JP2002521052A - Human calcium sensory receptor isoform protein - Google Patents

Abstract

  (57) [Summary] The present invention relates to human calcium sensory receptor isoform proteins and genes encoding these isoform proteins. Furthermore, the invention relates to a method for screening for agonists or antagonists of said isoforms, in particular for calcium receptor activity, the diagnostic use of these isoforms and the therapeutic use of agonists or antagonists. The present invention also relates to gene therapy using a gene encoding a molecule capable of down-regulating receptor activity, such as the receptor isoform protein or antisense sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to isoforms of the human calcium sensing receptor.
forms), and the genes encoding these isoforms. further,
The present invention relates to a method for screening for agonists or antagonists of the isoform proteins, particularly for calcium receptor activity, diagnostic uses of these isoform proteins and therapeutic uses of the agonists or antagonists. The present invention also relates to gene therapy using genes encoding isoforms or molecules of the receptor, such as antisense sequences, which are capable of down-regulating receptor activity.

BACKGROUND OF THE INVENTION Calcium is an extracellular messenger (Brown et al. (1995) Cell 83: 679-682).
). Serum calcium levels, 1,25-dihydroxyvitamin D _3, which is controlled by parathyroid hormone and calcitonin. Calcium sensory receptor (CaSR)
It has been identified in the bovine parathyroid gland (WO 94/18959; Brown et al. (1993) Nature 366: 575-
580). By cloning the cDNA encoding this receptor (CaSRa), a G protein-coupled receptor characterized by a large extracellular domain coupled to a seven-membrane spanning domain similar to that found in members of the family of G protein-coupled receptors was obtained. It became clear. This receptor has been shown to play an important role in Ca ⁺⁺ homeostasis by controlling parathyroid hormone secretion and renal tubular calcium reabsorption. This receptor recognizes calcium and other multivalent cations and is linked to the release of calcium from intracellular stores by altering phosphoinositide turnover. In addition to its abundant expression in parathyroid 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 types of CaSR, especially from bovine bone marrow cells, have also been described by House et al. ((199
7) J. Bone Min. Res. 12: 1959-1970) and in U.S. Patent Nos. 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).

[0003] Alternative splice forms of CaSRa have been identified in human medullary thyroid carcinoma and keratinocytes (see Freichel et al., Supra). A medullary thyroid carcinoma isoform protein called CaSRb has a 307 between nucleotides 186 and 495 corresponding to exon 2.
Includes base pair deletions. This deletion results in a shift in the reading frame at nucleotide 766 and premature termination. Translation of the CaSRb transcript was able to produce the extracellular portion of the receptor without a seven transmembrane anchor and a cytosolic tail. Thus, CaSRb is a secreted protein and its exact function has been determined, but by analogy to other known soluble receptors, it plays a role in regulating the interaction of native CaSR with its cationic ligand Can be played.

A second isoform of CaSRa has also been identified in keratinocytes (Oda et al. (1997
) FASEB J. 11 (9): A925; Abstract # 395). This type (CaSRc) lacks exon 4, which encodes a portion of the extracellular domain of the receptor that contains a region of acidic amino acids that can 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 by CaSR. Wild-type CaSR isoform proteins were expected to exhibit various pharmacological and signaling properties relative to wild-type. For example, various isoform proteins can differentially or specifically associate with known or unknown signaling pathways, including phosphoinositide turnover, calcium mobilization, protein kinase C activation, cAMP production, and other ion channel activities. Was. Alternatively, the modified isoform protein exhibits different cell surface expression, metabolic half-life, or intracellular trafficking when compared to the wild type, or behaves in a predominant negative or positive manner, thereby maintaining the same tissue. Exceeds the functionality of the wild-type receptor expressed in

The present invention focuses on this technical need, as described below. In particular,
Applicants provide evidence that CaSRb is also found in human kidney. Importantly, applicants found that in human kidney, nucleotides 1378-1608 and 1075-1386, respectively.
Two alternative CaSR transcripts (CaSRc and
d) was also identified. Citation of any reference herein shall not be construed as an admission that such reference is available as "prior art" to the present application.

SUMMARY OF THE INVENTION As noted above, the present invention relates to the identification of isoforms of the human calcium sensory receptor (CaSR). The present invention reveals the presence of multiple alternatively spliced CaSR transcripts in the human kidney. Two such isoform proteins identified, CaSRc and d, result from partial deletions of the wild-type sequence. CaSRc and d
Nucleotide deletion has no effect on the reading frame. Therefore, CaSRc and d
Will produce a receptor protein with a length of about 90-100 amino acids deleted from the extracellular domain. These deletions include the respective extracellular sequences that are rich in acidic residues (about 4%). The location and charge properties of the deleted CaSRc and d sequences suggest that these two CaSR splice variants may exhibit different cation-sensing properties than the wild-type receptor.

Accordingly, in a first aspect, the present invention is directed to an isolated nucleic acid encoding an isoform of the human calcium sensory receptor, comprising from about 2922 to about 3003 nucleotides and comprising SEQ ID NO: 11. Nucleic acids having a deletion of at least about 231 nucleotides are provided when compared to the indicated wild-type receptor. The term “about” or “approximately” means within 20%, preferably within 10%, more preferably within 5% of a specified value or range. Preferably, the deletion is in a region encoding the extracellular domain of the receptor.

[0008] In a preferred embodiment of the invention, the deletion comprises approximately the nucleotides of SEQ ID NO: 11
From 1075-1386. In another preferred embodiment, the deletion is from about nucleotides 1378-1608 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 acids of the invention have at least one property selected from: It can be amplified by the polymerase chain reaction (PCR) using oligonucleotide primers derived from SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11; Hybridizes with a nucleic acid having the nucleotide sequence set forth in NO: 7 or SEQ ID NO: 9; and is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, and allelic variants thereof. Encodes a polypeptide having a different amino acid sequence. Preferably, the nucleic acid of the present invention can be amplified by at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 6. In yet another preferred embodiment of the present invention, the isolated nucleic acid is SEQ ID NO: 8 (CaSRc)
Or, it encodes the amino acid sequence shown in SEQ ID NO: 10 (CaSRd), or a CaSR isoform protein containing an allelic variant thereof. Preferably, the isolated nucleic acid is
Includes the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9 or allelic variants thereof.

As will be readily appreciated by those skilled in the art, one effective method of preparing nucleic acids, particularly cDNAs, of the present invention is by PCR, a cDNA comprising a coding sequence for a CasR isoform protein.
Amplifying nucleic acids from a library. SEQ ID NO: 7, SEQ ID NO: 9 or SEQ
Various PCR primers corresponding to any desired segments from ID NO: 11
It can be used according to the invention. In certain embodiments described below, the isolated nucleic acids of the invention were amplified using PCR primers having the sequences set forth in SEQ ID NOS: 1-6. Alternatively, the nucleic acids of the invention can be isolated or identified, for example, with oligonucleotide probes of at least 10 bases, and under stringent conditions SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 9.
It hybridizes to a nucleotide having the sequence shown in NO: 11 or a complementary sequence. In a particular aspect, the oligonucleotides can be used in a method for detecting genomic DNA (Southern analysis) or a method for detecting expression of mRNA encoding CaSR isoform protein in cells (Northern analysis). In each case, the method comprises labeling a sample from the cell, for example, with a radioisotope or chromophore or fluorophore
Contacting with a detectable oligonucleotide, and genomic DNA or m in the sample
Detecting hybridization of the oligonucleotide with the RNA, including detecting the hybridization of the oligonucleotide with the RNA indicates the presence of the gene encoding the CaSR isoform in the genome, and hybridization with the mRNA. Detection indicates expression of mRNA encoding the CaSR isoform protein. Quantitative methods can also be used, for example, detecting the number of CaSR genes in the genome, or detecting an increase or decrease in mRNA expression levels.

The oligonucleotide of the present invention is an antisense oligonucleotide, ie,
An oligonucleotide that binds to mRNA encoding a CaSR isoform protein and prevents its translation in cells may be used. Such antisense molecules may be encoded by a vector that is expressed intracellularly, or may be synthetic oligonucleotides, preferably those that are resistant to intracellular nucleases by including non-phosphate linkages. In another aspect, the invention provides a vector comprising a nucleic acid encoding a human calcium sensory receptor isoform protein. Preferably, the nucleic acid is operatively linked to an expression control sequence that enables 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 can be a retrovirus, adenovirus, adenoassociated virus, herpes virus, or vaccinia virus.

In yet another aspect, the invention relates to a host cell transfected with a vector comprising a nucleic acid encoding an isoform of the human calcium sensory receptor. The host cell can be a bacterial, yeast, or mammalian cell. The host cells of the invention can be used to recombinantly produce a CaSR isoform protein.
The method involves culturing a host cell in a culture medium under conditions that allow for expression of the isoform protein. Accordingly, another aspect of the present invention is a method for expressing an isoform of the human calcium sensory receptor, comprising the following steps. Culturing the host cells in a culture medium under conditions that allow expression of the recombinant receptor; and identifying cells that express the receptor on their surface.

The present invention also relates to an isolated isoform of the human calcium sensory receptor, comprising about 974 to about 1001 amino acids, and compared to the wild-type receptor set forth in SEQ ID NO: 12. In some cases, isoform proteins having at least about 77 amino acid deletions are also provided. The deletion occurs at about amino acids 358-462 of SEQ ID NO: 12,
It can be from the extracellular domain of the receptor, such as from about amino acids 460-536 or about amino acids 358-536. In particular, the CaSR isoform protein of the present invention
Includes the amino acid sequence shown in Q ID NO: 8 or SEQ ID NO: 10, or allelic variants thereof.

The present invention advantageously provides methods for screening for molecules that modulate the activity of CaSR isoform proteins, and thus calcium levels. In particular, the present invention is a method of screening for agonists or antagonists of CaSR isoform protein activity, incubating a sample with CaSR isoform protein, measuring CaSR isoform protein activity, and measuring the activity without the sample. A method comprising comparing the activity with the case. Any of the screening techniques of the present technology, particularly high throughput screening methods, can be used. In certain embodiments, the method comprises screening the compound for its ability to affect intracellular calcium concentration. The compounds can be tested alone, with increased extracellular calcium concentrations, or in the presence of other agonists or antagonists of CaSR isoform protein activity. Intracellular calcium can be measured with a fluorescent indicator such as fura-2. The screening method of the present invention is a CaSR agonist (calcimimetics) or antagonist (
calcilytics).

