barr
Virus (EBV) can be used, such as pREP4 (BamHl, SfiI, XhoI, NotI, NheI,
HindIII, NheI, PvuIl, and KpnI
cloning site, constitutive Rous Sarcoma Virus Long Terminal Repeat (RSV-LTR)
promoter, hygromycin
selectable marker; Invitrogen), pCEP4 (BamH 1, SfiI, Xhol, NotI, NheI,
HindIII, NheI, PvuIl, and Kpn1
cloning site, constitutive human cytomegalovirus (hCMV) immediate early gene,
hygromycin selectable
marker; Invitrogen), pMEP4 (Kpnl, PvuI, NheI, HindIII, NotI, Xhol, SfiI, BamHl
cloning site, inducible
methallothionein IIa gene promoter, hygromycin selectable marker: Invitrogen),
pREP8 (BamHl, XhoI,
NotI, HindIII, NheI, and Kpnl cloning site, RSV-LTR promoter, histidinol
selectable marker; Invitrogen),
pREP9 (KpnI, NheI, HindIII, NotI, XhoI, Sfil, and BamHI cloning site, RSV-LTR
promoter, 6418
selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin
selectable marker, N-
terminal peptide purifiable via ProBond resin and cleaved by enterokinase;
Invitrogen). Selectable
mammalian expression vectors for use in the invention include pRc/CMV
(HindIII, BstXI, NotI, SbaI, and
Apa1 cloning site, 6418 selection; Invitrogen), pRc/RSV (HindIII, SpeI, BstXI,
NotI, XbaI cloning site,
1 S 6418 selection; Invitrogen), and others. Vaccinia virus mammalian
expression vectors (see, Kaufman,
1991, supra) for use according to the invention include but are not limited to
pSCI 1 (SmaI cloning site,
TK- and b-gal selection), pMJ601 (Salt, SmaI, AfII, Narl, BspMll, BamHI, ApaI,
NheI, SacII, KpnI, and
HindIII cloning site; TK- and b-gal selection), and pTKgptF 1 S (EcoRI, PstI,
SaII, AccI, HindII, SbaI,
BamHI, and Hpa cloning site, TK or XPRT selection).
Yeast expression systems can also be used according to the invention to
express isoforms of a
CaSR. For example, the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI,
EcoRI, BstXI, BamHl,
SacI, Kpn l , and HindIII cloning sit; Invitrogen) or the fusion pYESHisA, B,
C (XbaI, SphI, ShoI, NotI,
BstXI, EcoRI, BamHl, SacI, Kpnl, and HindIII cloning site, N-terminal peptide
purified with ProBond
resin and cleaved with enterokinase; Invitrogen), to mention just two, can be
employed according to the
invention.
Once a particular recombinant DNA molecule is identified and isolated, several
methods known in
the art may be used to propagate it. Once a suitable host system and growth
conditions are established,
recombinant expression vectors can be propagated and prepared in quantity. As
previously explained, the
expression vectors which can be used include, but are not limited to, the
following vectors or their
derivatives: human or animal viruses such as vaccinia virus or adenovirus;
insect viruses such as
baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and piasmid
and cosmid DNA vectors, to
name but a few.
In addition, a host cell strain may be chosen which modulates the expression
of the inserted
sequences, or modifies and processes the gene product in the specific fashion
desired. Different host cells
have characteristic and specific mechanisms for the translational and post-
translational processing and
CA 02336543 2001-O1-26
WO 00/06601 17 PCT/US99/17116 -
modification of proteins. Appropriate cell lines or host systems can be chosen
to ensure the desired
modification and processing of the foreign protein expressed. Expression in
yeast can produce a
biologically active product. Expression in eukaryotic cells can increase the
likelihood of "native" folding.
Moreover, expression in mammalian cells can provide a tool for reconstituting,
or constituting, CaSR
activity. Furthermore, different vector/host expression systems may affect
processing reactions, such as
proteolytic cleavages, to a different extent.
Vectors are introduced into the desired host cells by methods known in the
art, e.g., transfection,
electroporation, microinjection, transduction, cell fusion, DEAE dextran,
calcium phosphate precipitation,
lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter
(see, e.g., Wu et al., 1992, J.
Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624;
Hartmut et al., Canadian
Patent Application No. 2,012,311, filed March 15, 1990).
Soluble forms of the protein can be obtained by collecting culture fluid, or
solubilizing inclusion
bodies, e.g., by treatment with detergent, and if desired sonication or other
mechanical processes, as
described above. The solubilized or soluble protein can be isolated using
various techniques, such as
IS polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-
dimensional gel electrophoresis,
chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing
column chromatography),
centrifugation, differential solubility, immunoprecipitation, or by any other
standard technique for the
purification of proteins.
Antibodies to CaSR Isoforms
The invention provides an antibody which specifically binds a CaSR isoform.
Such
antibodies can be used diagnostically, to detect the presence and optionally
the quantity of the isoform in
cells. Antibodies of the invention, particularly single chain Fv antibodies
(scFv) can also be used
therapeutically, to suppress CaSR activity (see below). In a specific
embodiment, the antibody recognizes
an epitope which is not present in the wild type receptor, CaSRa. In another
specific embodiment,
exemplified infra, the antibody is polyclonal. Monoclonal antibodies, and
antibody fragments (in addition
to scFv antibodies) are also contemplated by this invention. Using the
antibody of the invention, one can
specifically detect expression of a CaSR isoform in a cell by contacting a
sample from the cell with the
antibody under conditions permitting binding of the antibody to the protein in
the sample, and detecting
binding of the antibody to a protein in the sample, wherein detection of
binding of the antibody to the
protein indicates expression of a CaSR isoform in the cell. Using quantitative
immunoassay or Western
blotting methods, it is possible to quantitate the amount of CaSR, and
particularly to detect increases or
decreases in the amount of CaSR relative to the cell at an earlier time, or to
normal cells.
According to the invention, a human CaSR isoform produced recombinantly or by
chemical
synthesis, and fragments or other derivatives or analogs thereof, including
fusion proteins, may be used as
CA 02336543 2001-O1-26
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WO 00/06601 PCTlUS99/17116 -
an antigen or immunogen to generate antibodies that recognize the polypeptide.
A molecule is "antigenic"
when it is capable of specifically interacting with an antigen recognition
molecule of the immune system,
such as an immunoglobulin (antibody) or T cell antigen receptor. An antigenic
polypeptide contains at
least about 5, and preferably at least about 10, amino acids. An antigenic
portion of a molecule can be that
portion that is immunodominant for antibody or T cell receptor recognition, or
it can be a portion used to
generate an antibody to the molecule by conjugating the antigenic portion to a
carrier molecule for
immunization. A molecule that is antigenic need not be itself immunogenic,
i.e., capable of eliciting an
immune response without a carrier. Preferably, the antigenic polypeptide
comprises an epitope and/or a
sequence not present in the wild-type CaSRa, and elicits antibodies which bind
to a CaSR isofonm.
Such antibodies include but are not limited to polyclonal, monoclonal,
chimeric, single chain, Fab
fragments, and an Fab expression library. The antibodies of the invention may
be cross reactive, e.g., they
may recognize CaSR isofarms from different species. Polyclonal antibodies have
greater likelihood of
cross reactivity. Alternatively, an antibody of the invention may be specific
for a single isoform of CaSR.
In a preferred embodiment, the antibodies are capable of specifically
recognizing an isoform of CaSR,
1 S and are not capable of recognizing the wild-type CaSR.
Various procedures known in the art may be used for the production of
polyclonal antibodies. For
the production of antibody, various host animals can be immunized by injection
with the CaSR isoform, or
a derivative (e.g., fragment or fusion protein) thereof, including but not
limited to rabbits, mice, rats,
sheep, goats, etc. In one embodiment, a poiypeptide or fragment thereof can be
conjugated to an
immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet
hemocyanin (KLH). Various
adjuvants may be used to increase the immunological response, depending on the
host species, including
but not limited to Freund's (complete and incomplete), mineral gels such as
aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole
limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such
as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum.
For preparation of monoclonal antibodies against the isoform, or fragment,
analog, or derivative
thereof, any technique that provides for the production of antibody molecules
by continuous cell lines in
culture may be used. These include but are not limited to the hybridoma
technique originally developed
by Kohler and Milstein [Nature 256:495-497 (1975)], as well as the trioma
technique, the human B-cell
hybridoma technique (Kozbor et al., Immunology Today 4:72 1983); Cote et al.,
Proc. Natl. Acad Sci.
U.S.A. 80:2026-2030 (1983)], and the EBV-hybridoma technique to produce human
monoclonal
antibodies [Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, lnc., pp. 77-96
(1985)]. In an additional embodiment of the invention, monoclonal antibodies
can be produced in germ-
free animals [international Patent Publication No. WO 89/12690, published 28
December 1989]. In fact,
according to the invention, techniques developed for the production of
"chimeric antibodies" [Morrison et
CA 02336543 2001-O1-26
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WO 00/06601 PCT/US99/17116 -
al., J. Bacteriol. 159:870 ( 1984); Neuberger et al., Nature 312:604-608 (
1984); Takeda et al., Nature
314:452-454 ( 1985)] by splicing the genes from a mouse antibody molecule
specific for a CaSR isoform
together with genes from a human antibody molecule of appropriate biological
activity can be used; such
antibodies are within the scope of this invention. Such human or humanized
chimeric antibodies are
preferred for use in therapy of human diseases or disorders (described infra),
since the human or
humanized antibodies are much less likely than xenogenic antibodies to induce
an immune response, in
particular an allergic response, themselves.
According to the invention, techniques described for the production of single
chain Fv (scFv)
antibodies [U.S. Patent Nos. 5,476,786 and 5,132,405 to Huston; U.S. Patent
4,946,778] can be adapted to
produce CaSR isoform-specific single chain antibodies. An additional
embodiment of the invention
utilizes the techniques described for the construction of Fab expression
libraries [Huse et al., Science
246:1275-1281 ( 1989)] to allow rapid and easy identification of monoclonal
Fab fragments with the
desired specificity for a CaSR isoform.
Antibody fragments which contain the idiotype of the antibody molecule can be
generated by
known techniques. For example, such fragments include but are not limited to:
the F(ab'), fragment which
can be. produced by pepsin digestion of the antibody molecule; the Fab'
fragments which can be generated
by reducing the disulfide bridges of the F(ab')Z fragment, and the Fab
fragments which can be generated
by treating the antibody molecule with papain and a reducing agent.
In the production of antibodies, screening for the desired antibody can be
accomplished by
techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked
immunosorbent assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin
reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or
radioisotope labels, for
example), western blots, precipitation reactions, agglutination assays (e.g.,
gel agglutination assays,
hemagglutination assays), complement fixation assays, immunofluorescence
assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody binding is
detected by detecting a label
on the primary antibody. In another embodiment, the primary antibody is
detected by detecting binding of
a secondary antibody or reagent to the primary antibody. In a further
embodiment, the secondary antibody
is labeled. Many means are known in the art for detecting binding in an
immunoassay and are within the
scope of the present invention. For exampie, to select antibodies which
recognize a specific epitope of a
CaSR isoform, one may assay generated hybridomas for a product which binds to
an isoform containing
such epitope. For selection of an antibody specific to an isoform from a
particular species of animal, one
can select on the basis of positive binding with a CaSR isoform expressed by
or isolated from cells of that
species of animal, but which does not bind the wild-type CaSR.
The foregoing antibodies can be used in methods known in the art relating to
the localization and
activity of the CaSR isoform, e.g., for Western blotting, imaging theisoform
in situ, measuring levels
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116
thereof in appropriate physiological samples, etc. using any of the detection
techniques mentioned above
or known in the art.
In a specific embodiment, antibodies that agonize or antagonize the activity
of CaSR, and, in
particular, are specific for a CaSR isoform, can be generated. Such antibodies
can be tested using the
assays described infra for identifying ligands. In particular, such antibodies
can be scFv antibodies
expressed intracellularly.
Screening Assays
Identification and isolation of a gene encoding isoforms of a CaSR of the
invention provides for
10 expression of these isoforms in quantities greater than can be isolated
from natural sources, or in indicator
cells that are specially engineered to indicate CaSR activity after
transfection or transformation of the
cells. Accordingly, in addition to rational design of agonists and antagonists
based on the structures of
CaSR isoforms, the present invention contemplates an alternative method for
identifying specific ligands
of CaSR isoforms using various screening assays known in the art.
15 Any screening technique known in the art can be used to screen for CaSR
agonists or antagonists.
The present invention contemplates screens for small molecule ligands or
ligand analogs and mimics, as
well as screens for natural ligands that bind to and agonize (calcimimetics)
or antagonize (calcilytics)
CaSR activity in vivo. For example, natural products libraries can be screened
using assays of the
invention for molecules that agonize or antagonize CaSR activity. The present
invention provides both the
20 means and methodology for identifying compounds capable of modulating CaSR
activity, including the
specific modulation of CaSR isoforms. Screening assays for calcimimetics and
calcilytics are discussed in
WO 94/18959 and US Pat. No. 5,763,569, the entire contents of which are
incorporated herein by
reference.
In a preferred screening assay compounds are assayed for their ability to
influence intracellular
calcium concentration. The compounds may be tested alone, in conjunction with
an elevation in
extracellular calcium concentration, or in the presence of other agonists or
antagonists of CaSR isoform
activity. Agonists include neomycin, di- and tri-valent cations (gadolinium,
calcium, magnesium,
strontium, barium, lanthanum), polyamines and other known calcimimetics.
Intracellular calcium is
measured with the fluorescent indicator, fura-2 (from Molecular Probes). For
example, HEK-293 cells
transfected with a nucleic acid encoding a CaSR isoform is loaded in buffer
containing O.SuM fura-2,
20mM HEPES, pH 7.35, 0.1% BSA, O.SmM CaClz, O.SmM MgCI,, 6.7mM KC1, 3mM
glucose and
142mM NaCI for 45 min at 37°C. The cells are washed and resuspended to
about 2 x 106 cells/ml in the
loading buffer without fura-2. For intracellular calcium measurement, cells
were placed in a quartz cuvette
equilibrated at 37°C. Fluorescence is detected using excitation
monochrometers centered at 340 and 380
nm and emission light collected at 505 nm.
CA 02336543 2001-O1-26
WO 00/06601 21 PCT1US99/17116 .
Knowledge of the primary sequence of CaSR isoforms, and the similarity of
their sequences with
proteins of known function, can provide an initial clue as the inhibitors or
antagonists of the proteir~
Identification and screening of antagonists is further facilitated by
determining structural features of the
protein, e.g., using X-ray crystallography, neutron diffraction, nuclear
magnetic resonance spectrometry,
and other techniques for structure determination. These techniques provide for
the rational design or
identification of agonists and antagonists.
Another approach uses recombinant bacteriophage to produce large libraries.