In yet another embodiment, the present invention provides a method for treating a patient suffering from a disease or disorder associated with abnormal calcium levels, such as in plasma, with a therapeutically effective amount of a compound capable of modulating the activity of a CaSR isoform protein. Pharmaceutical compositions and methods for treating by administration are provided. This compound is specific for the CaSR isoform protein. Such diseases include, for example, hyperparathyroidism (primary and secondary) and osteoporosis. Other diseases include Paget's disease, hypercalcemic malignancies, and hypertension. The compound may be calcimmetic or calcilytic identified using the screening assays of the present invention. Instead, the disease or disorder is treated with gene therapy.

In one embodiment, the patient's cells have been transfected with a vector encoding a CaSR isoform protein under conditions that allow expression of the isoform protein. Alternatively, if desired, the present invention provides a method of inhibiting CaSR activity in a cell of a patient, comprising reducing the level of CaSR in the cell. CaSR protein levels can be increased by introducing CaSR antisense nucleic acids into cells,
Antisense nucleic acids can hybridize to CaSR mRNA and be reduced. Instead, CaS
R protein levels can be measured using single-chain Fv antibodies (scFv) that specifically bind to CaSR isoform proteins or intracellular antibodies that encode nucleic acids
By introducing into cells at a level sufficient to bind and inactivate
Can be reduced.

Yet another object of the invention is to recover purified protein by transfection or fermentation of transduced cells, or to further control in vivo for in vitro testing or calcium homeostasis, eg, intracellular in vivo for gene therapy. And Ca
It is to provide high level expression of SR isoform protein. It is a particular object of the present invention to provide a screen for small molecule modulators of CaSR activity, particularly unique CaSR isoforms, such as agonists and antagonists. These and other objects are highlighted by the present invention and are described in further detail in the accompanying drawings and the following detailed description and examples.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based, in part, on the identification of an isoform of the human calcium sensory receptor, referred to herein as CaSR. Accordingly, the present invention provides nucleic acids encoding CaSR isoform proteins, purified proteins thereof, cells expressing CaSR, particularly nucleic acids encoding splice variant isoform proteins, and small molecules that act or antagonize the activity of CaSR. It relates to their use in screening natural products. The present invention can also be used to treat diseases or disorders associated with abnormal levels of calcium, including gene therapy applications (both coding and antisense molecule applications). In addition, anti-CaSR antibodies can be used for diagnostic and purification applications. These and other aspects of the invention, particularly for CaSR gene isolation, CaSR protein expression, production of anti-CaSR antibodies, particularly for gene therapy applications, encode screening assays and vectors for agonists and antagonists of CaSR activity. Delivery of CaSR is detailed in the following section. Section headings are for convenience of the reader only and are not limiting in any way.

Gene Encoding Calcium Sensory Receptor Isoform Protein The present invention contemplates isolating the gene encoding the human calcium sensory receptor isoform protein. Here, the term “gene” is a collection of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA methods within the skill of the art can be utilized. Such methods are explained fully in the literature. For example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual
, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York ("Sambrook et al., 1989"); DNA Cloning: A Practical Approach
, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ
Gait ed. 1984); Nucleic Acid Hybridization [BD Hames & SJHiggins e
ds. (1985)]; Transcription And Translation [BD Hames & SJ Higgins, eds
(1984)]; Animal Cell Culture [RI Freshney, ed. (1986)]; Immobilized
Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Mo
lecular Cloning (1984); FM Ausubel et al. (eds.), Current Protocols in Molec
See ular Biology, John Wiley & Sons, Inc. (1994).

Accordingly, as used herein, the following terms have the definitions set forth below. A "cloning vector" is a replicon, such as a plasmid, phage, or cosmid, to which other DNA segments have joined, resulting in replication of the joined segment. A “replicon” is any genetic element (eg, a plasmid, chromosome, virus) that functions as an autonomous unit of DNA in vivo replication, ie, is capable of replication under its own control. Cloning vectors are replicable in one cell type and can be expressed in other cells ("shuttle vectors").

“Cassette” refers to a segment of DNA that can be inserted into a vector at a specific restriction site. This segment of DNA encodes the polypeptide of interest, and the cassette and restriction sites are designed to ensure that the cassette is inserted in the proper reading frame for transcription and translation. Cells are capable of introducing foreign or heterologous DNA into such cells when introduced into the cell.
"Transfected" by A cell is transformed when the transfected DNA causes a phenotypic change. Transforming DNA is the chromosomal DNA that makes up the cell's genome.
It can be incorporated (conjugated) into it.

A “nucleic acid molecule” is a phosphate-polymerized form of a ribonucleoside (adenosine, guanosine, uridine or cytidine; an “RNA molecule”) or a deoxynucleoside (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any of these phosphate analogs, such as phosphorothioates and thioesters, in single-stranded form or double-stranded helix. Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, especially a DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and is not limited to any particular tertiary form. Thus, the term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (eg, restriction fragments), plasmids, and chromosomes. For the structure of a particular double-stranded DNA molecule, the sequence usually gives sequences only in the 5 ′ to 3 ′ direction along the untranslated strand of DNA (ie, the strand having a sequence homologous to the mRNA). It is described according to the rules of A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

If the nucleic acid molecule in single-stranded form can anneal to another nucleic acid molecule under the appropriate conditions of temperature and ionic strength of the solution, the nucleic acid molecule will be a cDNA, genomic DNA, or other nucleic acid such as RNA. The molecule is "hybridizable". (See Sambrook et al., Supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening of homologous nucleic acids, low stringency hybridization corresponding to a _Tm of 55- can be used (e.g., 5x SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher _Tm , for example, 40% formamide, 5x or 6x SCC. High stringency hybridization conditions correspond to the highest T _m, eg, 50% formamide, 5x or 6XSCC. Hybridization may involve base mismatches due to the stringency of hybridization, but requires that the two nucleic acids contain complementary sequences. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementarity of the variables known in the art. The higher the degree of identity or homology between two nucleic acid sequences, the higher the _Tm value for hybridization of nucleic acids having those sequences. Nucleic acid hybridization relative stability (corresponding to higher T _m) decreases in the following order: RNA: R
NA, DNA: RNA, DNA: DNA. For hybrids greater than 100 nucleotides in length, equations for calculating _Tm have been derived (see Sambrook et al., 9.50-0.51 supra). For hybridization with shorter nucleic acids, ie, oligonucleotides, the location of mismatches becomes important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., Supra, 11.7-11.8, supra). Preferably, the length of a hybridizable nucleic acid is at least about 10 nucleotides; preferably, at least about 15 nucleotides; more preferably, at least about 20 nucleotides.

In a specific embodiment, the term “standard hybridization conditions” refers to a 55 ° C.
T_mAnd the conditions as described above are used. In a preferred embodiment, T_mIs 60-C
In a more preferred embodiment, T_mIs 65-C. Here, the term “oligonucleotide” generally refers to at least 18 nucleotides.
Genomic DNA molecule, cDNA molecule, or CaSR or its
It is an mRNA molecule encoding an isoform protein. Oligonucleotides are, for example, ³² Nucleic acids conjugated with a P-nucleotide or a label such as biotin
Can be labeled with otide. In other embodiments, the labeled oligonucleotide is a CaSR or
Is used as a probe to detect the presence of the nucleic acid encoding the isoform protein.
Can be used. In another embodiment, the oligonucleotides (one or both of which are labeled)
) For cloning of CaSR isoform protein or CaSR isoform protein
Can be used as a PCR primer to detect the presence of nucleic acid encoding the quality
. In a further embodiment, an oligonucleotide of the invention has a CaSR DNA molecule.
Can form a triple helix. Generally, oligonucleotides are synthetically preferred.
Alternatively, it is prepared by a nucleic acid synthesizer. Therefore, the oligonucleotide is
It is regulated by the binding of non-naturally occurring phosphate ester analogs, such as
Can be manufactured.

A DNA “coding sequence” is a double-stranded DNA sequence that is transcribed and translated in vivo or in vitro into a polypeptide within a cell 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 includes, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic organisms (eg, mammals), and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, the polyadenylation signal and transcription termination sequence will usually be located 3 'to the coding sequence. In this case, the nucleic acid is "operably linked" to an expression control sequence that allows expression of the protein in an expression-competent host cell.

The transcription and translation control sequences are expression DNA control sequences that provide for the expression of the coding sequence in the host cell, such as promoters, enhancers, terminators and the like.
In eukaryotic cells, polyadenylation signals are regulatory sequences. The "promoter sequence" binds to the cellular RNA polymerase and is downstream (3 'direction).
) Is a DNA regulatory region capable of initiating transcription of the coding sequence. For purposes of defining the present invention, a promoter sequence is linked at its 3 'end to a transcription start site and is linked upstream (5'
Direction) and contains the minimum number of bases or elements required to initiate transcription at a detectable level above background. Transcription start site within the promoter sequence
(Conveniently defined, for example, by mapping with nuclease S1), and the protein binding domain (consensus sequence) responsible for the binding of RNA polymerase.

The coding sequence is “under control” of the transcriptional and translational control sequences in the cell when RNA polymerase transcribes the coding sequence into mRNA, and the trans-RNA is spliced (where the coding sequence replaces the intron). If included) and translated into the protein encoded by the coding sequence. Here, the term "homology" is used in all its grammatical forms and spelling variants.
For example, it refers to a relationship between proteins having a `` common evolutionary origin '', including proteins from immunoglobulin superfamily) and homologous proteins from heterologous (eg, myosin light chains, etc.) (Reeck et al., 1987, Cell 50). : 667). Such proteins (and their encoding genes) have sequence homology, as reflected by high sequence similarity. Thus, `` sequence similarity '' means, in all its grammatical forms, the identity or identity between the 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 normal usage and in the present application, the term "homology", when modified with an adverb such as "highly", means sequence similarity and not a common evolutionary source.