Using the "phage
method" [Scott and Smith, 1990, Science 249:386-390 ( 1990); Cwirla, et al.,
Proc. Natl. Acad. Sci.,
87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)], very large
libraries can be constructed
(106-10g chemical entities). A second approach uses primarily chemical
methods, of which the Geysen
method [Geysen et al., Molecular Immunology 23:709-715 ( 1986); Geysen et al.
J. Immunologic Method
102:259-274 ( 1987)] and the method of Fodor et al. (Science 251:767-773 (
1991 )] are examples. Furka et
al. [14th International Congress of Biochemistry, Volume S, Abstract FR:013 (
1988); Furka, Int. J. Peptide
Protein Res. 37:487-493 (1991)], Houghton [U.S. Patent No. 4,631,211, issued
December 1986] and
Rutter et al. [U.S. Patent No. 5,010,175, issued April 23, I 991 ] describe
methods to produce a mixture of
peptides that can be tested as agonists or antagonists.
In another aspect, synthetic libraries [Needels et al., Proc. Natl. Acad. Sci.
USA 90:10700-4
(1993); Ohlmeyer et al., Proc. Natl. Acad Sci. USA 90:10922-10926 (1993); Lam
et al., International
Patent Publication No. WO 92/00252; Kocis et al., International Patent
Publication No. WO 9428028,
each of which is incorporated herein by reference in its entirety], and the
like can be used to screen for
CaSR ligands according to the present invention.
The screening can be performed with recombinant cells that express a CaSR
isoform, or
alternatively, using purified protein, e.g., produced recombinantly, as
described above. For example, the
ability of a labeled, soluble CaSR isoform that includes the extracellular
calcium binding portion of the
molecule, can be used to screen libraries, as described in the foregoing
references.
In one embodiment, a CaSR isoform may be directly labeled. In another
embodiment, a labeled
secondary reagent may be used to detect binding of an isoform to a molecule of
interest, e.g., a molecule
attached to a solid phase support. Binding may be detected by in situ
formation of a chromophore by an
enryme label. Suitable enzymes include, but are not limited to, alkaline
phosphatase and horseradish
peroxidase. In a further embodiment, a two color assay, using two chromogenic
substrates with two
enzyme labels on different acceptor molecules of interest, may be used. Cross-
reactive and singly-reactive
ligands may be identified with a two-color assay.
Other labels for use in the invention include colored latex beads, magnetic
beads, fluorescent
labels (e.g., fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red
(TR), rhodamine, free or
chelated lanthanide series salts, especially Eu'', to name a few
fluorophores), chemiluminescent
CA 02336543 2001-O1-26
WO 00/06601 22 PCTNS99/17116
molecules, radio-isotopes, or magnetic resonance imaging labels. Two color
assays may be performed
with two or more colored latex beads, or fluorophores that emit at different
wavelengths. Labeled msy be
detected visually or by mechanical/optica) means. Mechanical/optical means
include fluorescence
activated sorting, i.e., analogous to FACS, and micromanipulator removal
means.
As exemplified herein, the level of the CaSR isoform can be evaluated by
metabolic labeling of
the proteins. As the metabolic labeling occurs during in vitro incubation of
the tissue biopsy in the
presence of culture medium supplemented with ['sS]-methionine, the level of
each of the markers detected
may be affected by the in vitro conditions. In addition to metabolic (or
biosynthetic) labeling with ['sS]-
methionine, the invention further contemplates labeling with ['4C]-amino acids
and ['H]-amino acids (with
the tritium substituted at non-labile positions). Thus, a sample or library of
compounds can be directly
analyzed after labeling of the proteins therein, e.g., by colorimetric
staining using silver, gold, coomassie
blue, or amido-schwartz, to mention a few techniques; isotopic labeling, e.g.,
with ['zP]-orthophosphate,
[~zsl]~ [ns~I]~ fluorescent or chemiluminescent tags; and immunologica)
detection with labeled antibody or
specific binding partner of a marker.
Pharmaceutical Compositions and Therapy
Diseases or disorders associated with calcium homeostasis, and therefore, the
CaSR, are known in
the art. Such diseases are related to the functional responses of cells to
calcium, such as parathyroid .
hormone secretion from parathyroid cells, calcitonin secretion by C-cells and
bone resorption by
osteoclasts. An example is hyperparathyroidism, which results in elevated
levels of parathyroid hormone
in the plasma. Therefore, a method of decreasing plasma parathyroid hormone
levels is a way of treating
hyperparathyroidism. Alternatively, increased levels of plasma calcitonin are
associated with inhibition of
bone resorption. Inhibition of bone resorption offers a way to treat
osteoporosis, for example. The present
invention provides both the means and methodology for identifying compounds
capable of modulating
CaSR activity, including the specific modulation of CaSR isoforms, and of
using these compounds for the
treatment of diseases or disorders associated with abnormal calcium levels.
Therefore, the present invention provides pharmaceutical compositions and
methods for the
treatment of a patient suffering from a disease or disorder associated with
abnormal calcium levels,
such as in the plasma, by the administration of a therapeutically effective
amount of a compound
capable of modulating the activity of a CaSR isoform. The term "patient"
includes both human and
other mammals.
"Pharmaceutical composition" refers to a composition comprising the compound
and at least
one component selected from the group comprising pharmaceutically acceptable
carriers, diluents,
adjuvants, excipients, or vehicles, such as preserving agents, fillers,
disintegrating agents, wetting
agents, emulsifying agents, suspending agents, sweetening agents, flavoring
agents, perfuming agents,
CA 02336543 2001-O1-26
23
WO 00/06601 PCTNS99/17116
antibacterial agents, antifungal agents, lubricating agents and dispensing
agents, depending on the
nature of the mode of administration and dosage forms. Examples of suspending
agents include '
ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or
mixtures of these
substances. Prevention of the action of microorganisms can be ensured by
various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid,
and the like. It may also
be desirable to include isotonic agents, for example sugars, sodium chloride
and the like. Prolonged
absorption of the injectable pharmaceutical form can be brought about by the
use of agents delaying
absorption, for example, aluminum monosterate and gelatin. Examples of
suitable carriers, diluents,
solvents or vehicles include water, ethanol, polyols, suitable mixtures
thereof, vegetable oils (such as
olive oil) and injectable organic esters such as ethyl oleate. Examples of
excipients include lactose,
milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate phosphate.
Examples of
disintegrating agents include starch, alginic acids and certain complex
silicates. Examples of
lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well
as high molecular weight
polyethylene glycols.
"Pharmaceutically acceptable" means it is, within the scope of sound medical
judgement,
suitable for use in contact with the cells of humans and lower animals without
undue toxicity,
irritation, allergic response and the like, and are commensurate with a
reasonable benefit/risk ratio.
"Phanmaceuticallly acceptable dosage forms" refers to dosage forms of the
compound of the
invention, and includes, for example, tablets, dragees, powders, elixirs,
syrups, liquid preparations,
including suspensions, sprays, inhalants tablets, lozenges, emulsions,
solutions, granules, capsules and
suppositories, as well as liquid preparations for injections, including
liposome preparations.
Techniques and formulations generally may be found in Remington's
Pharmaceutical Sciences, Mack
Publishing Co., Easton, PA, latest edition.
"Pharmaceutically acceptable salts" refers to the relatively non-toxic,
inorganic and organic
acid addition salts, and base addition salts, of compounds of the present
invention. These salts can be
prepared in situ during the final isolation and purification of the compounds.
In particular, acid
addition salts can be prepared by separately reacting the purified compound in
its free base form with
a suitable organic or inorganic acid and isolating the salt thus formed.
Exemplary acid addition salts
include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate,
nitrate, acetate, oxalate,
valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate,
phosphate, tosylate, citrate,
maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate,
lactiobionate,
sulphamates, malonates, salicylates, propionates, methylene-bis-b-
hydroxynaphthoates, gentisates,
isethionates, di-p-toluoyltartrates, methane-sulphonates, ethanesulphonates,
benzenesulphonates,
p-toluenesulphonates, cyclohexylsulphamates and quinateslaurylsulphonate
salts, and the like. (See,
for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 66:
p.l-19 (1977) which is
incorporated herein by reference.) Base addition salts can also be prepared by
separately reacting the
purified compound in its acid form with a suitable organic or inorganic base
and isolating the salt thus
CA 02336543 2001-O1-26
24
WO 00/06601 PCT/US99/17116 .
formed. Base addition salts include pharmaceutically acceptable metal aid
amine salts. Suitable metal
salts include the sodium, potassium, calcium, barium, zinc, magnesium; and
aluminum salts. The'
sodium and potassium salts are preferred. Suitable inorganic base addition
salts are prepared from
metal bases which include sodium hydride, sodium hydroxide, potassium
hydroxide, calcium
hydroxide, aluminium hydroxide, lithium hydroxide, magnesium hydroxide, zinc
hydroxide. Suitable
amine base addition salts are prepared from amines which have sufficient
basicity to form a stable
salt, and preferably include those amines which are frequently used in
medicinal chemistry because of
their low toxicity and acceptability for medical use. ammonia,
ethylenediamine, N-methyl-glucamine,
lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine,
chloroprocaine, diethanolamine,
procaine, N-benzylphenethylamine, diethylamine, piperazine,
tris(hydroxymethyl)-aminomethane,
tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine,
dehydroabietylamine,
N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium,
methylamine,
dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and
arginine, and
dicyclohexylamine, and the like.
"Solid dosage form" means the dosage form of the compound of the invention is
solid form,
for example capsules, tablets, pills, powders, dragees or granules. In such
solid dosage forms, the
compound of the invention is admixed with at least one inert customary
excipient (or carrier) such as
sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for
example, starches, lactose,
sucrose, glucose, mannitol and silicic acid, (b) binders, as for example,
carboxymethylcellulose,
alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants,
as for example, glycerol,
(d) disintegrating agents, as for example, agar-agar, calcium carbonate,
potato or tapioca starch,
alginic acid, certain complex silicates and sodium carbonate, (e) solution
retarders, as for example
paraffin, (f) absorption accelerators, as for example, quaternary ammonium
compounds, (g) wetting
agents, as for example, cetyl alcohol and glycerol monostearate, (h)
adsorbents, as for example, kaolin
and bentonite, (i) lubricants, as for example, talc, calcium stearate,
magnesium stearate, solid
polyethylene glycols, sodium lauryl sulfate, (j) opacifying agents, (k)
buffering agents , and agents
which release the compounds) of the invention in a certain part of the
intestinal tract in a delayed
manner.
The choice of vehicle and the content of active substance in the vehicle are
generally
determined in accordance with the solubility and chemical properties of the
active compound, the
particular mode of administration and the provisions to be observed in
pharmaceutical practice. For
example, excipients such as lactose, sodium citrate, calcium carbonate,
dicalcium phosphate and
disintegrating agents such as starch, alginic acids and certain complex
silicates combined with
lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be
used for preparing
tablets. To prepare a capsule, it is advantageous to use lactose and high
molecular weight
polyethylene glycols. When aqueous suspensions are used they can contain
emulsifying agents or
agents which facilitate suspension. Diluents such as sucrose, ethanol,
polyethylene glycol, propylene
glycol, glycerol and chloroform or mixtures thereof may also be used.
CA 02336543 2001-O1-26
WO 00/06601 PCTNS99/17116
The oily phase of the emulsions of this invention maybe constituted from known
ingredients
in a known manner. While the phase may comprise merely an emulsifier
(otherwise known as an'
emulgent), it desirably comprises a mixture of at least one emulsifier with a
fat or an oil or with both
a fat and an oil. Preferably, a hydrophilic emulsifier is included together
with a lipophilic emulsifier
which acts as a stabilizer. It is also preferred to include both an oil and a
fat. Together, the
emulsifiers) with or without stabilizers) make up the emulsifying wax, and the
way together with the
oil and fat make up the emulsifying ointment base which forms the oily
dispersed phase of the cream
formulations. Emulgents and emulsion stabilizers suitable for use in the
formulation of the present
invention include Tween~ 60, Span~ 80, cetostearyl alcohol, benzyl alcohol,
myristyl alcohol,
10 glyceryl mono-stearate and sodium lauryl sulfate.
If desired, the aqueous phase of the cream base may include, for example, a
least 30% w/w of
a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such
as propylene glycol,
butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol
(including PEG 400) and
mixtures thereof. The topical formulations may desirably include a compound
which enhances
15 absorption or penetration of the active ingredient through the skin or
other affected areas. Examples
of such dermal penetration enhancers include dimethyl sulphoxide and related
analogs.
The choice of suitable oils or fats for the formulation is based on achieving
the desired
cosmetic properties. Thus the cream should preferably be a non-greasy, non-
staining and washable
product with suitable consistency to avoid leakage from tubes or other
containers. Straight or
20 branched chain, mono.. or dibasic alkyl esters such as di-isopropyl
myristate, decyl oleate, isopropyl
palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain
esters known as
Crodamol CAP may be used, the last three being preferred esters. These may be
used alone or in
combination depending on the properties required. Alternatively, high melting
point lipids such as
white soft paraffin and/or liquid paraffin or other mineral oils can be used.
25 Solid compositions of may also be employed as fillers in soft and hard-
filled gelatin capsules
using such excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols,
and the like.
The pharmaceutical compositions can be administered in a suitable formulation
to humans
and animals by topical or systemic administration, including oral,
inhalational, rectal, nasal, buccal,
sublingual, vaginal, parenteral (including subcutaneous, intramuscular,
intravenous, intradermal,
intrathecal and epidural), intracisternal and intraperitoneal. It will be
appreciated that the preferred
route may vary with for example the condition of the recipient.
The formulations can be prepared in unit dosage form by any of the methods
well known in
the art of pharmacy. Such methods include the step of bringing into
association the active ingredient
with the carrier which constitutes one or more accessory ingredients. In
general the formulations are
prepared by uniformly and intimately bringing into association the active
ingredient with liquid
carriers or finely divided solid carriers or both, and then, if necessary,
shaping the product.
CA 02336543 2001-O1-26
WO 00/06601 26 PCT/US99/17116 -
"Formulations suitable for oral administration" may be presented as discrete
units such as
capsules, cachets or tablets each containing a predetermined amount of the
active ingredient; as a'
powder or granules; as solution or a suspension in an aqueous liquid or a non-
aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active
ingredient may also be
presented as a bolus, electuary or paste.
A tablet may be made by compression or moulding, optionally with one or more
accessory
ingredients. Compressed tables may be prepared by compressing in a suitable
machine the active
ingredient in a free-flowing form such as a powder or granules, optionally
mixed with a binder,
lubricant, inert diluent, preservative, surface active or dispersing agent.
Moulded tablets may be
made by moulding in a suitable machine a mixture ofthe powdered compounds
moistened with an
inert liquid diluent. The tablets may optionally be coated or scored and may
be formulated so as to
provide slow or controlled release of the active ingredient therein.
Solid compositions for rectal administration include suppositories formulated
in accordance
with known methods and containing at least one compound of the invention.