In certain embodiments, two DNA sequences are characterized in that at least about 50% (preferably at least about 75%, most preferably about 90% or 95%) of the nucleotides match over a defined length of the DNA sequence. "Substantially homologous" or "substantially similar". Sequences that are substantially homologous can be determined by comparing the sequences using a sequence data bank or standard software available in Southern hybridization experiments under stringent conditions, for example, as defined for the particular system. Can be identified. Defining appropriate hybridization conditions is within the skill of the art. For example, Maniatis et al .; DNA Cloning, Vols. I ⅈ Nucleic Acid Hybridiz
See ation. Similarly, in certain embodiments, the two amino acid sequences are the same as the 30
"Substantially homologous" or "substantially similar" when at least about% is the same, or when at least about 60% is similar (functionally the same). Preferably, similar or homologous sequences are, for example, GCG (Genetics Computer Group, Program Manual for the GCG Package, Versi
on 7, Madison, Wisconsin).

Here, the term “corresponding” means a sequence that is the same or different in its exact position as the molecule whose similarity or homology is measured, but different or similar. Alignment of nucleic acid or amino acid sequences can include spaces. Thus, the term "corresponding" refers to sequence similarity and not to numbering of amino acid residues or nucleotide bases. The gene encoding the CaSR isoform protein, whether genomic DNA or cDNA, can be isolated from human cDNA or genomic libraries. Methods for obtaining genes encoding CaSR isoform proteins are well known in the art, as described above (see, eg, Sambrook et al., 1989, supra).

Thus, any human cell may serve as a source of nucleic acids for molecular cloning of the CaSR isoform protein gene. DNA can be obtained from cloned DNA (e.g., a DNA `` library '') by standard methods known in the art, and is preferably a cDNA library prepared from tissue having high level expression of the protein (e.g., Chemical synthesis, cDNA cloning, or genomic D from brain, thyroid and kidney cDNA
Obtained by cloning NA or fragments thereof purified from the desired cells (see, eg, Sambrook et al., 1989, supra; Glover, DM (ed.), 1985, DNA Clonin
g: A Practical Approach, see MRL Press, Ltd., Oxford, UK Vol. I, II).
Clones derived from genomic DNA contain control and intron D in addition to the coding region.
Clones derived from cDNA may not contain intron sequences. In certain embodiments, the isoform proteins CaSRc and CaSRd were isolated from a human kidney cell library. Whatever the origin, the gene should be molecularly cloned into a vector suitable for propagation of the gene.

[0030] Once the DNA fragments have been generated, the identification of specific DNA fragments containing the gene encoding the CaSR isoform protein can be accomplished in a number of ways. For example, DNA fragments can be screened by nucleic acid hybridization to a labeled probe (B
enton and Davis, 1977, Science 196: 180; Grunstein and Hogness, 1975, Proc.
Natl. Acad. Sci. USA 72: 3961). Those DNA fragments that are substantially homologous to the probe will hybridize. As noted above, the higher the degree of homology, the more stringent hybridization conditions can be used. In certain embodiments, Northern hybridization conditions are used to identify mRNA splicing variants of the CaSR gene.

Further selections include properties of the gene, eg, the gene has an isoelectric, electrophoretic, amino acid composition, or partial amino acids of a CaSR isoform protein as disclosed herein. This can be done based on whether it encodes a protein product having a sequence. Thus, the presence of a gene can be detected by assays based on the physical, chemical, or immunological properties of the expression product. For example, cDNA clones or DNA clones that hybrid-select the appropriate mRNAs can be selected, e.g., similar or identical electrophoretic migration, isoelectric focusing or non-equilibrium pH gel electrophoretic behavior, proteolysis Generate a digestion map, or protein with known antigenic properties for CaSR. In certain embodiments, the isoform protein is recognized by a polyclonal antibody that does not recognize wild-type CaSR.

The present invention relates to genes encoding allelic variants, splicing variants, analogs, and derivatives of the CaSR isoform proteins of the present invention having the same or homologous functional activity as the isoform proteins (for example, cDNAs). The manufacture and use of derivatives and analogs related to CaSR isoform proteins are within the scope of the present invention. In certain embodiments, derivatives and analogs can exhibit functional activity, ie, one or more functional activities associated with the isoforms of the invention. In particular, such analogs can bind calcium. Alternatively, allelic variants may include variants that are unable to bind calcium. Derivatives can be made by altering the encoding nucleic acid sequence by substitutions, additions or deletions that result in a functionally equivalent molecule. Preferably, a derivative is created that has enhanced or increased functional activity relative to the native CaSR isoform protein.

Due to the degeneracy of the nucleotide coding sequence, other DNA sequences encoding amino acid sequences that are substantially identical to the gene encoding the CaSR isoform protein, including amino acid sequences containing single amino acid variants, It can be used to practice the invention. These are converted by, but not limited to, allelic variants, homologous genes from other species, and the substitution of various codons encoding the same amino acid residue within the sequence,
A nucleotide sequence containing all or a part of the CaSR isoform protein that has undergone a silent change in this way is included. Similarly, the derivatives of the present invention include, but are not limited to, the replacement of a functionally equivalent amino acid residue with a residue in the main amino acid sequence resulting in a conservative amino acid substitution. CaSR including conversion sequence
Derivatives containing all or part of the amino acid sequence of the isoform protein are included.
For example, one or more amino acid residues within a sequence are replaced by another amino acid of like polarity that acts as a functional equivalent, resulting in a silent change. Substitutions for an amino acid in the sequence may be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids, alanine, leucine, isoleucine,
Valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing an aromatic ring structure are phenylalanine, tryptophan, and tyrosine. As polar neutral amino acids, glycine, serine, threonine,
Cysteine, tyrosine, asparagine, and glutamine. Positive charge (
Basic) amino acids include arginine, lysine and histidine.
Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such changes are not expected to affect polyacrylamide gel electrophoresis or apparent molecular weight as determined by isoelectric point.

Particularly preferred substitutions are as follows: -Lys for Arg, and vice versa, so that a positive charge is maintained;-Glu for Asp, and vice versa, so that a negative charge is maintained;-Thr, such that free -OH is maintained And Gln to Asn such that free CONH ₂ is maintained. Also, amino acid substitutions can be introduced to change amino acids to have particularly favorable properties. For example, Cys can guide a potential site of disulfide bridges with another Cys. His can be introduced specifically as a “catalytic” site (ie, His can act as an acid or base and is the most common amino acid in biochemical catalysis). P
ro is introduced, especially due to its planar structure, and induces b-rotation of the protein structure.

[0035] Genes encoding the CaSR isoform proteins, derivatives and analogs of the present invention can be produced by various methods known in the art. The manipulations that produce those products can be triggered at the gene or protein level. For example, cloned gene sequences can be modified by any of the many strategies known in the art (Sambrook et al., 1989,
Supra). The sequence can be cleaved at the appropriate site by a restriction endonuclease and then optionally further enzymatically modified, isolated and ligated in vitro. Ca
In generating genes encoding SR isoform proteins, derivatives or analogs,
Care should be taken to ensure that the modified gene remains in the same translational reading frame as the CaSR gene (SEQ ID NO: 11), which is not interrupted by a translation termination signal, within the gene region encoding the desired activity. .

In addition, the CaSR isoform protein encoding the nucleic acid sequence may be mutated in vitro or in vivo to create and / or disrupt translation, initiation, and / or termination sequences, or create variants within the coding region, And / or create new restriction endonuclease sites or destroy existing restriction endonuclease sites, further promoting in vitro modifications. Preferably, such a mutant enhances the functional activity of the mutated gene product. Any mutagenesis method known in the art can be used,
Without limitation, 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.
USA 83: 710), the use of a TAB® linker (Pharmacia), and the like.
PCR is preferred for site-directed mutagenesis (Higuchi, 1989, "Using PCR to
Engineer DNA ", in PCR Technology: Principles and Applications for DNA A
(See mplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70.)
.

Then, the identified or isolated gene is inserted into an appropriate cloning vector. Many vector-host systems known in the art can 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, bacteriophages such as E. coli, lambda derivatives, or plasmids such as, for example, pBR322 derivatives or pUC plasmid derivatives, such as pGEX vectors, pmal-c, pFLAG, and the like. Insertion into a cloning vector can be achieved, for example, by ligating the DNA fragment into a cloning vector having complementary cohesive ends. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the DNA
The ends of the molecule may be modified enzymatically. Alternatively, any desired sites are created by linking nucleic acid sequences (linkers) to the DNA ends; these linking linkers comprise specifically chemically synthesized oligonucleotides that encode a restriction endonuclease recognition sequence. sell. Recombinant genes are introduced into host cells by transformation, transfection, infection, electroporation, and the like, producing multiple copies of the gene sequence. Preferably, the cloned gene is contained on a shuttle vector plasmid that is easy to purify for expansion of the cloning cell, eg E. coli, and, if desired, subsequent insertion into an appropriate expression cell line. For example, a shuttle vector, a vector that can replicate in one or more organisms, can be prepared by ligating sequences from an E. coli plasmid having a sequence that forms a yeast 2m plasmid in both E. coli and Saccharomyces cerevisiae. .

Expression of CaSR Isoform Proteins Nucleotide sequences encoding CaSR isoform proteins, or derivatives or analogs thereof, including chimeric proteins, can be expressed in a suitable expression vector, ie, transcription and translation of the inserted protein-coding sequence. Can be inserted into a vector containing the necessary elements. Here, such an element is called a “promoter”. Thus, the nucleic acid encoding the CaSR isoform of the present invention is operably linked to the promoter in the expression vector of the present invention. Both cDNA and genomic sequences can be cloned and expressed under the control of such control sequences. The expression vector preferably also includes an origin of replication. The necessary transcription and translation signals are provided on recombinant expression vectors, or they may be supplied by native genes encoding CaSR, CaSR isoform proteins and / or flanking regions thereof.

[0039] Possible host-vector systems include, but are not limited to, viral (
A mammalian cell line infected with a virus (e.g., a baculovirus); a microorganism such as a yeast containing a yeast vector; or a bacteriophage, DNA, plasmid Bacteria transformed with DNA or cosmid DNA. The expression elements of the vector are:
It depends on their length and specificity. Any of a number of suitable transcription and translation elements, depending on the host-vector system used, can be used.

[0040] The recombinant CaSR isoform proteins, derivatives, or analogs thereof of the present invention may be expressed chromosomally after integration of the coding sequence by recombination. In this regard, high levels of stable gene expression can be achieved using any of a number of amplification systems (Sambr
ook et al., 1989, supra). Cells having a recombinant vector containing a nucleic acid encoding a CaSR isoform protein are cultured in a suitable cell culture medium under conditions that provide for expression of the protein by the cells. Any of the previously described methods for inserting DNA fragments into cloning vectors can be used to construct expression vectors containing a gene consisting of appropriate transcription / translation control signals and protein coding sequences. These methods include in vitro recombinant DNA and synthetic methods and in vivo recombination (genetic recombination).