If desired, and for more effective distribution, the compounds can be
microencapsulated in, or
attached to, a slow release or targeted delivery systems such as a
biocompatible, biodegradable
polymer matrices (e.g. poly(d,l-lactide co-glycolide)), liposomes, and
microspheres and
subcutaneously or intramuscularly injected by a technique called subcutaneous
or intramuscular depot
to provide continuous slow release of the compounds) for a period of 2 weeks
or longer. The
compounds may be sterilized, for example, by flftration through a bacteria
retaining filter, or by
incorporating sterilizing agents in the form of sterile solid compositions
which can be dissolved in
sterile water, or some other sterile injectable medium immediately before use.
Actual dosage levels of active ingredient in the compositions of the invention
may be varied
so as to obtain an amount of active ingredient that is effective to obtain a
desired therapeutic response
for a particular composition and method of administration. The selected dosage
level therefore
depends upon the desired therapeutic effect, on the route of administration,
on the desired duration of
treatment and other factors.
Total daily dose of the compounds of this invention administered to a host in
single or divided
doses may be in amounts, for example, of from about 0.001 to about 100 mg/kg
body weight daily
and preferably 0.01 to 10 mg/kg/day. Dosage unit compositions may contain such
amounts of such
submultiples thereof as may be used to make up the daily dose. It will be
understood, however, that
the specific dose level for any particular patient will depend upon a variety
of factors including the
body weight, general health, sex, diet, time and route of administration,
rates of absorption and
excretion, combination with other drugs and the severity of the particular
disease being treated.
The amount of each component administered is determined by the attending
clinicians taking
into consideration the etiology and severity of the disease, the patient's
condition and age, the potency
of each component and other factors.
CA 02336543 2001-O1-26
WO 00/06601 2~ PCT/US99/17116 .
The formulations may be presented in unit-dose or multi-dose containers, for
example sealed
ampoules and vials with elastomeric stoppers, and may be stored in a freeze-
dried (lyophilized) '
condition requiring only the addition of the sterile liquid carrier, for
example water for injections,
immediately prior to use. Extemporaneous injection solutions and suspensions
may be prepared from
sterile powders, granules and tablets of the kind previously described.
Gene Therapy and Transgenic Vectors
The present invention also relates to gene therapy of diseases or disorders
associated with
abnormal levels of calcium. As discussed above, a "vector" is any means for
the transfer of a nucleic acid
according to the invention into a host cell. Preferred vectors are viral
vectors, such as retroviruses, herpes
viruses, adenoviruses, and adeno-associated viruses. Thus, a gene encoding a
CaSR, a CaSR isoform, a
polypeptide domain fragment thereof, or a nucleic acid encoding a CaSR
antisense sequence is introduced
in vivo, ex vivo, or in vitro using a viral vector or through direct
introduction of DNA. Expression in
targeted tissues can be effected by targeting the transgenic vector to
specific cells, such as with a viral
vector or a receptor ligand, or by using a tissue-specific promoter, or both.
Expression vectors of the invention can be used, as pointed out above, both to
transfect cells for
screening or biological testing of modulators of CaSR activity, or for
delivery of a CaSR nucleic acid, as
described above, or CaSR antisense gene in vivo or ex vivo for gene therapy,
e.g., to increase or decrease
the level of CaSR activity. A vector that expresses an anti-CaSR scFv can also
be introduced using the
techniques discussed below.
Viral vectors commonly used for in vivo or ex vivo targeting and therapy
procedures are DNA-
based vectors and retroviral vectors. Methods for constructing and using viral
vectors are known in the art
[see, e.g., Miller and Rosman, BioTechnigues 7:980-990 (1992)]. Preferably,
the viral vectors are
replication defective, that is, they are unable to replicate autonomously in
the target cell. In general, the
genome of the replication defective viral vectors which are used within the
scope of the present invention
lack at least one region which is necessary for the replication of the virus
in the infected cell. These
regions can either be eliminated (in whole or in part), be rendered non-
functional by any technique known
to a person skilled in the art. These techniques include the total removal,
substitution (by other sequences,
in particular by the inserted nucleic acid), partial deletion or addition of
one or more bases to an essential
(for replication) region. Such techniques may be performed in vitro (on the
isolated DNA) or in situ,
using the techniques of genetic manipulation or by treatment with mutagenic
agents. Preferably, the
replication defective virus retains the sequences of its genome which are
necessary for encapsulating the
viral particles.
DNA viral vectors include an attenuated or defective DNA virus, such as but
not limited to herpes
simplex virus (HSV), papillomavirus, Epstein Ban virus (EBV), adenovirus,
adeno-associated virus
CA 02336543 2001-O1-26
WO 00/06601 28 PCT/US99/17116
(AAV), vaccinia virus, and the like. Defective viruses, which entirely or
almost entirely lack viral genes,
are preferred. Defective virus is not replication competent after introduction
into a cell, and thus does not
lead to a productive viral infection. Use of defective viral vectors allows
for administration to cells in a
specific, localized area, without concern that the vector can infect other
cells. Thus, a specific tissue can
be specifically targeted. Examples of particular vectors include, but are not
limited to, a defective herpes
virus 1 (HSV 1 ) vector [Kaplitt et al., Molec. Cell. Neurosci. 2:320-330 (
1991 )], defective herpes virus
vector lacking a glyco-protein L gene [Patent Publication RD 371005 A], or
other defective herpes virus
vectors [international Patent Publication No. WO 94/21807, published September
29, 1994; International
Patent Publication No. WO 92/05263, published April 2, 1994]; an attenuated
adenovirus vector, such as
the vector described by Stratford-Perricaudet et al. [J. Clin. Invest. 90:626-
630 (1992); see also La Salle et
al., Science 259:988-990 ( 1993)]; and a defective adeno-associated virus
vector [Samulski et al., J. Yirol.
61:3096-3101 ( 1987); Samulski et al., J. Yirol. 63:3822-3828 ( 1989);
Lebkowski et al., Mol. Cell. BioL
8:3988-3996 ( 1988)].
Preferably, for in vivo administration, an appropriate immunosuppressive
treatment is employed in
conjunction with the viral vector, e.g., adenovirus vector, to avoid immuno-
deactivation of the viral vector
and transfected cells. For example, immunosuppressive cytokines, such as
interfeukin-12 (IL-12),
interferon-y (IFN-y), or anti-CD4 antibody, can be administered to block
humoral or cellular immune
responses to the viral vectors [see, e.g., Wilson, Nature Medicine (1995)]. In
addition, it is advantageous
to employ a viral vector that is engineered to express a minimal number of
antigens.
Naturally, the invention contemplates delivery of a vector that will express a
therapeutically
effective amount of a CaSR, or an antisense thereto, for gene therapy
applications. The phrase
"therapeutically effective amount" is used herein to mean an amount sufficient
to reduce by at least about
15 percent, preferably by at least 50 percent, more preferably by at least 90
percent, and most preferably
prevent, a clinically significant deficit in the activity, function and
response of the host. Alternatively, a
therapeutically effective amount is sufficient to cause an improvement in a
clinically significant condition
in the host.
Any vector, viral or non-viral, of the invention will preferably be introduced
in vivo in a
pharmaceutically acceptable vehicle or carrier. The phrase "pharmaceutically
acceptable" refers to
molecular entities and compositions that are physiologically tolerable and do
not typically produce an
allergic or similar untoward reaction, such as gastric upset, dizziness and
the like, when administered to a
human. Preferably, as used herein, the term "pharmaceutically acceptable"
means approved by a
regulatory agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more particularly in
humans. The term
"carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the
compound is administered.
Such pharmaceutical carriers can be sterile liquids, such as water and oils,
including those of petroleum,
CA 02336543 2001-O1-26
WO 00/06601 29 PCT/US99/17116
animal, vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the I;~ce.
Water or aqueous solution saline solutions and aqueous dextrose and glycerol
solutions are preferably
employed as carriers, particularly for injectable solutions. Suitable
pharmaceutical carriers are described
in "Remington's Pharmaceutical Sciences" by E.W. Martin.
Adenovirus vectors
In a preferred embodiment, the vector is an adenovirus vector. Adenoviruses
are eukaryotic DNA
viruses that can be modified to efficiently deliver a nucleic acid of the
invention to a variety of cell types.
Various serotypes of adenovirus exist. Of these serotypes, preference is
given, within the scope of the
present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5)
or adenoviruses of animal
origin (see W094/26914). Those adenoviruses of animal origin which can be used
within the scope of the
present invention include adenoviruses of canine, bovine, murine (example:
Mavl, Beard et al., Virology
75 {1990) 81), ovine, porcine, avian, and simian (example: SAV) origin.
Preferably, the adenovirus of
animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g.
Manhattan or A26/61
strain (ATCC VR-800), for example).
Preferably, the replication defective adenoviral vectors of the invention
comprise the ITRs, an
encapsidation sequence and the nucleic acid of interest. Still more
preferably, at least the EI region of the
adenoviral vector is non-functional. The deletion in the E1 region preferably
extends from nucleotides 455
to 3329 in the sequence of the Ad5 adenovirus (PvuII-BgIII fragment) or 382 to
3446 (Hinfil-Sau3A
fragment). Other regions may also be modified, in particular the E3 region
(W095/02697), the E2 region
(W094/28938), the E4 region (W094/28152, W094/12649 and W095/02697), or in any
of the late genes
LI-LS.
In a preferred embodiment. the adenoviral vector has a deletion in the E1
region (Ad I.0).
Examples of E 1-deleted adenoviruses are disclosed in EP 185,573, the contents
of which are incorporated
herein by reference. In another preferred embodiment, the adenoviral vector
has a deletion in the E1 and
E4 regions (Ad 3.0). Examples of El/E4-deleted adenoviruses are disclosed in
W095/02697 and
W096/22378, the contents of which are incorporated herein by reference. In
still another preferred
embodiment, the adenoviral vector has a deletion in the E 1 region into which
the E4 region and the nucleic
acid sequence are inserted (see FR94 13355, the contents of which are
incorporated herein by reference).
The replication defective recombinant adenoviruses according to the invention
can be prepared by
any technique known to the person skilled in the art (Levrero ei al., Gene 101
(1991) 195, EP 185 573;
Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by
homologous recombination
between an adenovirus and a piasmid which carries, inter alia, the DNA
sequence of interest. The
homologous recombination is effected following cotransfection of the
adenovirus and plasmid into an
appropriate cell line. The cell line which is employed should preferably (i)
be transformable by the said
elements. and (ii) contain the sequences which are able to complement the part
of the genome of the
CA 02336543 2001-O1-26
WO 00/06601 30 PCT/US99/17116
replication defective adenovirus, preferably in integrated form in order to
avoid the risks of recombination.
Examples of cell lines which may be used are the human embryonic kidney cell
line 293 (Graham et al., J.
Gen. Virol. 36 ( 1977) 59) which contains the lefr-hand portion of the genome
of an Ad5 adenovirus ( I 2%)
integrated into its genome, and cell lines which are able to complement the E1
and E4 functions, as
described in applications W094/26914 and W095/02697. Recombinant adenoviruses
are recovered and
purified using standard molecular biological techniques, which are well known
to one of ordinary skill in
the art.
Adeno-associated virus vectors
The adeno-associated viruses (AAV) are DNA viruses of relatively small size
which can integrate,
in a stable and site-specific manner, into the genome of the cells which they
infect. They are able to infect
a wide spectrum of cells without inducing any effects on cellular growth,
morphology or differentiation,
and they do not appear to be involved in human pathologies. The AAV genome has
been cloned,
sequenced and characterised. It encompasses approximately 4700 bases and
contains an inverted terminal
repeat (ITR) region of approximately 145 bases at each end, which serves as an
origin of replication for
the virus. The remainder of the genome is divided into two essential regions
which carry the
encapsulation functions: the left-hand part of the genome, which contains the
rep gene involved in viral
replication and expression of the viral genes; and the right-hand part of the
genome, which contains the
cap gene encoding the capsid proteins of the virus.
The use of vectors derived from the AAVs for transferring genes in vitro and
in vivo has been
described (see WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488
528). These
publications describe various AAV-derived constructs in which the rep and/or
cap genes are deleted and
replaced by a gene of interest. and the use of these constructs for
transferring the said gene of interest in
vitro (into cultured cells) or in vivo, (directly into an organism). The
replication defective recombinant
AAVs according to the invention can be prepared by cotransfecting a plasmid
containing the nucleic acid
sequence of interest flanked by two AAV inverted terminal repeat (ITR)
regions, and a plasmid carrying
the AAV encapsulation genes (rep and cap genes), into a cell line which is
infected with a human helper
virus (for example an adenovirus). The AAV recombinants which are produced are
then purified by
standard techniques.
The invention also relates, therefore, to an AAV-derived recombinant virus
whose eenome
encompasses a sequence encoding a nucleic acid encoding a CaSR flanked by the
AAV ITRs. The
invention also relates to a plasmid encompassing a sequence encoding a nucleic
acid encoding a CaSR
flanked by two ITRs from an AAV. Such a plasmid can be used as it is for
transferring the nucleic acid
sequence. with the plasmid, where appropriate, being incorporated into a
liposomal vector (pseudo-virus).
Retrovirus vectors
In another embodiment the gene can be introduced in a retroviral vector, e.g.,
as described in
CA 02336543 2001-O1-26
WO 00/06601 31 PCT/US99/17116
Anderson et al., U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33:153;
Temin et al., U.S. Patent~lo.
4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., 1988, J.
Virol. 62:1120; Temin et al.,
U.S. Patent No. 5,124,263; EP 453242, EP178220; Bernstein et al. Genet. Eng. 7
( 1985) 235; McCormick,
BioTechnology 3 (1985) 689; International Patent Publication No. WO 95/07358,
published March 16,
S 1995, by Dougherty et al.; and Kuo et al., 1993, Blood 82:845. The
retroviruses are integrating viruses
which infect dividing cells. The retrovirus genome includes two LTRs, an
encapsulation sequence and
three coding regions (gag, pol and envy. In recombinant retroviral vectors,
the gag, po! and env genes are
generally deleted, in whole or in part, and replaced with a heterologous
nucleic acid sequence of interest.
These vectors can be constructed from different types of retrovirus, such as,
HIV, MoMuLV ("murine
Moloney leukaemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey
sarcoma virus");
SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus.
Defective retroviral vectors
are disclosed in W09S/02697.
In general, in order to construct recombinant retroviruses containing a
nucleic acid sequence, a
plasmid is constructed which contains the LTRs, the encapsulation sequence and
the coding sequence.
1S This construct is used to transfect a packaging cell line, which cell line
is able to supply in traps the
retroviral functions which are deficient in the plasmid. In general, the
packaging cell lines are thus able to
express the gag, pol and env genes. Such packaging cell lines have been
described in the prior art, in
particular the cell line PA317 (US4,861,719); the PsiCRIP cell line
(W090/02806) and the GP+envAm-12
cell line (W089/07150). In addition, the recombinant retroviral vectors can
contain modifications within
the LTRs for suppressing transcriptional activity as well as extensive
encapsulation sequences which may
include a part of the gag gene (Bender et al., J. Virol. 61 ( 1987) 1639).
Recombinant retroviral vectors are
purified by standard techniques known to those having ordinary skill in the
art.
Retroviral vectors can be constructed to function as infections particles or
to undergo a single
round of transfection. In the former case, the virus is modified to retain all
of its genes except for those
2S responsible for oncogenic transformation properties, and to express the
heterologous gene. Non-infectious
viral vectors are prepared to destroy the viral packaging signal, but retain
the structural genes required to
package the co-introduced virus engineered to contain the heterologous gene
and the packaging signals.
Thus, the viral particles that are produced are not capable of producing
additional virus.
Targeted gene delivery is described in International Patent Publication WO
95/28494, published
October 1995.
Non-viral vectors
Alternatively, the vector can be introduced in vivo by lipofection. For the
past decade, there has
been increasing use of liposomes for encapsulation and transfection of nucleic
acids in vitro. Synthetic
cationic lipids designed to limit the difficulties and dangers encountered
with liposome mediated
3S transfection can be used to prepare liposomes for in vivo transfection of a
gene encoding a marker
CA 02336543 2001-O1-26
WO 00/06601 32 PCT/US99/17116
[Felgner, et. al., Proc. Natl. Acad Sci. U.S.A. 84:7413-7417 ( 1987); see
Mackey, et al., Proc. Natl. A~cad.
Sci. U.S.A. 85:8027-8031 ( 1988); Ulmer et al., Science 259:1745-1748 (
1993)]. The use of cationic lipids
may promote encapsulation of negatively charged nucleic acids, and also
promote fusion with negatively
charged cell membranes [Felgner and Ringold, Science 337:387-388 ( 1989)].
Particularly useful lipid
compounds and compositions for transfer of nucleic acids are described in
International Patent
Publications W095/18863 and W096/17823, and in U.S. Patent No. 5,459,127. The
use of lipofection to
introduce exogenous genes into the specific organs in vivo has certain
practical advantages. Molecular
targeting of liposomes to specific cells represents one area of benefit. It is
clear that directing transfection
to particular cell types would be particularly advantageous in a tissue with
cellular heterogeneity, such as
pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to
other molecules for the
purpose of targeting [see Mackey, et. al., supra]. Targeted peptides, e.g.,
hormones or neurotransmitters,
and proteins such as antibodies, or non-peptide molecules could be coupled to
liposomes chemically.
Other molecules are also useful for facilitating transfection of a nucleic
acid in vivo, such as a
cationic oligopeptide (e.g., International Patent Publication W095/21931),
peptides derived from DNA
I S binding proteins (e.g., International Patent Publication W096/25508), or a
cationic polymer (e.g.,
International Patent Publication W095/21931 ).
It is also possible to introduce the vector in vivo as a naked DNA piasmid.
Naked DNA vectors
for gene therapy can be introduced into the desired host cells by methods
known in the art, e.g.,
transfection, efectroporation, microinjection, transduction, cell fusion, DEAF
dextran, calcium phosphate
precipitation, use of a gene gun, or use of a DNA vector transporter [see,
e.g., Wu et al., J. Biol. Chem.
267:963-967 (1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut
et al., Canadian
Patent Application No. 2,012,311, filed March 15, 1990; Williams et al., Proc.
Natl. Acad Sci. USA
88:2726-2730 ( 1991 )]. Receptor-mediated DNA delivery approaches can also be
sued [Curiel et al., Num.
Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)].
This invention provides several embodiments for specifically inhibiting CaSR
activity in a
patient suffering from a disease or disorder associated with abnormal calcium
levels.
As a first embodiment, CaSR expression is inhibited by nucleic acids
comprising a sequence
complementary to the sequence encoding CaSR, or isoform thereof, and down-
regulating or blocking
its expression. A preferred embodiment comprises an antisense polynucleotide
molecule. Preparation
and use of antisense polynucleotides, DNA encoding antisense RNA molecules and
use of oligo and
genetic antisense is disclosed in WO 92/15680, the entire contents of which
are incorporated herein
by reference.
Antisense nucleic acids of the invention are preferably RNA capable of
specifically
hybridizing with all or part of the sequence selected from the group
consisting of SEQ ID No. 7, SEQ
ID No. 9, and SEQ ID No. 11 or the corresponding messenger RNA. The antisense
sequence of the
present invention may be derived from DNA sequences whose expression in the
cell produces RNA
CA 02336543 2001-O1-26
WO 00/06601 33 PCT/US99/17116 .
complementary to all or part of the CaSR. These antisense sequences can be
prepared by expression
of all or part of the sequence selected from the group consisting of SEQ ID
No. 7, SEQ ID No. 9, and
SEQ 1D No. 11 in the opposite orientation (EP 140 308). Any length of the
antisense sequence is
suitable for practice of the invention so long as it is capable of down-
regulating or blocking
expression of the CasR, or isoform thereof. Preferably, the antisense sequence
is at least 20
nucleotides in length.
In another aspect of this preferred embodiment the nucleic acid encodes
antisense RNA
molecules. In this embodiment, the nucleic acid is operably linked to signals
enabling expression of
the nucleic acid sequence, and is introduced into a cell utilizing,
preferably, recombinant vector
constructs, which will express the antisense nucleic acid once the vector is
introduced into the cell.
Examples of suitable vectors includes plasmids, adenoviruses, adeno-associated
viruses, retroviruses,
and herpes viruses as described above.
Suitable expression signals include transcriptional promoter and termination
sequences.
Among the promoter sequences useful for practice of this invention are
tetracycline-regulated
transcriptional modulators and CMV, SV-40, Ela, MLP, and LTR promoters.
Tetracycline-regulated
transcriptional modulators and CMV promoters are described in WO 96/01313, US
5,168,062 and
5,385,839, the entire contents of which are incorporated herein by reference.
The nucleic acid
constructs of this invention are capable of down-regulating or blocking
expression of a CaSR, or
isoform thereof, and are delivered, in a preferred aspect of the invention,
locally to cells capable of
regulating calcium levels in a patient.
A second embodiment of the present invention's method of specifically
inhibiting human
CaSR activity, or an isoform thereof, at selected sites, comprises inhibiting
CaSR function by
expression of a nucleic acid sequence encoding an intracellular binding
protein capable of selectively
interacting with the CaSR, or isoform thereof, within a transfected cell. WO
94/29446 and WO
94/02610, the entire contents of which are incorporated herein by reference,
disclose cellular
transfection with genes encoding an intracellular binding protein. An
intracellular binding protein
includes any protein capable of selectively interacting, or binding, with a
CaSR, or isoform thereof, in
the cell in which it is expressed and of neutralizing the function of bound
CaSR. Preferably, the
intracellular binding protein is an antibody or a fragment of an antibody.
More preferably, the
antibody or fragment thereof binds the cytoplasmic domain of the CaSR. Most
preferably, the
intracellular binding protein is a single chain antibody capable of inhibiting
cellular calcium sensing.
WO 94/02610 discloses preparation of antibodies and identification of the
nucleic acid
encoding a particular antibody. Using the CaSR or an isoform thereof, a
monoclonal antibody
specific for the cytoplasmic domain is prepared by according to techniques
known to those skilled in
the art. A vector comprising the nucleic acid encoding an intracellular
binding protein, or a portion
thereof, and capable of expression in a host cell is subsequently prepared for
use in the method of this
invention. Suitable vectors and methods of delivering nucleic acids encoding
intracellular binding
proteins to cells containing a CaSR include those discussed above for delivery
of antisense nucleic
CA 02336543 2001-O1-26
WO 00/06601 34 PCT/US99/17116 .
acids.
In a preferred aspect of this second embodiment, the nucleic acid sequence
encoding a G''aSR
intracellular binding protein additionally comprises a sequence encoding a
localization signal for
targeting the intracellular binding protein to the cellular location of CaSR
and/or a sequence enabling
insertion of the intracellular binding protein in the plasma membrane. The
localization signal or
insertion sequence can be located anywhere on the intracellular binding
protein, so long as it does not
interfere with binding to the CaSR or isoform thereof. Examples of
localization signals are disclosed
in WO 94/02610. Preferably, the localization signal targets the intracellular
binding protein to the
plasma membrane.
The present invention may be better understood by reference to the following
non-limiting
Examples, which are provided as exemplary of the invention.
EXAMPLES
IS
Material and Methods
General Materials and Methods:
Bacterial strain. The strain TG 1 of Escherichia toll of the genotype supE,
hsdDS, thi, D(lac-
proAB), F'[tra D36 pro A+B+ IacIq lacZDMISJ may be used as a means to amplify
and isolate the
recombinant plasmids utilized.
It may be cultivated on:
LB medium: -NaCI (5 g/1) (Difco)
-Bacto-tryptone ( 10 g/1) (Difco)
-Yeast extract (5 g/I) (Difco)
This medium is rendered solid by the addition de 20 g/I of agar (Difco).
Ampicillin (100 pg/ml) permits
selection of the bacteria that have received the plasmids that carry the gene
imparting resistance to this
antibiotic as a marker.
Plasmids.
Bluescript series vectors (Stratagene), may used. These vectors permit cloning
to be performed
just like the pMTL series (Chambers et al.; Gene 1988, 68, pp 139-149).
In addition, the vectors pCDNA3 (lnvitrogen) and derivative vectors (pSG42 and
pCNWB), which
permit the expression of proteins in mammal cells under the control of the CMV
promoter, may be used.
Also, the vectors pCRII or pCR2.1 (Invitrogen), which permit cloning of PCR
fragments, may be
used.
CA 02336543 2001-O1-26
WO 00/06601 35 PCT/US99/17116
The genetic engineering techniques used to clone and insert cDNAs into these
plasmids empty
routine protocols (Maniatis T. et al., "Molecular Cloning, a Laboratory
Manual," Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F.M. et al. (eds.),
"Current Protocols in Molecular
Biology," John Wiley & Sons, New York, 1987).
Preparation of the plasmid DNA. Large quantities of DNA may be prepared using
Promega's
rapid DNA preparation kit in accordance with the manufacturer's instructions.
Small quantities of DNA
may be prepared in the following manner: bacteria containing the plasmid are
cultivated for at least 4
hours in 2 ml of LB medium in a shaker with agitation. They are then
centrifuged for 2 minutes at 14,000
rpm in Eppendorf tubes, then the concentrate is put back in suspension in 100
pl of solution I (50 mM of
glucose, 25 mM of Tris-HC1 pH 8 buffer, 10 mM of EDTA pH 8), lysed with 200 pl
of solution II (0.2 M
of NaOH, 1 % SDS). The lysis solution is then neutralized with 150 pl of
solution III (3 M of potassium
acetate, 11.5% (v/v) glacial acetic acid). After agitation of the tubes until
a flocculent precipitate is
obtained, 1 SO pl of a mixture of phenol/chloroform (50% phenol and 50%
chloroform saturated in water)
is added, and the entire mixture is agitated for 30 seconds. The aqueous phase
containing the DNA is
1 S recovered after centrifugation for 2 minutes at 14,000 rpm. The DNA is
then precipitated via the addition
of 0.5 volume of isopropanol, then centrifuged for 5 minutes at 14.000 rpm and
air-dried in order to finally
be dissolved in 20 pl of TE-RNAse (solution of 10 mM of Tris-HCI and 1 mM of
EDTA with 50 ~cg/ml of
RNAse).
Enzyme amplification of DNA by Polymerase Chain Reaction (PCR). PCR reactions
may be
carried out in a final volume of 100 pl in the presence of the double stranded
DNA, dNTP (0.2 mM), PCR
buffer (10 mM of Tris-HCL pH 8.5, 1 mM of MgCl2, 5 mM of KCI, gelatin 0.01 %),
0.5 pg of each of the
oligonucleotides, and 2.5 IU of Ampli Taq DNA polymerase (Perkin Elmer) with
or without formamide
(5%). The mixture iss covered with 2 drops of paraffin oil to limit
evaporation of the sample. The
equipment used may be Appligene's "Crocodile II." Unless otherwise specified,
denaturation is effected
at a temperature of 90°C for denaturation of the helix, a temperature
for hybridization of the
oligonucleotides to the denatured (single-stranded) DNA that iss 5 to 10
degrees lower than the
temperature for the separation of the oligonucleotides, and a temperature of
72°C for elongation by the
enzyme. The fragments obtained by PCR, which are used for cloning, are
systematically resequenced
once they were cloned, so as to verify the absence of any mutations that might
have occurred during the
amplification.
The ofigodeoxynucleotides may be chemically synthesized according to the
phosphoramidite
method by utilizing I3-cyanoethyl protector groups. After synthesis. the
protector groups are eliminated by
treatment with ammonia, and two precipitations with butanol permit
purification and concentration of the
oligodeoxynucleotides. The DNA concentration may be determined by measuring
the optical density at
260 nm.
CA 02336543 2001-O1-26
WO 00/Ob601 36 PCT/US99/17116 -
Legations. Legation reactions may be carried out at +14°C for one night
in a final volume of l;Q pl
in the presence of 100 to 200 ng of vector, 0.5 to 2 pg of insert, 40 IU of
enzyme T4 DNA ligase
(Biolabs), and a legation buffer (50 mM of Tris-HCI pI-I 7.8; 10 mM of MgCl2;
10 mM of DTT; 1 mM of
ATP). The negative control is formed by the legation of the vector in the
absence of the insert.
The filling of the prominent 5' ends is carried out, as needed, before
legation via the Klenow
fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's
specifications. The
destruction of the prominent 3' ends is accomplished in the presence of DNA
Polymerase of the T4 phage
(Biolabs) used according to the manufacturer's recommendations.
Transformation of bacteria. The entire legation volume ( 10 pl) may be used to
transform bacteria,
which may be rendered competent by the method of Chung et al. ( 1988, Proc.
Natl. Acad. Sci. 86:2172-
2175). The bacteria are placed in culture in a liquid LB medium for several
hours in an incubator with
agitation at 37°C until an OD of 0.6 was obtained at 600 nm. The medium
is then centrifuged at 6,000 rpm
for 10 mn. The bacteria are rendered competent by dissolving the bacterial
concentrate in a volume of TSB
(LB medium + 100 g/l of PEG 4000, 5% DMSO, l0 mM of MgCl2, 10 mM of MgS04)
corresponding to
1 /10 of the volume of the medium of the initial culture. After incubation at
4°C for 30 to 60 minutes, 200
Irl of bacteria are placed in contact with the legation products for 15
minutes on ice. After the addition of
200 pl of LB [medium], the bacteria are incubated for 30 mn at 37°C,
then spread out on an LB +
ampicillin medium.