[0041] Expression of a gene can be controlled by any promoter / enhancer element known in the art, but these control elements must be functional in the host chosen for expression. Promoters that can be 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 Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445)
, The regulatory sequence 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., 197
8, Proc. Natl. Acad. Sci. USA 75: 3727-3731) or the tac promoter (DeBo
er et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25); Scientific American,
See also 1980, 242: 74-94, "Useful proteins from recombinant bacteria"; Gal
4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, promoter elements from yeast or other bacteria such as alkaline phosphatase promoter; and exhibit tissue specificity, and are used in transfected animals Animal transcription regulatory region: elastase I gene regulatory region active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, C
old Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987, Hepato
7: 425-515); Insulin gene regulatory region active in pancreatic β cells (Hanahan,
1985, Nature 315: 115-122), immunoglobulin gene regulatory region active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 5
33-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444), a mouse mammalian tumor virus control region active in testis, breast, lymphatic system and mast cells (Leder et al.
, 1986, Cell 45: 485-495), a liver-active albumin gene regulatory region (Pinkert et al.).
, 1987, Genes and Devel. 1: 268-276), α-fetal protein gene regulatory region active in liver (Krumlauf et al., 1985, Mol.Cell. Biol. 5: 1639-1648; Hammer et al., 1987,
Science 235: 53-58), α1-antitrypsin gene regulatory region active in liver (Kels
ey et al., 1987, Genes and Devel. 1: 161-171), a β-globin gene regulatory region active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986,
Cell 46: 89-94), myelin protein gene regulatory region active in oligodendroglial cells of the brain (Readhead et al., 1987, Cell 48: 703-712), myosin light chain-2 gene active in skeletal muscle Regulatory regions (Sani, 1985, Nature 314: 283-286), and gonadotropin-releasing hormone gene regulatory regions active in the hypothalamus (Mason et al., 1986, Science 234:
1372-1378);

The expression vector containing the nucleic acid encoding the CaSR isoform protein of the present invention comprises:
Five general approaches can be identified: (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selectable marker gene function, (d) appropriate Analysis with various restriction endonucleases and (e) expression of the inserted sequence. In the first approach, the nucleic acid is amplified by PCR, providing for detection of the amplified product. In the second approach, the presence of a foreign gene inserted into the expression vector is detected by nucleic acid hybridization using a probe containing a sequence homologous to the inserted marker gene. In a third approach, a recombinant vector / host system is used in which a particular “selectable marker” gene function (eg, b-galactosidase activity,
Identification and selection based on the presence or absence of thymidine kinase activity, resistance to antibodies, transformed phenotype, occlusion in baculovirus, and the like. In another example, when a nucleic acid encoding a CaSR isoform protein is inserted within the "selectable marker" gene sequence of the vector, a recombinant containing the nucleic acid is detected by its lack of gene function. In a fourth approach, a recombinant expression vector is identified by digestion with an appropriate restriction endonuclease. In the fifth approach,
The recombinant expression vector is used to determine the activity, biochemical, or immunological properties of the gene product expressed by the recombinant, such that the expressed protein is functionally active in a conformation. Can be identified on condition that

A wide variety of host / expression vector recombination can be used to express the DNA sequences of the present invention.
For example, useful expression vectors consist of stainable, non-stainable and synthetic DNA sequences.
Suitable vectors include derivatives of SV40 and known bacterial plasmids, such as the E. coli plasmid col El, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Ge
ne 67: 31-40), plasmids such as pMB9 and their derivatives, RP4; phage DNA
S, e.g., many derivatives of phage 1, e.g. NM989, and other phage DNA, e.g., M13 and filamentous single-stranded phage DNA; yeast plasmids such as 2m plasmids or derivatives thereof; vectors useful in insect or mammalian cells. Such vectors include those useful in eukaryotic cells; modified vectors derived from a combination of plasmid and phage DNAs, such as plasmids utilizing phage DNA or other expression control sequences; and the like.

For example, in a baculovirus expression system, but not limited to, pVL941 (Bam
H1 cloning site; Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI,
XmaIII, BglII, and PstI cloning sites; Invitrogen), pVL1392 (BglII, P
stI, NotI, XmaIII, EcoRI, XbaI, SmaI, and BamH1 cloning site; Summer
s and Invitrogen), and pBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII
Cloning sites, allowing blue / white recombinant screening; non-fusion transfer vectors such as, but not limited to, pAc700 (BamH1 and Kpn
I cloning site, BamH1 recognition site starts with start codon; Summers), pAc701
and pAc702 (similar reading frame as pAc700), pAc360 (cloning site 36 base pairs downstream of the BamH1 polyhedron start codon; Invitrogen (195)), and pBlueBacHisA,
B, C (3 with BamH1, BglII, PstI, NcoI, and HindIII cloning sites
Both different reading frames, N-terminal peptides for ProBond purification and blue / white recombination screening of plaques; fusion transfer vectors such as Invitrogen (220)) can be used.

[0045] Mammalian expression vectors contemplated for use in the present invention include vectors having an inducible promoter such as the dihydrofolate reductase (DHFR) promoter, for example, any expression vector having a DHFR expression vector, or pED (clone PstI, SalI, SbaI, SmaI, and Eco with vectors expressing both gene and DHFR
RI cloning site; Kaufman, Current Protocols in Molecular Biology, 16.
12 (1991)) and DHFR / methotrexate co-amplification vector.
Alternatively, pEE14 (HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning sites, the vector expresses glutamine synthetase and the cloned gene; Cel
glutamine synthetase / methionine sulfoximine co-amplification vectors such as ltech). In another embodiment, the following vectors for episomal expression under the control of Epstein-Barr virus (EBV) can be used: pREP4 (BamH1, SfiI,
XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning sites, constitutive Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI , NheI, HindII
I, NheI, PvuII, and KpnI cloning sites, constitutive human cytomegalovirus (hCMV) immediate early gene, hygromycin selectable marker; Invitrogen), pME
P4 (KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible metallothionein IIa gene promoter, hygromycin selectable marker: Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI, and KpnI)
Cloning site, RSV-LTR promoter, Histidinol selectable marker; Inv
itrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamHI cloning sites, RSV-LTR promoter, G418 selectable marker; Invitrogen), and
pEBVHis (RSV-LTR promoter, hygromycin selectable marker, N-terminal peptide purifiable by ProBond resin and cleaved by enterokinase;
trogen). Mammalian expression vectors that can be selected for use in the present invention include pRc / C
MV (HindIII, BstXI, NotI, SbaI, and ApaI cloning sites, G418 selection; In
vitrogen), pRc / RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G
418 selection; Invitrogen). Vaccine viral mammalian expression vectors (see Kaufman, 1991, supra) for use in the present invention include, but are not limited to, pSC11 (SmaI cloning site, TK- and b-gal selection), pMJ601 (SalI, SmaI,
AflI, NarI, BspMII, BamHI, ApaI, NheI, SacII, KpnI, and HindIII cloning sites; TK- and b-gal selections), and pTKgptF1S (EcoRI, PstI, SalI, AccI, H
indII, SbaI, BamHI, and Hpa cloning sites, TK or XPRT selection).

The use of a yeast expression system in accordance with the present invention also allows expression of CaSR isoforms. For example, for just two, the unfused pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, and
HindIII cloning site; Invitrogen) or fusion pYESHis A, B, C (XbaI, SphI
, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning sites, an N-terminal peptide purified by ProBond resin and cleaved with entetokinase; Invitrogen) can be used according to the invention.

[0047] Once a particular recombinant DNA molecule has been identified and isolated, it can be propagated by several methods known in the art. Once suitable host systems and growth conditions are established, recombinant expression vectors can be propagated and prepared in large quantities. As described above, expression vectors that can be used are not limited,
Includes the following vectors or derivatives thereof: human or animal viruses, such as vaccinia virus or adenovirus; insect viruses, such as baculovirus; yeast vectors; bacteriophage vectors, to name just a few.
Lambda), and plasmid and cosmid DNA vectors.

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 manner desired. Various host cells have features and specific mechanisms for translation and post-translational protein processing and modification. Selecting the appropriate cell line or host system can ensure the 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 "natural" folding. Furthermore, expression in mammalian cells can provide a means of remodeling, or building, CaSR activity. In addition, different vector / host expression systems may affect processing reactions, such as proteolytic cleavage, to different extents.

Vectors can be prepared by methods known in the art, for example, transfection, electroporation,
Microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), gene gun, or use of a DNA vector transporter (e.g., Wu et al., 1992, J. Biol. Chem. 267: 963-967). ; Wu and
Wu, 1988, J. Biol. Chem. 263: 14621-14624; see Hartmut et al., Canadian Patent Application 2,012,311 filed March 1990) into the desired host cell.

The soluble form of the protein may be obtained by collecting the culture broth or dissolving the inclusion bodies, for example by treatment with detergents and the desired ultrasound or other mechanical methods as described above. it can. Solubilized or soluble proteins can be obtained by polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, two-dimensional gel electrophoresis, chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography), Isolation can be achieved using a variety of methods such as centrifugation, absorbance differential solubility, immunoprecipitation, or any other standard method for protein purification.

Antibodies to CaSR isoforms The present invention provides antibodies that specifically bind to CaSR isoforms. Such antibodies are used in diagnosis to detect the presence and optionally the amount of the isoform in the cell. Among the antibodies of the present invention, particularly single-chain Fv antibodies (scFv)
Is also used in therapy and suppresses CaSR activity (see below). In a specific example, the antibody recognizes an epitope that is not present on the wild-type receptor, CaSRa. Monoclonal antibodies and antibody fragments (in addition to scFv antibodies) are also contemplated by the present invention. The antibody binds to the protein in the sample,
When the antibody of the present invention is used under conditions that allow detection of the binding of the antibody to the protein in the sample, the CaSR isoform in the cell can be obtained by contacting the sample taken from the cell with the antibody. It is possible to specifically detect the expression of the form, and detecting that the antibody binds to the protein indicates that the CaSR isoform is expressed in the cell.
The Western blot method, which is a quantitative immunoassay, makes it possible to quantify CaSR and detect an increase or decrease in the amount of CaSR, especially when compared to the first time point of the same cell or normal cells.

According to the present invention, a human CaSR isoform produced by genetic recombination or chemical synthesis and a fragment or another derivative or analog thereof are used to produce an antibody that recognizes its polypeptide, including a fusion protein. Used as an antigen, ie, an immunogen. A molecule is an antigen when it specifically interacts with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or a T cell antigen receptor. Antigenic polypeptides contain at least about 5, and preferably at least 10, amino acids. The antigenic portion of the molecule may be immunodominant for antibody or T cell receptor recognition, or used to produce antibodies against the molecule by attaching the antigenic portion to a carrier molecule involved in immunization. It can be said that it is the part that is done. Molecules that serve as antigens do not need to be immunogenic on their own and cannot elicit an immune response without a carrier. Desirably, the antigenic polypeptide has an epitope and / or wild-type C
It is a sequence that is not present in aSRa and induces antibodies that bind to the CaSR isoform.

[0053] Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fab expression libraries. The antibodies of the present invention exhibit cross-reactivity and may, for example, recognize different CaSR isoforms of different origin. In the case of polyclonal antibodies, the likelihood of cross-reactivity is greater. Alternatively, the antibodies of the invention may be specific for a single isoform of CaSR. In a preferred embodiment, the antibody is
It can specifically recognize CaSR isoforms, but cannot recognize wild-type CaSR.

Various methods well known to those skilled in the art are used to favor polyclonal antibodies. In order to produce antibodies, various host animals such as rabbits, mice, rats, sheep, goats, etc. can be immunized by inoculating CaSR isoforms or derivatives thereof, but not limited thereto. . In one embodiment, the polypeptide or fragment can be conjugated to an immunogenic carrier such as, for example, bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants are used depending on the host to enhance the immunological response, such as Freund (complete adjuvant and incomplete adjuvant), mineral gels such as aluminum hydroxide, lysolecithin, pronic polyols, polyvalent anions, and peptides. , Oily emulsions, surfactants such as keyhole limpet hemocyanin, dinitrophenol, and adjuvants that are likely to be applied to humans, such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum, but are not limited to these. Not necessarily.

To generate monoclonal antibodies to isoforms, fragments, analogs, and derivatives, techniques for producing antibody molecules by continuous cell line culture are used. Such techniques include trioma techniques and human B cell fusion techniques [Kozbor et al., Immunology Today 4:72 1983]; Cote et al., Proc. Natl. Acad.
Sci. USA 80: 2026-2030 (1983)] and EB virus cell fusion technology for producing human monoclonal antibodies [Cole et al., In Monoclonal Antibodies and Cance
r Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)], Kohler and Milstei
n, including, but not limited to, the cell fusion technology first developed by Nature [Nature 256: 495-497 (1975)]. In an additional embodiment of the present invention, the monoclonal antibody is a sterile animal [International Patent Publication No. WO 89/12
690, published 28 December 1989]. In fact, according to the present invention, CaSR
A "chimeric antibody" is obtained by splicing the gene of a mouse antibody molecule specific to an isoform with the gene of a human antibody molecule exhibiting appropriate biological activity.
[Morrison et al., J. Bacteriol. 159: 870 (1984); Neuberger et al., Nature 312: 604-
608 (1984); Takeda et al., Nature 314: 452-454 (1985)], and such antibodies are also within the scope of the invention. Compared to human or humanized chimeric antibodies compared to xenogeneic antibodies, such human or humanized chimeric antibodies are used in the treatment of human diseases, as they are very unlikely to cause an immune response, especially an allergic reaction. This is desirable (see below).

According to the present invention, single-chain Fv (scFv) antibody antibodies [US Patent No. 5,476,78
6 and 5,132,405 to Huston; US Pat. No. 4,946,778] can be applied to generate CaSR isoform-specific single chain antibodies. In an additional embodiment of the present invention, a monoclonal antibody having the desired specificity for a CaSR isoform is obtained by utilizing the technique described for Fab expression libraries [Huse et al., Science 246: 1275-1281 (1989)].
Fab fragments can be identified quickly and easily. Antibody fragments which contain the idiotype of the antibody molecule can be produced by known techniques. For example, such fragments include F (ab ′) 2 fragments produced by pepsin digestion of antibody molecules, Fab ′ fragments produced by reducing disulfide bonds of F (ab ′) 2 fragments, papain, and reducing agents. Examples include, but are not limited to, Fab fragments produced by processing antibody molecules.

When producing an antibody, screening for a desired antibody can be performed by, for example, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, antibody labeling method, immunoradiometric assay, in-gel precipitation reaction, immunodiffusion method (for example, In situ immunoassay (using colloidal gold, enzyme or radioisotope labeling), Western blotting, sedimentation, agglutination (eg, in-gel agglutination, hemagglutination), complement binding assay, immunofluorescence, Performed by techniques known in the art, such as protein A assays, and immunoelectrophoresis. In some embodiments, the binding of the antibody is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting the binding of a secondary antibody or reagent to the primary antibody. In yet another embodiment, the secondary antibody is labeled. In the field,
Many methods for detecting binding in immunoassays are known and are within the scope of the present invention. For example, to select antibodies that recognize a specific epitope of the CaSR isotope, hybridomas may be measured for products that bind to isoforms containing such epitopes. When selecting antibodies specific for isoforms from a particular animal species,
Selection can be based on binding positively to CaSR isoforms or CaSR isoforms isolated therefrom but not to wild-type CaSR.

[0058] Methods known in the art relating to the localization and activity of CaSR isoforms, such as, for example, western blotting, in situ imaging of isoforms, and assessing levels in appropriate physiological samples therefor. The antibody can be used while measuring its level in a suitable physiological sample using the detection techniques described above or techniques known in the art. In a specific example, an antibody that enhances the activity of CaSR or an antibody against it, and particularly an antibody specific to the CaSR isoform can be prepared. Such antibodies can be tested using the ligand identification assays described below. In particular, such antibodies include scFv antibodies expressed in cells.

Screening Methods In comparison to cells isolated from natural sources, indicator cells that have been specially engineered to show CaSR activity after nucleic acid infection or cell transformation,
Identification and isolation of genes encoding SR isoforms results in high expression levels of these isoforms. As a result, in addition to the theoretical design of agonists and antagonists based on the structure of the CaSR isoform, the present invention provides for the use of various screening methods known in the art to specifically identify CaSR isoforms. Consider a variant to identify the ligand. To screen for CaSR agonists or antagonists, any of the screening techniques known in the art may be used. The present invention provides for the selection of intrinsic ligands that bind to CaSR activity in vivo to activate (calcimimetics) or inhibit (calcilytics), It is intended to separate ligands or ligand homologs from analogs. For example, using the assays of the present invention, natural product libraries are screened for molecules that agonize or antagonize CaSR activity. The present invention provides both techniques and methodologies for identifying compounds that can modulate CaSR activity, including modulation specific to CaSR isoforms. Screening methods for calsimimetics and calcilytics are discussed in WO 94/18959 and US Pat. No. 5,763,569, the entire contents of which are incorporated by reference.

In a preferred screening method, compounds are evaluated for their ability to affect intracellular calcium levels. In some cases, the compound is tested alone in connection with increasing extracellular calcium concentration, and in the other, it is tested in the presence of an agonist or antagonist of CaSR isoform activity. Agonists can include neomycin, divalent and trivalent cations (gadolinium, calcium, magnesium, strontium, barium, lanthanum), polyamines, and other known calsimimetics. Intracellular calcium is measured using a fluorescent indicator Hula-2 (manufactured by Molecular Probe). For example, HEK- into which a nucleic acid encoding a CaSR isoform has been introduced.
293 cells were 0.5 uM fura-2, 20 mM HEPES, pH 7.35, 0.1% BSA, 0.5 mM CaCl2, 0.5
Buffer 37 mM mM MgCl2, 6.7 mM KCl, 3 mM glucose, and 142 mM NaCl.
Put into ° C for 45 minutes. The cells are washed and resuspended in loading buffer without Fra-2 to approximately 2 x 106 cells / ml. For measurement of intracellular calcium, cells were placed in a quartz cuvette kept at 37 ° C. Fluorescence is detected using an excitation monochromator focused at 340 and 380 nm, and emission light collected at 505 nm.

The similarity between the primary sequence of the CaSR isoform and the sequence of the protein, which is a known function, provides a first clue to the protein as an inhibitor or antagonist. Determining the structural characteristics of the protein, such as by using X-ray crystallography, neutron diffraction, magnetic resonance analysis, and other techniques for structure determination, further exacerbates the identification and screening of antagonists. These techniques provide a theoretical design for agonists and antagonists and allow identification. Another approach uses recombinant bacteriophage to create large libraries. "Phage method" [Scott and Smith, 1990, Science 249: 386-39
0 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87: 6378-6382 (1990); Devin et al.,
Science, 249: 404-406 (1990)], a very large library can be constructed (chemicals 106-108 items). The second approach mainly uses chemical methods, such as the Geysen method [Geysen et al., Molecular Imm.
unology 23: 709-715 (1986); Geysen et al. J. Immunologic Method 102: 259-274 (1
987)], and Fodor et al. [Science 251: 767-773 (1991)]. Furka and others
[14th International Congress of Biochemistry, Volume 5, 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. 5,010,1
No. 75, issued April 23, 1991] describes a method for preparing a mixture of peptides that can be tested as agonists or antagonists.

In another embodiment, a synthetic library [Needels et al., Proc. Natl. Acad. Sci.
SA 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; K
ocis et al., International Patent Publication No. WO 9428028, each of which is incorporated in its entirety into the reference material], and analogs thereof, which, in accordance with the present invention, screen for CaSR ligands. Can be used. Using recombinant cells expressing the CaSR isoform, or alternatively,
For example, screening may be performed using a protein synthesized and then purified by genetic recombination as described above. As described in the above references, a labeled water-soluble CaSR isoform containing, for example, the extracellular calcium-binding protein of the molecule may be used for library selection.