Separation and extraction of the DNA. The separation of the DNA is performed
by
electrophoresis as a function of their size. In order to do this, different
gels are used depending on the size
of the fragments to be separated:
-1% agarose gel (Gibco BRL) in a TBE buffer (90 mM of Tris base; 90 mM of
borate: 2 mM of
EDTA) to separate large DNA fragments (greater than 500 bp);
-2% NuSieve agarose gel (FMC Bioproducts) in a TBE buffer to separate small
fragments (less
than S00 bp).
Migration on agarose gel or on polyacrylamide gel is carried out in a TBE
buffer and in the
presence of a molecular weight marker (1 Kb ladder. Gibco BRL). The DNA was
mixed with 1/10 of the
deposit volume of blue (200 g/I of Ficoll, 0.5 g/I of bromophenol blue, 50 mM
of EDTA) before being
deposited on the gel. After migration at 100 Volts and staining with ethidium
bromide (concentration 0.5
pg/ml of gel), the bands are viewed under a UV lamp.
Extraction of the DNA from the band of an agarose gel is carried out by means
of electroelution as
follows: the piece of gel containing the DNA fragment s cut out with a scalpel
and placed in a dialysis tube
closed with two clamps and containing 100 to 500 pl of TBE. The entire mixture
is placed in an
electrophoresis tank, where it is subjected to an electrical field of 100
Volts. After being removed from the
gel, the DNA is then purified by means of two extractions with
phenol/chloroform followed by two
CA 02336543 2001-O1-26
WO 00/06601 37 PCT'/US99/17116
extractions with chloroform. then precipitated in the presence of 0.3 M of
sodium acetate and 2.5 volume
of absolute alcohol. After centrifugation (5 mn at 14,000 rpm), the DNA
concentrate is dried and then
dissolved in 20 pl of water.
Fluorescent sequencing oJplasmid DNA. The sequencing may be carried out
according to
Sanger's method using 4 dideoxyribonucleotides possessing a different
fluorescent marker. The
incorporation of one of these dideoxyribonucleotides causes a halt in the
replication by the poiymerase
Taq of the DNA to be sequenced. This reaction yields DNA fragments of various
sizes, all of which are
terminated at 3' by one of the 4 dideoxyribonucleotides. One pg of a plasmid
and 4 picomoles of a primer
are added to 9.5 pl of a "premix" supplied by Applied Biosystems under the
trademark PRISM~. The
final volume is 20 ul in order to perform a PCR for 25 cycles, broken down
into a denaturation phase at
96°C for 30 seconds, a hybridization phase at 50°C for 15
seconds, and an elongation phase at 60°C for 4
minutes. DNA fragments obtained after amplification are purified on an
exclusion column (Chromaspin-
30 from Clontech) and are then dried in a Speed Vac. All of the dried material
is dissolved in 5 pl of a
mixture made up of 24 pl of EDTA (SO mM) and 120 pl of deionized formamide.
After denaturation at
96°C for 3 minutes, 3 to S pl are deposited on an electrophoresis gel.
The different DNA fragments are
separated according to their size and then successively passed in front of a
laser reader of the ABI 370
DNA sequencer (Applied Biosystems), where the different fluorescent
chromophores are detected.
EXAMPLE 1: PCR amplification of CaSR splice variants
First-strand cDNA, isolated from total RNA from normal human adult tissue, was
purchased
from lnvitrogen. The RNA was subsequently treated with DNase (RNase-free) to
eliminate genomic
DNA contamination. Ten micrograms (l0ug) of the RNA is primed with an Oligo
(dT) primer and
reverse transcribed with MMLV reverse transcriptase. The reaction is stopped
by incubating at 65°C
for 10 minutes. The eDNA is in 40u1 of RT buffer. (IxRT Buffer: SOmM Tris HC1,
pH 8.3, 75 mM
KCI, 3mM MgCl2, IOmM DTT).
CA 02336543 2001-O1-26
WO 00/06601 38 PCT/US99/17116
The following oligonucleotide primers were used to identify CaSR isoforms:
Primer Sequence Wild tune
CaSR position
1-AS 5'-CATGGGTCAATTCACTGTCAT-3' 3383 - 3403 (SEQ
ID
NO:1 )
3-AS 5'-GCCAGATCACACAGATGACAA-3' 2207 - 2227 (SEQ
ID
N0:2)
4-AS S'-GGCATAGACGTTGTAATACCC-3' 1525 - 1545 (SEQ
ID
N0:3)
5-AS 5'-TGTGGACAGACTTCCTGGGAT-3' 1013 - 1033 (SEQ
ID
N0:4)
5-S 5'-ATCCCAGGAAGTCTGTCCACA-3' 1013 - 1033 (SEQ
ID
NO:S)
7-S 5'-ACTCCTAGCTGTCTCATCCCT-3' -44 - -24 (SEQ
ID
N0:6)
Splice variant CaSRc was amplified with Perkin Elmer's AmpliTaq Gold. The
final reaction mix
consisted of IOmM Tris-HC1, pH 8.3, SOmM KC1, I.SmM MgCl2, 0.001 (w/v)
gelatin, 0.8mM dATP,
0.8mM dCTP, 0.8mM dGTP, 0.8mM dTTP, 2.5 Units AmpIiTaq Gold, 0.4uM Primer 3-
AS, 0.4uM
Primer 5-S, and 2u1 Invitrogen human kidney cDNA / 100u1 reaction. Reaction
conditions are: hold
at 95°C for 9 minutes; 40 cycles of 94°C - 30 seconds,
60°C - 1 minute; and hold at 60°C for 10
minutes.
CaSRb was amplified using the same conditions as CaSRc, except that 0.4uM
Primer S-AS
and 0.4uM Primer 7-S were used in place of Primers 3-AS and S-S.
Splice variant CaSRd was amplified using the same components as CaSRc with the
following
exceptions: 0.4uM Primer I-AS and 0.4uM Primer 7-S were used in place of
Primers 3-AS and 5-S,
and 3ul instead of 2u1 of Invitrogen human kidney cDNA / 100u1 reaction was
used. Reaction
conditions were: hold at 95°C for 9 minutes; 20 cycles of 94°C -
30 seconds, 64°C - 3 minutes; 20
cycles of 94°C - 30 seconds, 64°C - 3 minutes (increment 10
seconds / cycle); hold at 60°C for 10
minutes. SOuI of this reaction mix was then electrophoresed on a 1 % agarose
gel and the bands in the
3.0 - 3.4kb region were excised and extracted using Qiagen's Gel Extraction
kit and eluted in SOuI
ddH20. 1 ul of this extract was used as a template for the next reaction which
contained the same
components as the previous reaction with the following exceptions: 0.4uM
Primer 4-AS was used
instead of Primer 1-AS, and the template was replaced. Reaction conditions for
the first and second
CA 02336543 2001-O1-26
WO 00/06601 39 PCT/US99/17116 .
amplifications were identical.
All PCR products were cloned into pCR2.1 according to Invitrogen's protocols.
Sequenciltg
was performed with an automated DNA sequencer.
EXAMPLE 2: Expression of CaSRb and CaSRc in human kidn~
The strategy of searching for CaSR splice variants involved the use of primer
pairs to scan
different parts of of the sensor cDNA. Human kidney first strand cDNA was used
as template and was
amplified with either primer pair 3AS/SS or SAS/7S. Electrophoretic resolution
of the PCR mixture
obtained with primer pair 3AS/SS revealed the presence of a product with the
expected size ( l.2kb)
of the wild type CaSR and one with lower molecular mass of approximately
1.Okb. The primer pair
SAS/7S also yielded two visible PCR products with estimated size of 1.0 and
0.7kb. The l.Okb
product corresponded to the expected size of the wild type CaSR. The PCR
products from both
primer pairs were ligated into pCR 2.1 and multiple clones were selected for
analysis by restriction
digestion with EcoRl to release the insert. Clones bearing the putative wild
type CaSR insert and
those with smaller insert size were sequenced. The results of these
experiments confirmed the
presence of the correct CaSR sequence in the putative wild type clones. On the
other hand, clones
with shorter insert were found to contain either a deletion from nucleotide
186-495 or from nucleotide
1378-1608. The 186-495 deleted CaSR corresponds to CaSRb described originally
in medullary
thyroid carcinoma. The 1378-1608 deleted CaSR is designated as CaSRc (Figure
1). Unlike CaSRb,
the deletion in CaSRc does not cause a shift in reading frame.
Example 3: Expression of CaSRd in human kidney
Following an initial PCR enrichment of human kidney first strand cDNA with
primer pair
IAS/7S, products in the 3.0 - 3.4 kb range were gel purified and were
amplified further with primer
pair 4AS/7S. The PCR products were isolated by TA cloning and sequenced. One
of these clones was
found to contain a deletion from nucleotide 1075-1386 (Figure 1). The 1075-
1386 deleted CaSR is
designated CaSRd. No change in reading frame was detected in this
alternatively spliced CaSR
transcript.
Example 4: Stable exaression of isoform CaSRd in HEK-293 cells
Full length CaSRd was cloned into the mammalian expression vector pCEP4 (from
lnvitrogen). The CaSRd DNA used for transfection was prepared using the Qiagen
plasmid
CA 02336543 2001-O1-26
WO 00/06601 40 PCT/US99/17116 _
preparation kit. LipofectAMINE (Life Technologies, Inc.) was used as a carrier
for transfection.
Transfection of HEK-293 cells with CaSRd DNA was performed according to the
general protoccft
described in the LipofectAMINE transfection kit. The CaSRd DNA and
lipofectamine complex (1 ml)
was overiayed onto HEK-293 cells (90% confluent) in 6 well plates. After 5 hr.
at 37°C, 1 ml of
DMEM containing 20% fetal bovine serum, penicillin and streptomycin was added
to each well. After
incubating at 37°C for 16 hours, the media was replaced with 2 ml of
DMEM containing 10% bovine
serum albumin, penicillin and streptomycin and the cultures were incubated
further for 8 hours at
37°C. CaSRd transfectants were isolated by selection in the presence of
hygromycin following limited
dilution. The cells in each well were trypsinized, and cultures were diluted
in 100 ml of DMEM
containing 10% fetal bovine serum, 200 ug/ml of hygromycin, penicillin and
streptomycin. 1 ml
aliquots of the diluted cultures were added to each well of several 24 well
tissue culture plates. After 4
weeks in culture, wells containing a single colony were identified and each
cell clone was expanded
into a T75 flask. The expression of CaSRd in each cell clone was monitored by
Northern analysis. A
clone, 21/2 with the highest expression level was used for functional analysis
as described below.
The function of isoform CaSRd was assayed by its ability to increase
intraceltular
concentration in response to elevation in extracellular calcium concentrations
and other agonists. The
wild type receptor has been shown to increase intracellular calcium
concentration when extracellular
calcium concentrations were raised. Intracellular calcium was measured with
the fluorescent
indicator, fura-2 (from Molecular Probes). HEK-293 cells transfected with
CaSRd was loaded in
buffer containing O.SuM fura-2, 20mM HEPES, pH 7.35, 0.1% BSA, O.SmM CaCl2,
O.SmM MgClz,
6.7mM KC1, 3mM glucose and 142mM NaCI for 45 min at 37°C. The cells
were washed and
resuspended to 2 x 106 cells/ml in the loading buffer without furs-Z. For
intracellular calcium
measurement, cells were placed in a quartz cuvette equilibrated at
37°C. Excitation monochrometers
were centered at 340 and 380 nm with emission light collected at 505 nm.
Different CaSR agonists
were added to different final concentrations to activate the CaSR. Usually a
final concentration of
IOmM external CaClz is sufficient to activate the wild type receptor
maximally. Results indicated that
CaSRd did not respond to agonists such as Ca~, Mg++ and neomycin but did
respond to gadolinium
(Table 1). In the presence of a CaSR potentiating compound, NPS568 (WO
94/18959, Fox et al.,
(1993) J. Bone Min. Res. 8: 5181; Abstract #260) CaSRd responded to calcium,
magnesium and
neomycin.
Table I
Effects of extracellular Ca+", NPS568 and gadolinium on intracellular calcium
level in CaSRd
expressing HEK-293 cells
CA 02336543 2001-O1-26
WO 00/06601 41 PCT/US99/17116 .
Increase in furs-2 fluorescence (cps x 106)
4
Expt 1 Expt 2
20mM Caz+ 0.2 ' 0
20mM Ca2' + 1.5 1.7
2uM NPS568
lOuM NPS568 0.2 0.1
100uM gadolinium 2.0
The present invention is not to be limited in scope by the specific
embodiments described herein.
Indeed, various modifications of the invention in addition to those described
herein will become apparent
to those skilled in the art from the foregoing description and the
accompanying figures. Such
modifications are intended to fall within the scope of the appended claims.
It is further to be understood that all base sizes or amino acid sizes, and
all molecular weight or
molecular mass values, given for nucleic acids or polypeptides are
approximate, and are provided for
description.
Various publications are cited herein, the disclosures of which are
incorporated by reference in their
entireties.
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1
SEQUENCE LISTING
4
<110> Yu, Kin Tak
Thrower, Larry W.
Labaudiniere, Richard F.