In one embodiment, the CaSR isoform is directly labeled. In another embodiment, a labeled secondary agent is used to detect binding of the isoform to the molecule of interest, eg, to attach the molecule to a solid support. Binding is detected by chromophore formation by enzymatic labeling in situ. Suitable enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase. In yet another embodiment, a two-color analysis is performed using two chromophores with enzyme labels on different receptor molecules of interest. Two-color analysis distinguishes between cross-reactive and single-reactive ligands. Labels used in the present invention may also include colored latex beads, magnet beads, fluorescent labels (eg, fluorescent isocyanate (FITC), phycoerythrin (PE),
Texas red (TR), rhodamine, a chelate or free lanthanide-based salt, especially Eu3 +, especially named for some chromophores), chemiluminescent molecules, radioisotopes or magnetic resonance labels. Two-color analysis is performed using two or more colored latex beads or chromophores that emit different wavelengths. The label is detected visually or by mechanical / optical methods. Mechanical / optical methods include excitation fluorescence sorting, ie, FACS and minimanipulator removal.

As illustrated herein, levels of CaSR isoforms are assessed by the metabolic level of the protein. The level of detection of each marker is affected by in vitro conditions during in vitro sensitization of biopsy-removed tissue in media supplemented with [ ³⁵ S] -methionine. In addition to metabolic (or biosynthetic) labeling with [ ³⁵ S] -methionine, the present invention contemplates labeling with [ ¹⁴ C] -amino acids and [ ³ H] -amino acids. Thus, a sample or library of compounds can be analyzed directly after labeling the protein, for example, by colorimetric staining with silver, gold, Coomassie blue, or amide-cyvalz, Isotope labeling with [ ³² P] -orthophosphate, [ ¹²⁵ I], [ ¹³¹ I], etc .;
Fluorescent or chemiluminescent tags; immunological detection using labeled antibodies or specific binding partners of markers.

Pharmaceutical Compositions and Therapy In the art, disorders and diseases associated with calcium homeostasis, and thus CaSR, are known. Such diseases are associated with functional cellular responses to calcium, such as parathyroid hormone secretion from parathyroid cells, calcitonin secretion by C cells, and bone resorption by osteoclasts. Taking hyperparathyroidism as an example, this disease results in elevated plasma parathyroid hormone levels. Therefore, a method of lowering plasma parathyroid hormone concentration is one of the treatment methods for hyperparathyroidism. Alternatively, increasing plasma calcitonin concentration is accompanied by suppression of bone resorption. Inhibition of bone resorption provides one treatment for osteoporosis. The present invention provides methods and methodologies for identifying compounds that can regulate CaSR activity, such as specific regulation of CaSR isoforms, and can be used for the treatment of diseases and disorders associated with abnormal calcium levels. It shows both. As a result, the present invention provides a pharmaceutical composition for treating a patient suffering from a disease or disorder associated with abnormal calcium concentration in plasma or the like by administering a therapeutically effective amount of a compound capable of regulating the activity of a CaSR isoform. It provides things and treatment methods. The term "patient" is intended to include both humans and other animals.

“Pharmaceutical composition” refers to a compound of the present invention and a preservative, filler, disintegrant, wetting agent, emulsifier, suspending agent, sweetener, flavor, fragrance, antibacterial, antifungal, At least one of the components selected from the group of pharmaceutically acceptable carriers, diluents, adjuvants, adjuvants or vehicles, depending on the nature of the dosage form and dosage form, such as, lubricants, and dispensing agents. A compound containing one type of component is meant. Examples of suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, or mixtures of these substances. Control of microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It is also desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectables can be obtained by using agents which delay the absorption, such as aluminum monostearate and gelatin. Examples of suitable carriers, diluents, solvents or vehicles include water, ethanol, polyols, mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. included. Examples of excipients include lactose, lactose, sodium citrate, calcium carbonate, dicalcium phosphate phosphate. Examples of disintegrants include starch, alginic acid, and certain silicate complexes. Examples of the lubricant include magnesium stearate, sodium lauryl sulfate, and talc in addition to the high molecular weight polyethylene glycol. "Pharmaceutically acceptable" is within the scope of sound medical judgment and is suitable for use in contact with cells of humans and lower animals, and
It does not cause irritation, allergic reactions, and the like, meaning that there is a reasonable benefit-to-risk ratio.

“Pharmaceutically acceptable dosage form” refers to dosage forms of the compounds of the present invention, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, suspensions, It includes sprays, inhalation tablets, troches, emulsions, solutions, granules, capsules, and suppositories, as well as other liquid preparations for injection, including liposomal preparations. Technology and synthesis are from Remington's Pharmaceutical Sciences, Mac
k Publishing Co., Easton, PA.

“Pharmaceutically acceptable salt” refers to relatively less toxic inorganic, organic, and base addition salts with respect to compounds of the present invention. Such salts are formed during the final separation and purification of the compound. In particular, acid addition salts are formed by separately reacting a purified compound in the form of the free base with a suitable organic or inorganic acid and separating the salt formed thereafter. Typical acid addition salts include 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, Glucoheptanate, lactiobionate, sulfamate, malonate, salicylate, propionate, methylene-bis-b-hydroxynaphthoate, gentisate, isethionate, di-p -Toluoyl tartrate, methane-sulfonate, ethanesulfonate, benzenesulfonate, p-
Toluene sulfonate, cyclohexyl sulfamate, and quinate lauryl sulfonate, and the like (eg, SM Berge et al., "Pharmaceutical Salts," J. Pharm. Sci. , 66: p.
1-19 (1977)). Also, base addition salts are prepared by separately reacting a purified compound in the acid form with a suitable organic or inorganic base and separating the salt formed therefrom, Contains chemically acceptable metals and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. Sodium and potassium salts are preferred. Suitable inorganic base addition salts are
It is made from metal bases such as sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide and zinc hydroxide. Suitable amine base addition salts are those frequently used in medicinal chemistry due to their low toxicity and pharmaceutically acceptable because they are made from amines that have sufficient basicity to make stable salts Includes Ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine,
Piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, trimitylamine, dibenzylamine, ephenamine,
Basic amino acids such as dehydroabiethylamine, N-ethylpiperazine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, lysine, and arginine, dicyclohexylamine, and the like.

“Solid dosage form” means that the dosage form of the compound of the present invention is solid, and includes, for example, capsules, tablets, pills, powders, dragees or granules. In such solid dosage forms, the compound of the invention may contain at least one inert conventional adjuvant (or carrier), such as sodium citrate or dicalcium phosphate, i.e., (a) starch, lactose, Fillers and bulking agents such as sugar, glucose, mannitol, and silicic acid; (b) binders such as carboxymethylcellulose, alignate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants such as glycerol ( d) disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicate complexes, sodium carbonate,
(E) solution retarders such as paraffin (f) absorption enhancers such as quaternary ammonium compounds (g) wetting agents such as cetyl alcohol and glycerol monostearate (h)
Adsorbents such as kaolin and bentonite (i) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol and sodium lauryl sulfate (j) Opacifier (k) Buffer, and delayed in certain parts of the intestinal tract In combination with agents that release the compounds of the invention in any suitable manner.

The choice of excipient and the content of the active substance in the excipient are generally based on the solubility and chemical properties of the active substance, the particularity of the mode of administration and the pharmacologically recognized regulations. Is determined. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate combined with lubricants such as magnesium stearate, sodium lauryl sulfate, and talc, and starch, alginic acid, and certain silicate complexes Disintegrants, such as the body, may be used to produce tablets. For the manufacture of capsules, it is convenient to use lactose and high molecular weight polyethylene glycols. When an aqueous suspension is used, it may contain an agent that promotes suspension, such as an emulsion. Diluents such as sucrose, ethanol, polyethylene glycol, glycerol, and chloroform, or mixtures thereof, may also be used.

The oil phase of the emulsion according to the invention is, in a known manner, composed of known components.
The phase may contain only emulsifiers (otherwise known as emission enhancers), but preferably at least one emulsifier and a mixture of fat or oil or both fat and oil It is better to contain Preferably, the hydrophilic emulsifier is included with a lipophilic emulsifier that acts as a stabilizer. It is also desirable that it contains both oil and fat. Taken together, the emulsifiers, whether or not they contain stabilizers, form emulsifiable waxes and, when oil and fat coexist, form an emulsified basecoat that forms the oil dispersion phase of the cream formulation. In the preparation of the present invention, Tween® 60, Spa
nR 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearic acid, and sodium lauryl sulfate. If desired, the aqueous phase of the cream base may include, for example, polyhydric alcohols, ie, polypropylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol, and polyethylene glycol (including PEG 400), mixtures thereof, and the like. , At least 30% w / w of alcohols having two or more hydroxyl groups. Formulations for topical use preferably include a compound that enhances absorption or penetration of the active ingredient through the skin or other areas that affect the skin. Examples of such skin penetration enhancers include dimethyl sulfoxide and analogs related thereto.

The selection of an appropriate oil or fat for the product is based on the desired cosmetic properties. The cream is therefore desirably non-greasy and non-dyeing,
The product should be one that can be washed while having a stable concentration that does not leak from tubes or other containers. Straight or branched chain, such as di-isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a mixture of branched esters known as Crodamol CAP , Mono- or dibasic alkyl esters are used, the last three being preferred esters. These may be used alone or in combination depending on the required properties. Alternatively, high melting point liquids such as white petrolatum and / or liquid paraffin or other mineral oils may be used. In soft and hard-filled gelatin capsules, the solid components may be used as fillers with excipients such as lactose, ie, lactose, in addition to high molecular weight polyethylene glycols and the like.

Pharmaceutical compositions include oral, inhalation, rectal, nasal, buccal, sublingual, vaginal, parenteral (subcutaneous, intramuscular, intravenous, intradermal, Internal administration,
It is administered in a formulation suitable for humans and animals by local or systemic administration, such as intrathecal and epidural administration), intracisternal administration and intraperitoneal administration. It will be appreciated that the preferred route of administration may vary with the condition of the recipient and the like. The formulations are made into unit dosages using 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 so that the active ingredient is homogeneously and carefully combined with the liquid carrier, or finely divided solid carrier, or both, and, if necessary, subsequently shape the product. I have.

“Formulations suitable for oral administration” are each predefined as powders or granules; aqueous or non-aqueous liquid solutions or suspensions; oil-in-water or water-in-oil liquid emulsifiers. It is made up of discrete units such as capsules, cachets or tablets containing the active ingredient. The active ingredient is prepared as a large pill, a lozenge, or a paste. Tablets are made by compression or molding, optionally with auxiliary ingredients. Compressed tablets are made by compressing the active ingredient in a fluid state, such as a powder or granules, with a suitable machine, optionally with binders, lubricants, inert diluents, preservatives, surfactants Or mixed with a dispersant. Molded tablets are made by pouring the mixture of the powdered compound moistened with an inert liquid diluent into a suitable machine. The tablets are optionally coated and are designed so that the active ingredient therein is released in a slow and controlled manner.

Solid compositions for rectal administration include suppositories prepared according to known methods and containing at least one compound of the invention. Where necessary and for more effective distribution of the compound, the compound may be incorporated into a biocompatible, biodegradable polymer matrix (eg, poly (d, 1-lactide co-) in a slow release or targeted delivery system. Subcutaneous injection using a technique called subcutaneous or intramuscular depots, such as glycolide)), liposomes, and microspheres, which are placed in microcapsules or attached and slowly released continuously over 2 weeks or more. Or it may be injected intramuscularly. The compound may be sterilized by, for example, filtration through a bacteria-removing filter, or by incorporating a bactericide in the form of a sterile solid composition that can be dissolved in sterile water or other injectable solution immediately before use.

The actual dosage levels for the active ingredients in the compositions of the present invention may be varied in order to obtain the required amount of active ingredient to achieve the desired therapeutic response due to the particular composition and mode of administration. There is. Therefore, the selected dosage level depends on the desired therapeutic effect, the route of administration, the desired duration of administration, and other factors. The compounds of the present invention are administered to a host in single or divided doses, for example, from about 0.001 to about 100 mg / kg body weight per day, preferably from 0.01 to 10 mg / kg / day. To meet the daily dose, unit doses of the compositions thus include such submultiples of the daily dose. However, for special patients, such as body weight, general condition, gender, diet, time and route of administration, absorption and elimination rates, combinations with other drugs, and the severity of the particular disease being treated, etc. It will be appreciated that various factors will set particular dosage levels. The dosage of each component is determined by the attending physician, taking into account the cause and severity of the disease, the condition and age of the patient, the effectiveness of each component, and other factors. The formulation is placed in a unit dose or multi-dose container, for example, a sealed ampoule and a vial with a rubber stopper, and lyophilized (freeze only) in which a sterile liquid carrier such as water for injection is added immediately before use. (Dry) condition. Extemporaneous inoculation solutions and suspensions are prepared from sterile powders, granules, and tablets of the kind previously described.

Gene Therapy and Transgenic Vectors The present invention is also involved in gene therapy for diseases involving abnormal calcium levels. As mentioned above, a "vector" is a means for transferring a nucleic acid into a host cell according to the present invention. Preferred vectors are retrovirus, herpes virus,
Viral vectors such as adenovirus and adeno-associated virus. Thus, the gene encoding CaSR, CaSR isoform, its polypeptide domain fragment, or the nucleic acid encoding the CaSR antisense sequence can be used in vivo, half-vivo, depending on whether a viral vector is used or the DNA is directly introduced. ,
Or introduced in vitro. Whether expression in target tissues can direct the transgenic vector to specific cells, such as by using a viral vector or receptor ligand, using a tissue-specific promoter, or using both. Affected by

The expression vectors of the present invention may be used in vivo or for the purpose of screening or biological testing for modulators of CaSR activity, or for gene therapy purposes such as increasing or decreasing CaSR activity levels, as indicated above. To deliver a CaSR nucleic acid or a CaSR antisense gene ex vivo, the cells can be used to transfect nucleic acids. In addition, a vector expressing the anti-CaSR scFv can be introduced using the above-described technique. Viral vectors commonly used for targeting and therapeutic purposes in vivo or ex vivo are DNA base vectors and retroviral vectors. Methods for synthesizing and using viral vectors are well known in the art [see, eg, Miller and Rosman, BioTechniques 7: 980-990 (1992)]. Preferably, the viral vector is replication defective, ie, the viral vector cannot replicate automatically in the host cell. Generally, the genome of the replication-defective viral vector used within the scope of the present invention has at least one deletion in a region required for virus replication in infected cells. These areas may be removed (in whole or in part) by techniques known to those skilled in the art, or may stop functioning. These techniques include total removal, substitution (by another sequence, especially an inserted nucleic acid), partial deletion, or the addition of one or more bases to an essential region (for replication). Such techniques are available in vitro (on isolated DNA) or in situ.
It is performed by using a genetic engineering technique or by treating with a mutagenic substance. Preferably, the replication deficient virus maintains the genomic sequence required for virus particle encapsulation.

DNA viral vectors include attenuated or defective DNA such as herpes simplex virus (HSV), papilloma virus, EB virus (EBV), adenovirus, adeno-associated virus (AAV) vaccinia virus, and the like. Viruses include, but are not limited to. Preferably, the defective virus lacks all or most of the viral genes. Defective viruses cannot replicate after insertion into cells and do not lead to productive virus infection. By using a defective viral vector, the vector can be administered to cells located in a particular localized location without the risk of infecting other cells. In this way, specific tissues can be specifically targeted. Examples of special vectors include the defective herpesvirus 1 (HSV1) vector [Kaplitt et al., Molec.
Cell. Neurosci. 2: 320-330 (1991)], a defective herpesvirus vector lacking the glycoprotein L gene [Patent Publication RD 371005 A], or another defective herpesvirus vector [International Patent Publication No. WO 94/21807, 1994] Published October 29; International Patent Publication No. WO 92/05263, issued April 2, 1994]; Stratford-Perricaudet et al. [J. Clin.
Invest. 90: 626-630 (1992); see also La Salle et al., Science 259: 988-990 (1993)].
An attenuated adenovirus vector, such as the vector described by
Defective adeno-associated virus vector [Samulski et al., J. Virol. 61: 3096-3101 (1987)
; Samulski et al., J. Virol. 63: 3822-3828 (1989); Lebkowski et al., Mol. Cell. Bio.
l. 8: 3988-3996 (1988)]

Preferably, for in vivo administration, appropriate immunosuppressive treatment is performed in conjunction with a viral vector, such as an adenovirus vector, to avoid immune inactivation of the cells containing the viral vector and nucleic acid. To block a humoral or cellular immune response to a viral vector, for example, administer an immunosuppressive cytokine such as interleukin-12 (IL-12), interferon-g (IFN-g), or an anti-CD4 antibody Good [see eg Wilson, Nature Medicine (1995)]
. Furthermore, it is convenient to use a viral vector that can minimize the number of expressed antigens. Of course, the present invention contemplates the delivery of, or antisense to, vectors expressing a therapeutically effective amount of CaSR for gene therapy applications. The phrase "therapeutically effective amount" herein inhibits at least about 15%, preferably at least 50%, more preferably at least 90%, and most preferably, in terms of host activity, function, or response, It means that the amount is sufficient to prevent a significant loss. Alternatively, a therapeutically effective amount is an amount sufficient to effect improvement if the host is in a clinically severe condition.

The vectors of the invention, whether viral or not, should preferably be introduced into the body, preferably in a pharmaceutically acceptable excipient or carrier. The phrase "pharmaceutically acceptable", when administered to a human, is physiologically tolerable and typically results in an allergic reaction, or increased gastric peristalsis,
It means that the molecular structure does not cause similar adverse reactions such as dizziness and the like. Preferably, as used herein, the term "pharmaceutically acceptable" is recognized by federal or state government regulators for use in animals, particularly humans, or It is meant to be listed in other recognized pharmacopeias. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers may be water and sterile liquids, such as petroleum, animal, vegetable, and synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or saline, dextrose, and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are
It is described in "Remington's Pharmaceutical Sciences" by EW Martin.

Adenovirus Vector In a preferred embodiment, the vector is an adenovirus. Adenovirus is
It is a eukaryotic DNA virus that is modified to efficiently deliver the nucleic acid of the present invention to various cells. There are different serotypes of adenovirus. Of these serotypes, within the scope of the present invention, a human-derived adenovirus of type 2 or type 5 (Ad
2 or Ad 5) or the use of animal-derived adenovirus (see WO94 / 26914). The animal-derived adenovirus used in the scope of the present invention is derived from dogs, cows, mice (eg, Mav1, Beard et al., Virology 75 (1990) 81), sheep, pigs, chickens, and monkeys (eg, SAV). Adenovirus.
Preferably, the animal derived adenovirus is a canine adenovirus, more preferably a CAV2 adenovirus (eg, Manhattan strain or A26 / 61s strain (ATCC VR-800)).

Preferably, the replication-defective adenovirus vector of the invention comprises ITRs, encapsulation sequences and comprises the relevant nucleic acid sequences. More preferably, at least the E1 region of the adenovirus vector does not function. The deletion in the E1 region is preferably
The sequence of the Ad5 adenovirus extends to nucleotides 455-3329 (PvuII-BglII fragment) or 382-3446 (HinfII-Sau3A fragment).
In other regions, particularly, any region is modified in the E3 region (WO95 / 02697), the E2 region (WO94 / 28938), the E4 region (WO94 / 28152, WO94 / 12649 and WO95 / 02697), or the genes L1-L5 described below. ing. In a preferred embodiment, the adenovirus vector has a deletion in the E1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed in EP 185,573, the contents of which are incorporated herein by reference. In another preferred embodiment, the adenovirus vector has deletions in the E1 and E4 regions (Ad 3.0). Examples of E1 / E4-deleted adenoviruses are disclosed in WO95 / 02697 and WO96 / 22378, the contents of which are incorporated herein by reference. In yet another preferred embodiment, the adenovirus vector has a deletion in the E4 region and the E1 region into which the nucleic acid sequence has been inserted (see FR94 13355, the contents of which are incorporated herein by reference). ).

The replication-defective recombinant adenovirus according to the invention is produced by techniques known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J
3 (1984) 2917). In particular, it is produced by homologous recombination between an adenovirus and a plasmid carrying a DNA sequence of particular interest. Homologous recombination is effected following co-transduction of the adenovirus and the plasmid into the appropriate cell line. The cell line used is preferably (i) a part that can be modified by said elements, and (ii) a part of the genome of the replication-defective adenovirus, preferably intact, in order to avoid the risk of recombination. Is included.
An example of a cell line used is the human fetal kidney cell line 293 (Graham et al., J. Gen. Virol.
1977) 59) and patent applications WO 94/26914 and WO 95/02697
And a cell line that can supplement E4 function. Recombinant adenovirus is recovered and purified using standard molecular biology techniques well known to those skilled in the art.