<120> ISOFORMS OF HUMAN CALCIUM SENSING RECEPTOR
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CA 02336543 2001-O1-26
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2
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Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala Leu Thr Trp His
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Thr Ser Ala Tyr Gly Pro Asp Gln Arg Ala Gln Lys Lys Gly Asp Ile
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Gln Asp Leu Lys Ser Arg Pro Glu Ser Val Glu Cys Ile Arg Tyr Asn
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Phe Arg Gly Phe Arg Trp Leu Gln Ala Met Ile Phe Ala Ile Glu Glu
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Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg
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Ser Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe
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Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala Val Val.Gly Ala
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Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe
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Tyr Ile Pro Gln Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn
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Lys Asn Gln Phe Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His
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Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu
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4
720
Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser
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Glu Leu Ile Ser Gln Tyr Ser Asp Glu Glu Glu Ile Gln His Val Val
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Glu Val Ile Gln Asn Ser Thr Ala Lys Val Ile Val Val Phe Ser Ser
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Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val Arg Arg Asn Ile
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Thr Gly Lys Ile Trp Leu Ala Ser Glu Ala Trp Ala Ser Ser Ser Leu
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Ile Ala Met Pro Gln Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe
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Ala Leu Lys Ala Gly Gln Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys
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Val His Pro Arg Lys Ser Val His Asn Gly Phe Ala Lys Glu Phe Trp
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Glu Glu Thr Phe Asn Cys His Leu Gln Glu Gly Ala Lys Gly Pro Leu
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Pro Val Asp Thr Phe Leu Arg Gly His Glu Glu Ser Gly Asp Arg Phe
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Ser Asn Ser Ser Thr Ala Phe Arg Pro Leu Cys Thr Gly Asp Glu Asn
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Ser Tyr Asn Val Tyr Leu Ala Val Tyr Ser Ile Ala His Ala Leu Gln
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6
Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu
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7
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4
Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala Tyr Ala Ser Thr Tyr
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Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala
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Pro Ser Ser Ser Ile Ser Ser Lys Ser Asn Ser Glu Asp Pro Phe Pro
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Gln Pro Glu Arg Gln Lys Gln Gln Gln Pro Leu Ala Leu Thr Gln Gln
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Glu Gln Gln Gln Gln Pro Leu Thr Leu Pro Gln Gln Gln Arg Ser Gln
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Thr Phe Ser Leu Ser Phe Asp Glu Pro Gln Lys Asn Ala Met Ala His
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900 905 910
4
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2784
Arg Asn Ser Thr His Gln Asn Ser Leu Glu Ala Gln Lys Ser Ser Asp
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Thr Leu Thr Arg His Gln Pro Leu Leu Pro Leu Gln Cys Gly Glu Thr
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2880
Asp Leu Asp Leu Thr Val Gln Glu Thr Gly Leu Gln Gly Pro Val Gly
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2928
Gly Asp Gln Arg Pro Glu Val Glu Asp Pro Glu Glu Leu Ser Pro Ala
965 970 975
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2976
Leu Val Val Ser Ser Ser Gln Ser Phe Val Ile Ser Gly Gly Gly Ser
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995 1000
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Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg
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Val His Pro Arg Lys Ser Val His Asn Gly Phe Ala Lys Glu Phe Trp
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Glu Glu Thr Phe Asn Cys His Leu Gln Glu Gly A1a Lys Gly Pro Leu
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355 360 365
Pro Val Asp Thr Phe Leu Arg Gly His Glu Glu Ser Gly Asp Arg Phe
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Ser Asn Ser Ser Thr Ala Phe Arg Pro Leu Cys Thr Gly Asp Glu Asn
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Cys Ala Asp Ile Lys Lys Val Glu Ala Trp Gln Val Pro Phe Ser Asn
450 455 460
Cys Ser Arg Asp Cys Leu Ala Gly Thr Arg Lys Gly Ile Ile Glu Gly
465 470 475 480
Glu Pro Thr Cys Cys Phe Glu Cys Val Glu Cys Pro Asp Gly Glu Tyr
485 490 495
Ser Asp Glu Thr Asp Ala Ser Ala Cys Asn Lys Cys Pro Asp Asp Phe
500 505 510
Trp Ser Asn Glu Asn His Thr Ser Cys Ile Ala Lys Glu Ile Glu Phe
515 520 525
Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu Thr Leu Phe Ala Val
530 535 540
Leu Gly Ile Phe Leu Thr Ala Phe Val Leu Gly Val Phe Ile Lys Phe
545 550 555 560
Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu
565 570 575
Leu Leu Phe Ser Leu Leu Cys Cys Phe Ser Ser Ser Leu Phe Phe Ile
580 585 590
Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu Arg Gln Pro Ala Phe Gly
595 600 605
Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg
610 615 620
Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Phe His Arg Lys
625 630 635 640
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116 .
11
Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu Val Phe Leu Cys Thr Phe
645 650 655
4
Met Gln Ile Val Ile Cys Val Ile Trp Leu Tyr Thr Ala Pro Pro Ser
660 665 670
Ser Tyr Arg Asn Gln Glu Leu Glu Asp Glu Ile Ile Phe Ile Thr Cys
675 680 685
His Glu Gly Ser Leu Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys
690 695 700
Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu
705 710 715 720
Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile
725 730 735
Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala Tyr Ala Ser Thr Tyr
740 745 750
Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ala Ser
755 760 765
Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys Ile Tyr Ile Ile Leu
770 775 780
Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala
785 790 795 800
Ala His Ala Phe Lys Val Ala Ala Arg Ala Thr Leu Arg Arg Ser Asn
805 810 815
Val Ser Arg Lys Arg Ser Ser Ser Leu Gly Gly Ser Thr Gly Ser Thr
820 825 830
Pro Ser Ser Ser Ile Ser Ser Lys Ser Asn Ser Glu Asp Pro Phe Pro
835 840 845
Gln Pro Glu Arg Gln Lys Gln Gln Gln Pro Leu Ala Leu Thr Gln Gln
850 855 860
Glu Gln Gln Gln Gln Pro Leu Thr Leu Pro Gln Gln Gln Arg Ser Gln
865 870 875 880
Gln Gln Pro Arg Cys Lys Gln Lys Val Ile Phe Gly Ser Gly Thr Val
885 890 895
Thr Phe Ser Leu Ser Phe Asp Glu Pro Gln Lys Asn Ala Met Ala His
900 905 910
Arg Asn Ser Thr His Gln Asn Ser Leu Glu Ala Gln Lys Ser Ser Asp
915 920 925
CA 02336543 2001-O1-26
WO 00/06601 PCTNS99/17116 .
12
Thr Leu Thr Arg His Gln Pro Leu Leu Pro Leu Gln Cys Gly Glu Thr
4
930 935 940
Asp Leu Asp Leu Thr Val Gln Glu Thr Gly Leu Gln Gly Pro Val Gly
945 950 955 960
Gly Asp Gln Arg Pro Glu Val Glu Asp Pro Glu Glu Leu Ser Pro Ala
965 970 975
Leu Val Val Ser Ser Ser Gln Ser Phe Val Ile Ser Gly Gly Gly Ser
980 985 990
Thr Val Thr Glu Asn Val Val Asn Ser
995 1000
<210> 9
<211> 2922
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(2922)
<400> 9
atg gca ttt tat agc tgc tgc tgg gtc ctc ttg gca ctc acc tgg cac 48
Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala Leu Thr Trp His
1 5 10 15
acc tct gcc tac ggg cca gac cag cga gcc caa aag aag ggg gac att 96
Thr Ser Ala Tyr Gly Pro Asp Gln Arg Ala Gln Lys Lys Gly Asp Ile
20 25 30
atc ctt ggg ggg ctc ttt cct att cat ttt gga gta gca get aaa gat
144
Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp
35 40 45
caa gat ctc aaa tca agg ccg gag tct gtg gaa tgt atc agg tat aat
192
Gln Asp Leu Lys Ser Arg Pro Glu Ser Val Glu Cys Ile Arg Tyr Asn
50 55 60
ttc cgt ggg ttt cgc tgg tta cag get atg ata ttt gcc ata gag gag
240
Phe Arg Gly Phe Arg Trp Leu Gln Ala Met Ile Phe Ala Ile Glu Glu
65 70 75 80
ata aac agc agc cca gcc ctt ctt ccc aac ttg acg ctg gga tac agg
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116
13
288
Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg
85 90 95
ata ttt gac act tgc aac acc gtt tct aag gcc ttg gaa gcc acc ctg
336
Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu
100 105 110
agt ttt gtt get caa aac aaa att gat tct ttg aac ctt gat gag ttc
384
Ser Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe
115 120 125
tgc aac tgc tca gag cac att ccc tct acg att get gtg gtg gga gca
432
Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala
130 135 140
act ggc tca ggc gtc tcc acg gca gtg gca aat ctg ctg ggg ctc ttc
480
Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe
145 150 155 160
tac att ccc cag gtc agt tat gcc tcc tcc agc aga ctc ctc agc aac
528
Tyr Ile Pro Gln Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn
165 170 175
aag aat caa ttc aag tct ttc ctc cga acc atc ccc aat gat gag cac
576
Lys Asn Gln Phe Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His
180 185 190
cag gcc act gcc atg gca gac atc atc gag tat ttc cgc tgg aac tgg
624
Gln Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe Arg Trp Asn Trp
195 200 205
gtg ggc aca att gca get gat gac gac tat ggg cgg ccg ggg att gag
672
Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu
210 215 220
aaa ttc cga gag gaa get gag gaa agg gat atc tgc atc gac ttc agt
720
Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser
225 230 235 240
gaa ctc atc tcc cag tac tct gat gag gaa gag atc cag cat gtg gta
768
Glu Leu Ile Ser Gln Tyr Ser Asp Glu Glu Glu Ile Gln His Val Val
245 250 255
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116
14
gag gtg att caa aat tcc acg gcc aaa gtc atc gtg gtt ttc tcc agt
816
Glu Val Ile Gln Asn Ser Thr Ala Lys Val Ile Val Val Phe Ser Ser
260 265 270
ggc cca gat ctt gag ccc ctc atc aag gag att gtc cgg cgc aat atc
864
Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val Arg Arg Asn Ile
275 280 285
acg ggc aag atc tgg ctg gcc agc gag gcc tgg gcc agc tcc tcc ctg
912
Thr Gly Lys Ile Trp Leu Ala Ser Glu Ala Trp Ala Ser Ser Ser Leu
290 295 300
atc gcc atg cct cag tac ttc cac gtg gtt ggc ggc acc att gga ttc
960
Ile Ala Met Pro Gln Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe
305 310 315 320
get ctg aag get ggg cag atc cca ggc ttc cgg gaa ttc ctg aag aag
1008
Ala Leu Lys Ala Gly Gln Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys
325 330 335
gtc cat ccc agg aag tct gtc cac aat ggt ttt gcc aag gag ttt tgg
1056
Val His Pro Arg Lys Ser Val His Asn Gly Phe Ala Lys Glu Phe Trp
340 345 350
gaa gaa aca ttt aac tgc cac cta cgg cat cta aac ttt aca aac aat
1104
Glu Glu Thr Phe Asn Cys His Leu Arg His Leu Asn Phe Thr Asn Asn
355 360 365
atg ggg gag cag gtg acc ttt gat gag tgt ggt gac ctg gtg ggg aac
1152
Met Gly Glu Gln Val Thr Phe Asp Glu Cys Gly Asp Leu Val Gly Asn
370 375 380
tat tcc atc atc aac tgg cac ctc tcc cca gag gat ggc tcc atc gtg
1200
Tyr Ser Ile Ile Asn Trp His Leu Ser Pro Glu Asp Gly Ser Ile Val
385 390 395 400
ttt aag gaa gtc ggg tat tac aac gtc tat gcc aag aag gga gaa aga
1248
Phe Lys Glu Val Gly Tyr Tyr Asn Val Tyr Ala Lys Lys Gly Glu Arg
405 410 415
ctc ttc atc aac gag gag aaa atc ctg tgg agt ggt ttc tcc agg gag
1296
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116 -
Leu Phe Ile Asn Glu Glu Lys Ile Leu Trp Ser Gly Phe Ser Arg Glu
420 425 430
gtg ccc ttc tcc aac tgc agc cga gac tgc ctg gca ggg acc agg aaa
1344
Val Pro Phe Ser Asn Cys Ser Arg Asp Cys Leu Ala Gly Thr Arg Lys
435 440 445
ggg atc att gag ggg gag ccc acc tgc tgc ttt gag tgt gtg gag tgt
1392
Gly Ile Ile Glu Gly Glu Pro Thr Cys Cys Phe Glu Cys Val Glu Cys
450 455 460
cct gat ggg gag tat agt gat gag aca gat gcc agt gcc tgt aac aag
1440
Pro Asp Gly Glu Tyr Ser Asp Glu Thr Asp Ala Ser Ala Cys Asn Lys
465 470 475 480
tgc cca gat gac ttc tgg tcc aat gag aac cac acc tcc tgc att gcc
1488
Cys Pro Asp Asp Phe Trp Ser Asn Glu Asn His Thr Ser Cys Ile Ala
485 490 495
aag gag atc gag ttt ctg tcg tgg acg gag ccc ttt ggg atc gca ctc
1536
Lys Glu Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu
500 505 510
acc ctc ttt gcc gtg ctg ggc att ttc ctg aca gcc ttt gtg ctg ggt
1584
Thr Leu Phe Ala Val Leu Gly Ile Phe Leu Thr Ala Phe Val Leu Gly
515 520 525
gtg ttt atc aag ttc cgc aac aca ccc att gtc aag gcc acc aac cga
1632
Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg
530 535 540
gag ctc tcc tac ctc ctc ctc ttc tcc ctg ctc tgc tgc ttc tcc agc
1680
Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Leu Cys Cys Phe Ser Ser
545 550 555 560
tcc ctg ttc ttc atc ggg gag ccc cag gac tgg acg tgc cgc ctg cgc
1728
Ser Leu Phe Phe Ile Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu Arg
565 570 575
cag ccg gcc ttt ggc atc agc ttc gtg ctc tgc atc tca tgc atc ctg
1776
Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu
580 585 590
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116 -
16
gtg aaa acc aac cgt gtc ctc ctg gtg ttt gag gcc aag atc ccc acc
1824
s
Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr
595 600 605
agc ttc cac cgc aag tgg tgg ggg ctc aac ctg cag ttc ctg ctg gtt
1872
Ser Phe His Arg Lys Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu Val
610 615 620
ttc ctc tgc acc ttc atg cag att gtc atc tgt gtg atc tgg ctc tac
1920
Phe Leu Cys Thr Phe Met Gln Ile Val Ile Cys Val Ile Trp Leu Tyr
625 630 635 640
acc gcg ccc ccc tca agc tac cgc aac cag gag ctg gag gat gag atc
1968
Thr Ala Pro Pro Ser Ser Tyr Arg Asn Gln GIu Leu Glu Asp Glu Ile
645 650 655
atc ttc atc acg tgc cac gag ggc tcc ctc atg gcc ctg ggc ttc ctg
2016
Ile Phe Ile Thr Cys His Glu Gly Ser Leu Met Ala Leu Gly Phe Leu
660 665 670
atc ggc tac acc tgc ctg ctg get gcc atc tgc ttc ttc ttt gcc ttc
2064
Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe
675 680 685
aag tcc cgg aag ctg ccg gag aac ttc aat gaa gcc aag ttc atc acc
2112
Lys Ser Arg Lys Leu Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile Thr
690 695 700
ttc agc atg ctc atc ttc ttc atc gtc tgg atc tcc ttc att cca gcc
2160
Phe Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala
705 710 715 720
tat gcc agc acc tat ggc aag ttt gtc tct gcc gta gag gtg att gcc
2208
Tyr Ala Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala
725 730 735
atc ctg gca gcc agc ttt ggc ttg ctg gcg tgc atc ttc ttc aac aag
2256
Ile Leu Ala Ala Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys
740 745 750
atc tac atc att ctc ttc aag cca tcc cgc aac acc atc gag gag gtg
2304
Ile Tyr Ile Ile Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116 .
I7
755 760 765
4
cgt tgc agc acc gca get cac get ttc aag gtg get gcc cgg gcc acg
2352
Arg Cys Ser Thr Ala Ala His Ala Phe Lys Val Ala Ala Arg Ala Thr
770 775 780
ctg cgc cgc agc aac gtc tcc cgc aag cgg tcc agc agc ctt gga ggc
2400
Leu Arg Arg Ser Asn Val Ser Arg Lys Arg Ser Ser Ser Leu Gly Gly
785 790 795 800
tcc acg gga tcc acc cct tcc tcc tcc atc agc agc aag agc aac agc
2448
Ser Thr Gly Ser Thr Pro Ser Ser Ser Ile Ser Ser Lys Ser Asn Ser
805 810 815
gaa gac cca ttc cca cag ccc gag agg cag aag cag cag cag ccg ctg
2496
Glu Asp Pro Phe Pro Gln Pro Glu Arg Gln Lys Gln Gln Gln Pro Leu
820 825 830
gcc cta acc cag caa gag cag cag cag cag ccc ctg acc ctc cca cag
2544
Ala Leu Thr Gln Gln Glu Gln Gln Gln Gln Pro Leu Thr Leu Pro Gln
835 840 845
cag caa cga tct cag cag cag ccc aga tgc aag cag aag gtc atc ttt
2592
Gln Gln Arg Ser Gln Gln Gln Pro Arg Cys Lys Gln Lys Val Ile Phe
850 855 860
ggc agc ggc acg gtc acc ttc tca ctg agc ttt gat gag cct cag aag
2640
Gly Ser Gly Thr Val Thr Phe Ser Leu Ser Phe Asp Glu Pro Gln Lys
865 870 875 880
aac gcc atg gcc cac agg aat tct acg cac cag aac tcc ctg gag gcc
2688
Asn Ala Met Ala His Arg Asn Ser Thr His Gln Asn Ser Leu Glu Ala
885 890 895
cag aaa agc agc gat acg ctg acc cga cac cag cca tta ctc ccg ctg
2736
Gln Lys Ser Ser Asp Thr Leu Thr Arg His Gln Pro Leu Leu Pro Leu
900 905 910
cag tgc ggg gaa acg gac tta gat ctg acc gtc cag gaa aca ggt ctg
2784
Gln Cys Gly Glu Thr Asp Leu Asp Leu Thr Val Gln Glu Thr Gly Leu
915 920 925
caa gga cct gtg ggt gga gac cag cgg cca gag gtg gag gac cct gaa
CA 02336543 2001-O1-26
WO 00/06601 PCTNS99/17116 .
18
2832
Gln Gly Pro Val Gly Gly Asp Gln Arg Pro Glu Val Glu Asp Pro Glu
4
930 935 940
gag ttg tcc cca gca ctt gta gtg tcc agt tca cag agc ttt gtc atc
2880
Glu Leu Ser Pro Ala Leu Val Val Ser Ser Ser Gln Ser Phe Val Ile
945 950 955 960
agt ggt gga ggc agc act gtt aca gaa aac gta gtg aat tca
2922
Ser Gly Gly Gly Ser Thr Val Thr Glu Asn Val Val Asn Ser
965 970
<210> 10
<211> 974
<212> PRT
<213> Homo Sapiens
<400> 10
Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala Leu Thr Trp His
1 5 10 15
Thr Ser Ala Tyr Gly Pro Asp Gln Arg Ala Gln Lys Lys Gly Asp Ile
20 25 30
Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp
35 40 45
Gln Asp Leu Lys Ser Arg Pro Glu Ser Val Glu Cys Ile Arg Tyr Asn
50 55 60
Phe Arg Gly Phe Arg Trp Leu Gln Ala Met Ile Phe Ala Ile Glu Glu
65 70 75 80
Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg
85 90 95
Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu
100 105 110
Ser Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe
115 120 125
Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala
130 135 140
Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe
145 150 155 160
Tyr Ile Pro Gln Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn
165 170 175
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116 .
19
Lys Asn Gln Phe Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His
180 185 190
Gln Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe Arg Trp Asn Trp
195 200 205
Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu
210 215 220
Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser
225 230 235 240
Glu Leu Ile Ser Gln Tyr Ser Asp Glu Glu Glu Ile Gln His Val Val
245 250 255
Giu Val Ile Gln Asn Ser Thr Ala Lys Val Ile Val Val Phe Ser Ser
260 265 270
Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val Arg Arg Asn Ile
275 280 285
Thr Gly Lys Ile Trp Leu Ala Ser Glu Ala Trp Ala Ser Ser Ser Leu
290 295 300
Ile Ala Met Pro Gln Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe
305 310 315 320
Ala Leu Lys Ala Gly Gln Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys
325 330 335
Val His Pro Arg Lys Ser Val His Asn Gly Phe A1a Lys Glu Phe Trp
340 345 350
Glu Glu Thr Phe Asn Cys His Leu Arg His Leu Asn Phe Thr Asn Asn
355 360 365
Met Gly Glu Gln Val Thr Phe Asp Glu Cys Gly Asp Leu Val Gly Asn
370 375 380
Tyr Ser Ile Ile Asn Trp His Leu Ser Pro Glu Asp Gly Ser Ile Val
385 390 395 400
Phe Lys Glu Val Gly Tyr Tyr Asn Val Tyr Ala Lys Lys Gly Glu Arg
405 410 415
Leu Phe Ile Asn Glu Glu Lys Ile Leu Trp Ser Gly Phe Ser Arg Glu
420 425 430
Val Pro Phe Ser Asn Cys Ser Arg Asp Cys Leu Ala Gly Thr Arg Lys
435 440 445
Gly Ile Ile Glu Gly Glu Pro Thr Cys Cys Phe Glu Cys Val Glu Cys
CA 02336543 2001-O1-26
WO 00/06601 PGT/US99/17116 -
450 455 460
Pro Asp Gly Glu Tyr Ser Asp Glu Thr Asp Ala Ser Ala Cys Asn Lys
465 470 475 480
Cys Pro Asp Asp Phe Trp Ser Asn Glu Asn His Thr Ser Cys Ile Ala
485 490 495
Lys Glu Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu
500 505 510
Thr Leu Phe Ala Val Leu Gly Ile Phe Leu Thr Ala Phe Val Leu Gly
515 520 525
Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg
530 535 540
Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Leu Cys Cys Phe Ser Ser
545 550 555 560
Ser Leu Phe Phe Ile Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu Arg
565 570 575
Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu
580 585 590
Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr
595 600 605
Ser Phe His Arg Lys Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu Val
610 615 620
Phe Leu Cys Thr Phe Met Gln Ile Val Ile Cys Val Ile Trp Leu Tyr
625 630 635 640
Thr Ala Pro Pro Ser Ser Tyr Arg Asn Gln Glu Leu Glu Asp Glu Ile
645 650 655
Ile Phe Ile Thr Cys His Glu Gly Ser Leu Met Ala Leu Gly Phe Leu
660 665 670
Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe
675 680 685
Lys Ser Arg Lys Leu Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile Thr
690 695 700
Phe Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala
705 710 715 720
4
Tyr Ala Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala
725 730 735
CA 02336543 2001-O1-26
WO 00/06601 PCTNS99/17116 .
21
Ile Leu Ala Ala Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys
740 745 750
4
Ile Tyr Ile Ile Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val
755 760 765
Arg Cys Ser Thr Ala Ala His Ala Phe Lys Val Ala Ala Arg Ala Thr
770 775 780
Leu Arg Arg Ser Asn Val Ser Arg Lys Arg Ser Ser Ser Leu Gly Gly
785 790 795 800
Ser Thr Gly Ser Thr Pro Ser Ser Ser Ile Ser Ser Lys Ser Asn Ser
805 810 815
Glu Asp Pro Phe Pro Gln Pro Glu Arg Gln Lys Gln Gln Gln Pro Leu
820 825 830
Ala Leu Thr Gln Gln Glu Gln Gln Gln Gln Pro Leu Thr Leu Pro Gln
835 840 845
Gln Gln Arg Ser Gln Gln Gln Pro Arg Cys Lys Gln Lys Val Ile Phe
850 855 860
Gly Ser Gly Thr Val Thr Phe Ser Leu Ser Phe Asp Glu Pro Gln Lys
865 870 875 880
Asn Ala Met Ala His Arg Asn Ser Thr His Gln Asn Ser Leu Glu Ala
885 890 895
Gln Lys Ser Ser Asp Thr Leu Thr Arg His Gln Pro Leu Leu Pro Leu
900 905 910
Gln Cys Gly Glu Thr Asp Leu Asp Leu Thr Val Gln Glu Thr Gly Leu
915 920 925
Gln Gly Pro Val Gly Gly Asp Gln Arg Pro Glu Val Glu Asp Pro Glu
930 935 940
Glu Leu Ser Pro Ala Leu Val Val Ser Ser Ser Gln Ser Phe Val Ile
945 950 955 960
Ser Gly Gly Gly Ser Thr Val Thr Glu Asn Val Val Asn Ser
965 970
<210> 11
<211> 3234
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116 .
<222> (1)..(3234)
<400> 11
atg gca ttt tat agc tgc tgc tgg gtc ctc ttg gca ctc acc tgg cac 48
Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala Leu Thr Trp His
1 5 10 15
acc tct gcc tac ggg cca gac cag cga gcc caa aag aag ggg gac att 96
Thr Ser Ala Tyr Gly Pro Asp Gln Arg Ala Gln Lys Lys Gly Asp Ile
20 25 30
atc ctt ggg ggg ctc ttt cct att cat ttt gga gta gca get aaa gat
144
Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp
35 40 45
caa gat ctc aaa tca agg ccg gag tct gtg gaa tgt atc agg tat aat
192
Gln Asp Leu Lys Ser Arg Pro Glu Ser Val Glu Cys Ile Arg Tyr Asn
50 55 60
ttc cgt ggg ttt cgc tgg tta cag get atg ata ttt gcc ata gag gag
240
Phe Arg Gly Phe Arg Trp Leu Gln Ala Met Ile Phe Ala Ile Glu Glu
65 70 75 80
ata aac agc agc cca gcc ctt ctt ccc aac ttg acg ctg gga tac agg
288
Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg
85 90 95
ata ttt gac act tgc aac acc gtt tct aag gcc ttg gaa gcc acc ctg
336
Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu
100 105 110
agt ttt gtt get caa aac aaa att gat tct ttg aac ctt gat gag ttc
384
Ser Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe
115 120 125
tgc aac tgc tca gag cac att ccc tct acg att get gtg gtg gga gca
432
Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala
130 135 140
act ggc tca ggc gtc tcc acg gca gtg gca aat ctg ctg ggg ctc ttc
480
Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe
145 150 155 160
CA 02336543 2001-O1-26
WO 00/06601 PCTNS99/17116 -
tac att ccc cag gtc agt tat gcc tcc tcc agc aga ctc ctc agc aac
528
s
Tyr Ile Pro Gln Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn
165 170 175
aag aat caa ttc aag tct ttc ctc cga acc atc ccc aat gat gag cac
576
Lys Asn Gln Phe Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His
180 185 190
cag gcc act gcc atg gca gac atc atc gag tat ttc cgc tgg aac tgg
624
Gln Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe Arg Trp Asn Trp
195 200 205
gtg ggc aca att gca get gat gac gac tat ggg cgg ccg ggg att gag
672
Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu
210 215 220
aaa ttc cga gag gaa get gag gaa agg gat atc tgc atc gac ttc agt
720
Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser
225 230 235 240
gaa ctc atc tcc cag tac tct gat gag gaa gag atc cag cat gtg gta
768
Glu Leu Ile Ser Gln Tyr Ser Asp Glu Glu Glu Ile Gln His Val Val
245 250 255
gag gtg att caa aat tcc acg gcc aaa gtc atc gtg gtt ttc tcc agt
816
Glu Val Ile Gln Asn Ser Thr Ala Lys Val Ile Val Val Phe Ser Ser
260 265 270
ggc cca gat ctt gag ccc ctc atc aag gag att gtc cgg cgc aat atc
864
Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val Arg Arg Asn Ile
275 280 285
acg ggc aag atc tgg ctg gcc agc gag gcc tgg gcc agc tcc tcc ctg
912
Thr Gly Lys Ile Trp Leu Ala Sex Glu Ala Trp Ala Ser Ser Ser Leu
290 295 300
atc gcc atg cct cag tac ttc cac gtg gtt ggc ggc acc att gga ttc
960
Ile Ala Met Pro Gln Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe
305 310 315 320
get ctg aag get ggg cag atc cca ggc ttc cgg gaa ttc ctg aag aag
1008
Ala Leu Lys Ala Gly Gln Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys
CA 02336543 2001-O1-26
WO 00/06601 PCTNS99/17116
24
325 330 335
4
gtc cat ccc agg aag tct gtc cac aat ggt ttt gcc aag gag ttt tgg
1056
Val His Pro Arg Lys Ser Val His Asn Gly Phe Ala Lys Glu Phe Trp
340 345 350
gaa gaa aca ttt aac tgc cac ctc caa gaa ggt gca aaa gga cct tta
1104
Glu Glu Thr Phe Asn Cys His Leu Gln Glu Gly Ala Lys Gly Pro Leu
355 360 365
cct gtg gac acc ttt ctg aga ggt cac gaa gaa agt ggc gac agg ttt
1152
Pro Val Asp Thr Phe Leu Arg Gly His Glu Glu Ser Gly Asp Arg Phe
370 375 380
agc aac agc tcg aca gcc ttc cga ccc ctc tgt aca ggg gat gag aac
1200
Ser Asn Ser Ser Thr Ala Phe Arg Pro Leu Cys Thr Gly Asp Glu Asn
385 390 395 400
atc agc agt gtc gag acc cct tac ata gat tac acg cat tta cgg ata
1248
Ile Ser Ser Val Glu Thr Pro Tyr Ile Asp Tyr Thr His Leu Arg Ile
405 410 415
tcc tac aat gtg tac tta gca gtc tac tcc att gcc cac gcc ttg caa
1296
Ser Tyr Asn Val Tyr Leu Ala Val Tyr Ser Ile Ala His Ala Leu Gln
420 425 430
gat ata tat acc tgc tta cct ggg aga ggg ctc ttc acc aat ggc tcc
1344
Asp Ile Tyr Thr Cys Leu Pro Gly Arg Gly Leu Phe Thr Asn Gly Ser
435 440 445
tgt gca gac atc aag aaa gtt gag gcg tgg cag gtc ctg aag cac cta
1392
Cys Ala Asp Ile Lys Lys Val Glu Ala Trp Gln Val Leu Lys His Leu
450 455 460
cgg cat cta aac ttt aca aac aat atg ggg gag cag gtg acc ttt gat
1440
Arg His Leu Asn Phe Thr Asn Asn Met Gly Glu Gln Val Thr Phe Asp
465 470 475 480
gag tgt ggt gac ctg gtg ggg aac tat tcc atc atc aac tgg cac ctc
1488
Glu Cys Gly Asp Leu Val Gly Asn Tyr Ser Ile Ile Asn Trp His Leu
485 490 495
tcc cca gag gat ggc tcc atc gtg ttt aag gaa gtc ggg tat tac aac
CA 02336543 2001-O1-26
WO 00/06601 PCTNS99/17116 -
1536
Ser Pro Glu Asp Gly Ser Ile Val Phe Lys Glu Val Gly Tyr Tyr Asn
4
500 505 510
gtc tat gcc aag aag gga gaa aga ctc ttc atc aac gag gag aaa atc
1584
Va1 Tyr Ala Lys Lys Gly Glu Arg Leu Phe Ile Asn Glu Glu Lys Ile
515 520 525
ctg tgg agt ggg ttc tcc agg gag gtg ccc ttc tcc aac tgc agc cga
1632
Leu Trp Ser Gly Phe Ser Arg Glu Val Pro Phe Ser Asn Cys Ser Arg
530 535 540
gac tgc ctg gca ggg acc agg aaa ggg atc att gag ggg gag ccc acc
1680
Asp Cys Leu Ala Gly Thr Arg Lys Gly Ile Ile Glu Gly Glu Pro Thr
545 550 555 560
tgc tgc ttt gag tgt gtg gag tgt cct gat ggg gag tat agt gat gag
1728
Cys Cys Phe Glu Cys Val Glu Cys Pro Asp Gly Glu Tyr Ser Asp Glu
565 570 575
aca gat gcc agt gcc tgt aac aag tgc cca gat gac ttc tgg tcc aat
1776
Thr Asp Ala Ser Ala Cys Asn Lys Cys Pro Asp Asp Phe Trp Ser Asn
580 585 590
gag aac cac acc tcc tgc att gcc aag gag atc gag ttt ctg tcg tgg
1824
Glu Asn His Thr Ser Cys Ile Ala Lys Glu Ile Glu Phe Leu Ser Trp
595 600 605
acg gag ccc ttt ggg atc gca ctc acc ctc ttt gcc gtg ctg ggc att
1872
Thr Glu Pro Phe Gly Ile Ala Leu Thr Leu Phe Ala Val Leu Gly Ile
610 615 620
ttc ctg aca gcc ttt gtg ctg ggt gtg ttt atc aag ttc cgc aac aca
1920
Phe Leu Thr Ala Phe Val Leu Gly Val Phe Ile Lys Phe Arg Asn Thr
625 630 635 640
ccc att gtc aag gcc acc aac cga gag ctc tcc tac ctc ctc ctc ttc
1968
Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe
645 650 655
tcc ctg ctc tgc tgc ttc tcc agc tcc ctg ttc ttc atc ggg gag ccc
2016
Ser Leu Leu Cys Cys Phe Ser Ser Ser Leu Phe Phe Ile Gly Glu Pro
660 665 670
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116 -
cag gac tgg acg tgc cgc ctg cgc cag ccg gcc ttt ggc atc agc ttc
4
2064
Gln Asp Trp Thr Cys Arg Leu Arg Gln Pro Ala Phe Gly Ile Ser Phe
675 680 685
gtg ctc tgc atc tca tgc atc ctg gtg aaa acc aac cgt gtc ctc ctg
2112
Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu
690 695 700
gtg ttt gag gcc aag atc ccc acc agc ttc cac cgc aag tgg tgg ggg
2160
Val Phe Glu Ala Lys Ile Pro Thr Ser Phe His Arg Lys Trp Trp Gly
705 710 715 720
ctc aac ctg cag ttc ctg ctg gtt ttc ctc tgc acc ttc atg cag att
2208
Leu Asn Leu Gln Phe Leu Leu Val Phe Leu Cys Thr Phe Met Gln Ile
725 730 735
gtc atc tgt gtg atc tgg ctc tac acc gcg ccc ccc tca agc tac cgc
2256
Val Ile Cys Val Ile Trp Leu Tyr Thr Ala Pro Pro Ser Ser Tyr Arg
740 745 750
aac cag gag ctg gag gat gag atc atc ttc atc acg tgc cac gag ggc
2304
Asn Gln Glu Leu Glu Asp Glu Ile Ile Phe Ile Thr Cys His Glu Gly
755 760 765
tcc ctc atg gcc ctg ggc ttc ctg atc ggc tac acc tgc ctg ctg get
2352
Ser Leu Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala
770 775 780
gcc atc tgc ttc ttc ttt gcc ttc aag tcc cgg aag ctg ccg gag aac
2400
Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn
785 790 795 800
ttc aat gaa gcc aag ttc atc acc ttc agc atg ctc atc ttc ttc atc
2448
Phe Asn Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile
805 810 815
gtc tgg atc tcc ttc att cca gcc tat gcc agc acc tat ggc aag ttt
2496
Val Trp Ile Ser Phe Ile Pro Ala Tyr Ala Ser Thr Tyr Gly Lys Phe
820 825 830
gtc tct gcc gta gag gtg att gcc atc ctg gca gcc agc ttt ggc ttg
2544
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116
~'I
Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ala Ser Phe Gly Leu
835 840 845
s
ctg gcg tgc atc ttc ttc aac aag atc tac atc att ctc ttc aag cca
2592
Leu Ala Cys Ile Phe Phe Asn Lys Ile Tyr Ile Ile Leu Phe Lys Pro
850 855 860
tcc cgc aac acc atc gag gag gtg cgt tgc agc acc gca get cac get
2640
Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala His Ala
865 870 875 880
ttc aag gtg get gcc cgg gcc acg ctg cgc cgc agc aac gtc tcc cgc
2688
Phe Lys Val Ala Ala Arg Ala Thr Leu Arg Arg Ser Asn Val Ser Arg
885 890 895
aag cgg tcc agc agc ctt gga ggc tcc acg gga tcc acc cct tcc tcc
2736
Lys Arg Ser Ser Ser Leu Gly Gly Ser Thr Gly Ser Thr Pro Ser Ser
900 905 910
tcc atc agc agc aag agc aac agc gaa gac cca ttc cca cag ccc gag
2784
Ser Ile Ser Ser Lys Ser Asn Ser Glu Asp Pro Phe Pro Gln Pro Glu
915 920 925
agg cag aag cag cag cag ccg ctg gcc cta acc cag caa gag cag cag
2832
Arg Gln Lys Gln Gln Gln Pro Leu Ala Leu Thr Gln Gln Glu Gln Gln
930 935 940
cag cag ccc ctg acc ctc cca cag cag caa cga tct cag cag cag ccc
2880
Gln Gln Pro Leu Thr Leu Pro Gln Gln Gln Arg Ser Gln Gln Gln Pro
945 950 955 960
aga tgc aag cag aag gtc atc ttt ggc agc ggc acg gtc acc ttc tca
2928
Arg Cys Lys Gln Lys Val Ile Phe Gly Ser Gly Thr Val Thr Phe Ser
965 970 975
ctg agc ttt gat gag cct cag aag aac gcc atg gcc cac agg aat tct
2976
Leu Ser Phe Asp Glu Pro Gln Lys Asn Ala Met Ala His Arg Asn Ser
980 985 990
acg cac cag aac tcc ctg gag gcc cag aaa agc agc gat acg ctg acc
3024
Thr His Gln Asn Ser Leu Glu Ala Gln Lys Ser Ser Asp Thr Leu Thr
995 1000 1005
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116 .
cga cac cag cca tta ctc ccg ctg cag tgc ggg gaa acg gac tta gat
3072
Arg His Gln Pro Leu Leu Pro Leu Gln Cys Gly Glu Thr Asp Leu Asp
1010 1015 1020
ctg acc gtc cag gaa aca ggt ctg caa gga cct gtg ggt gga gac cag
3120
Leu Thr Val Gln Glu Thr Gly Leu Gln Gly Pro Val Gly Gly Asp Gln
1025 1030 1035 1040
cgg cca gag gtg gag gac cct gaa gag ttg tcc cca gca ctt gta gtg
3168
Arg Pro Glu Val Glu Asp Pro Glu G1u Leu Ser Pro Ala Leu Val Val
1045 1050 1055
tcc agt tca cag agc ttt gtc atc agt ggt gga ggc agc act gtt aca
3216
Ser Ser Ser Gln Ser Phe Val Ile Ser Gly Gly Gly Ser Thr Val Thr
1060 1065 1070
gaa aac gta gtg aat tca
3234
Glu Asn Val Val Asn Ser
1075
<210> 12
<211> 1078
<212> PRT
<213> Homo Sapiens
<400> 12
Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala Leu Thr Trp His
1 5 10 15
Thr Ser Ala Tyr Gly Pro Asp Gln Arg Ala Gln Lys Lys Gly Asp Ile
20 25 30
Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp
35 40 45
Gln Asp Leu Lys Ser Arg Pro Glu Ser Val Glu Cys Ile Arg Tyr Asn
50 55 60
Phe Arg Gly Phe Arg Trp Leu Gln Ala Met Ile Phe Ala Ile Glu Glu
65 70 75 80
Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg
85 90 95
Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu
100 105 110
CA 02336543 2001-O1-26
WO 00/06601 PCT/US99/17116 .
Ser Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe
lI5 120 125
4
Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala
130 135 140
Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe
145 150 155 160
Tyr Ile Pro Gln Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn
165 170 175
Lys Asn Gln Phe Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His
180 185 190
Gln Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe Arg Trp Asn Trp
195 200 205
Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu
210 215 220
Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser
225 230 235 240
Glu Leu Ile Ser Gln Tyr Ser Asp Glu Glu Glu Ile Gln His Val Val
245 250 255
Glu Val Ile Gln Asn Ser Thr Ala Lys Val Ile Val Val Phe Ser Ser
260 265 270
Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val Arg Arg Asn Ile
275 280 285
Thr Gly Lys Ile Trp Leu Ala Ser Glu Ala Trp Ala Ser Ser Ser Leu
290 295 300
Ile Ala Met Pro Gln Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe
305 310 315 320
Ala Leu Lys Ala Gly Gln Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys
325 330 335
Val His Pro Arg Lys Ser Val His Asn Gly Phe Ala Lys Glu Phe Trp
340 345 350
Glu Glu Thr Phe Asn Cys His Leu Gln Glu Gly Ala Lys Gly Pro Leu
355 360 365
Pro Val Asp Thr Phe Leu Arg Gly His Glu Glu Ser Gly Asp Arg Phe
370 375 380
Ser Asn Ser Ser Thr Ala Phe Arg Pro Leu Cys Thr Gly Asp Glu Asn
385 390 395 400
CA 02336543 2001-O1-26
WO 00/06601 PCTNS99/17116 -
Ile Ser Ser Val Glu Thr Pro Tyr Ile Asp Tyr Thr His Leu Arg Ile
4
405 410 415
Ser Tyr Asn Val Tyr Leu Ala Val Tyr Ser Ile Ala His Ala Leu Gln
420 425 430
Asp Ile Tyr Thr Cys Leu Pro Gly Arg Gly Leu Phe Thr Asn Gly Ser
435 440 445
Cys Ala Asp Ile Lys Lys Val Glu Ala Trp Gln Val Leu Lys His Leu
450 455 460
Arg His Leu Asn Phe Thr Asn Asn Met Gly Glu Gln Val Thr Phe Asp
465 470 475 480
Glu Cys Gly Asp Leu Val Gly Asn Tyr Ser Ile Ile Asn Trp His Leu
485 490 495
Ser Pro Glu Asp Gly Ser Ile Val Phe Lys Glu Val Gly Tyr Tyr Asn
500 505 510
Val Tyr Ala Lys Lys Gly Glu Arg Leu Phe Ile Asn Glu Glu Lys Ile
515 520 525
Leu Trp Ser Gly Phe Ser Arg Glu Val Pro Phe Ser Asn Cys Ser Arg
530 535 540
Asp Cys Leu Ala Gly Thr Arg Lys Gly Ile Ile Glu Gly Glu Pro Thr
545 550 555 560
Cys Cys Phe Glu Cys Val Glu Cys Pro Asp Gly Glu Tyr Ser Asp Glu
565 570 575
Thr Asp Ala Ser Ala Cys Asn Lys Cys Pro Asp Asp Phe Trp Ser Asn
580 585 590
Glu Asn His Thr Ser Cys Ile Ala Lys Glu Ile Glu Phe Leu Ser Trp
595 600 605
Thr Glu Pro Phe Gly Ile Ala Leu Thr Leu Phe Ala Val Leu Gly Ile
610 615 620
Phe Leu Thr Ala Phe Val Leu Gly Val Phe Ile Lys Phe Arg Asn Thr
625 630 635 640
Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe
645 650 655
Ser Leu Leu Cys Cys Phe Ser Ser Ser Leu Phe Phe Ile Gly Glu Pro
660 665 670
Gln Asp Trp Thr Cys Arg Leu Arg Gln Pro Ala Phe Gly Ile Ser Phe
CA 02336543 2001-O1-26
WO 00/06601 PCTNS99/17116
31
675 680 685
Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu
690 695 700
Val Phe Glu Ala Lys Ile Pro Thr Ser Phe His Arg Lys Trp Trp Gly
705 710 715 720
Leu Asn Leu Gln Phe Leu Leu Val Phe Leu Cys Thr Phe Met Gln Ile
725 730 735
Val Ile Cys Val Ile Trp Leu Tyr Thr Ala Pro Pro Ser Ser Tyr Arg
740 745 750
Asn Gln Glu Leu Glu Asp Glu Ile Ile Phe Ile Thr Cys His Glu Gly
755 760 765
Ser Leu Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala
770 775 780
Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn
785 790 795 800
Phe Asn Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile
805 810 815
Val Trp Ile Ser Phe Ile Pro Ala Tyr Ala Ser Thr Tyr Gly Lys Phe
820 825 830
Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ala Ser Phe Gly Leu
835 840 845
Leu Ala Cys Ile Phe Phe Asn Lys Ile Tyr Ile Ile Leu Phe Lys Pro
850 855 860
Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala His Ala
865 B70 875 880
Phe Lys Val Ala Ala Arg Ala Thr Leu Arg Arg Ser Asn Val Ser Arg
885 890 895
Lys Arg Ser Ser Ser Leu Gly Gly Ser Thr Gly Ser Thr Pro Ser Ser
900 905 910
Ser Ile Ser Ser Lys Ser Asn Ser Glu Asp Pro Phe Pro Gln Pro Glu
915 920 925
Arg Gln Lys Gln Gln Gln Pro Leu Ala Leu Thr Gln Gln Glu Gln Gln
930 935 940
Gln Gln Pro Leu Thr Leu Pro Gln Gln Gln Arg Ser Gln Gln Gln Pro
945 950 955 960
CA 02336543 2001-O1-26
WO 00/06601 PCT1US99/17116 .
Arg Cys Lys Gln Lys Val Ile Phe Gly Ser Gly Thr Val Thr Phe Ser
965 970 975
Leu Ser Phe Asp Glu Pro Gln Lys Asn Ala Met Ala His Arg Asn Ser
980 985 990
Thr His Gln Asn Ser Leu Glu Ala Gln Lys Ser Ser Asp Thr Leu Thr
995 1000 1005
Arg His Gln Pro Leu Leu Pro Leu Gln Cys Gly Glu Thr Asp Leu Asp
1010 1015 1020
Leu Thr Val Gln Glu Thr Gly Leu Gln Gly Pro Val Gly Gly Asp Gln
1025 1030 1035 1040
Arg Pro Glu Val Glu Asp Pro Glu Glu Leu Ser Pro Ala Leu Val Val
1045 1050 1055
Ser Ser Ser Gln Ser Phe Val Ile Ser Gly Gly Gly Ser Thr Val Thr
1060 1065 1070
Glu Asn Val Va1 Asn Ser
1075