Adeno-Associated Virus Vectors Adeno-associated virus vectors (AAV) are relatively small DNA viruses that can bind to the genome of infected cells in a stable, site-specific manner. They are capable of infecting a wide range of cells without affecting cell growth, morphology, or differentiation, and are not considered to be involved in human disease. The AAV genome has been cloned, sequenced and characterized. The AAV genome contains about 4700 bases, with about 145 bases at each end, an inverted terminal repeat (ITR) sequence region, which is the source of replication for the virus. The rest of the genome is divided into two essential regions that carry the encapsulation function, but the left-hand part of the genome contains rep genes involved in viral replication and expression of viral genes; Contains the cap gene encoding the capsid protein of the virus.

The use of vectors derived from AAVs has been described for in vitro and in vivo gene transfer (WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,9).
41, EP 488 528). These publications describe various AAV-derived constructs in which the rep and / or cap genes have been deleted and replaced with genes of interest, in vitro (in cultured cells) or in vivo (directly in bacteria). The use of these constructs to transfer the gene of interest is described. The replication-defective recombinant AAVs according to the invention comprise a plasmid containing an interesting nucleic acid sequence flanked by two AAV inverted terminal repeat (ITR) regions, an AAV encapsulated gene (rep gene and
A plasmid carrying the cap gene) can be made by co-transfection into a cell line infected with a human helper virus (eg, an adenovirus). The resulting AAV recombinant is purified by standard techniques.

Thus, the present invention relates to an AAV-derived recombinant virus having a genome comprising a sequence encoding a nucleic acid encoding CaSR flanked by AAV ITRs. Further, the present invention relates to a plasmid containing a sequence encoding a nucleic acid encoding CaSR sandwiched between two ATR-derived ITRs. Such a plasmid is suitable for binding to a liposomal vector (pseudovirus) and can therefore be used to carry nucleic acid sequences.

[0088] In another embodiment the retroviral vector, sometimes the gene is introduced into the retroviral vector,
For example, Anderson et al., U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33: 153; Temin.
Et al., U.S. Pat.No. 4,650,764; Temin et al., U.S. Pat.No. 4,980,289; Markowitz et al.
1988, J. Virol. 62: 1120; Temin et al., U.S. Pat.No. 5,124,263; EP 453242, EP
178220; Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology.
3 (1985) 689; International Patent Publication No.WO 95/07358, publishe
d March 16, 1995, by Dougherty et al .; and Kuo et al., 1993, Blood 82: 845. Retroviruses are integrated viruses that infect dividing cells. The retroviral genome consists of two LTRs, an encapsulation sequence, and 3
It has two coding regions (gag, pol and env). In general, the recombinant retrovirus will have the gag, pol and env genes deleted in the front or part and replaced with a heterologous nucleic acid sequence of interest. These vectors contain HIV, MoMuLV ("murine Moloney
Leukemia virus "MSV (" murine Moloney sarcoma virus "), HaSV (" Harvey sar
coma virus "); SNV (" spleen necrosis virus "); RSV (" Rous sarcoma virus ")
And different types of retroviruses, such as Friend virus. Defective retroviruses are disclosed in WO95 / 02697.

Generally, to make a recombinant retrovirus with a nucleic acid sequence, a plasmid containing the LTR, the encapsulation sequence, and the coding sequence is assembled. This construct,
For use in transferring into a packaging cell line, the cell line can provide this construct with retroviral functions lacking a plasmid in trans. Thus, in general, the packaged cell lines are gag, pol, and en
v gene can be expressed. Such packaged cell lines are particularly suitable for cell line P
A317 (US Patent No. 4,861,719); PsiCRIP cell line (WO90 / 02806) and GP + envAm-
12 cell line (WO89 / 07150) described in the prior art. In addition, the recombinant retroviral vector has a large encapsulation sequence containing part of the gag gene, along with the LTR
There are modifications to suppress transcriptional activity (Bender et al., J. Virol. 61 (1987) 16
39). Recombinant retroviral vectors are isolated by standard techniques known to those skilled in the art.

A retroviral vector functions as an infectious particle or is structured to carry out one round of nucleic acid infection. In the former case, the virus is modified to express a heterologous gene, leaving its own gene, except for the genes involved in oncogenic transformation properties. Uninfected viral vectors are engineered to disrupt the viral packaging signal, but retain the heterologous gene and the structural genes necessary to encapsulate the co-transfected virus that has been engineered to contain the packaging signal. As a result, the virus particles produced cannot produce the next virus. Delivery of targeted genes is described in International Patent Publication WO 95/2, published October 1995.
8494.

Non-Viral Vectors Alternatively, the vectors are introduced in vivo by lipofection. In the past decade, the use of liposomes for in vitro encapsulation and nucleic acid infection has increased. Synthetic cationic lipids designed to reduce the difficulty and risk of encountering liposomes that mediate nucleic acid infection are used to prepare liposomes for transfer of markers-encoding genes in vivo [Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987); Mac
Key et al., Proc. Natl. Acad. Sci. USA 85: 8027-8031 (1988); Ulmer et al., Scien.
ce 259: 1745-1748 (1993)]. The use of cationic lipids facilitates the encapsulation of negatively charged nucleic acids and also facilitates fusion with negatively charged cell membranes [Felg
ner and Ringold, Science 337: 387-388 (1989)]. Particularly useful lipid compounds and structures related to nucleic acid infections are described in International Patent Publications WO95 / 18863 and WO96 / 17823, and U.S. Pat.
No. 5,459,127. There are practical advantages to using lipofection to introduce foreign genes into specific organs in vivo. One of the advantages is the liposome target molecule for specific cells. In tissues with cellular heterogeneity, such as the pancreas, liver, kidney, and brain, it is clear that it is particularly advantageous to direct nucleic acid infection to special cell types. Lipids chemically bind to other molecules for targeting purposes [Mackey et al., Supra]. Targeted hormones, peptides such as neurotransmitters, and proteins such as antibodies, or non-peptide molecules are chemically bound to liposomes.

In addition, cationic oligopeptides (International Patent Publication WO95 / 21931), peptides derived from DNA binding proteins (for example, International Patent Publication WO96 / 25508), cationic polymers (International Patent Publication WO95 / 21931), and the like, There are molecules useful for promoting nucleic acid infection in vivo. It is also possible to introduce the vector in vivo, such as a naked DNA plasmid. Naked DNA vectors for gene therapy include nucleic acid infection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. It is introduced into the desired host cell by methods known in the art. [
For example, Wu et al., J. Biol. Chem. 267: 963-967 (1992); Wu and Wu, J. Biol. Chem. 26
3: 14621-14624 (1988); Hartmut et al., Canadian Patent Application No. 2,012,311, March 1990.
Submitted on 15th; see Williams et al., Proc. Natl. Acad. Sci. USA 88: 2726-2730 (1991).
There is also a need for a receptor-mediated DNA delivery approach [Curiel et al., Hum. Gene.
Ther. 3: 147-154 (1992); Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987)]
.

The present invention provides several examples for specifically inhibiting CaSR activity in patients suffering from a disorder associated with abnormal calcium levels. As a first example, CaSR expression is suppressed by a nucleic acid containing a sequence complementary to a sequence encoding CaSR, or the expression of an isoform thereof is controlled or blocked. A preferred embodiment includes an antisense polynucleotide molecule. Creation and use of antisense polynucleotides, DNA encoding antisense RNA molecules,
The use of oligo and genetic antisense is disclosed in WO 92/15680,
The entire contents of which are incorporated herein by reference.

[0094] The antisense nucleic acids of the present invention preferably comprise SEQ ID No. 7, SEQ ID No. 9 and SE.
It is an RNA that can specifically hybridize with the entire sequence or a part thereof selected from the group consisting of Q ID No. 11, or the corresponding messenger RNA. The antisense sequences of the present invention are those whose expression in a cell is derived from a DNA sequence that makes a complementary RNA to the whole CaSR or a part thereof. These antisense sequences are generated by expression of the entire sequence or a portion thereof selected from the group consisting of SEQ ID No. 7, SEQ ID No. 9, and SEQ ID No. 11 in opposite orientations (EP 140 308). Regardless of the length of the antisense sequence, it can be said that the antisense sequence is suitable for practicing the present invention as long as the antisense sequence suppresses or blocks the expression of CasR or its isoform. Preferably, the antisense sequence is at least 20 nucleotides in length.

[0095] In another embodiment of this preferred embodiment, the nucleic acid encodes an antisense RNA molecule. In this example, the nucleic acid is linked to a signal that allows expression of the nucleic acid sequence and is introduced into the cell being used, preferably a recombinant vector construct, but once the vector is introduced into the cell, the antisense nucleic acid Is expressed. As mentioned above,
Examples of suitable vectors include plasmids, adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses. Suitable expression signals include transcriptional promoters and termination sequences. Among the promoter sequences, useful in the practice of the present invention are the tetracycline-controlled transcriptional regulator, CMV promoter, SV-40 promoter, E1a promoter, MLP promoter, and LTR promoter. Tetracycline-regulated transcriptional regulators and the CMV promoter are described in WO 96/01313, US Pat. Nos. 5,168,062 and 5,385,839, the entire contents of which are incorporated herein by reference. The nucleic acid construct of the present invention controls or blocks the expression of CaSR or its idle form, and in a preferred embodiment of the present invention, is restricted to cells capable of controlling the calcium level of a patient. Delivered.

A second embodiment of the method of the present invention relating to specifically inhibiting human CaSR activity or an isoform thereof at a selected site includes the step of selectively transducing C cells in transfected cells.
Including CaSR function by expression of a nucleic acid sequence encoding an intracellular binding protein capable of interacting with aSR or its isoform. WO
The entire contents of 94/29446 and WO 94/02610 are hereby incorporated by reference and disclose intracellular nucleic acid infection using genes encoding intracellular binding proteins. Intracellular binding protein means a protein capable of selectively interacting with or binding to CaSR or an isoform thereof in cells expressing the same, and further neutralizing the function of the bound CaSR. are doing. Preferably, the intracellular binding protein is an antibody or a fragment of an antibody. More preferably, the antibody or fragment thereof binds to the cytoplasmic region of CaSR. Most desirably, the intracellular binding protein is a single chain antibody capable of inhibiting intracellular calcium sensing.

[0097] WO 94/02610 discloses the production of antibodies and the isolation of nucleic acids encoding particular antibodies. Using CaSR or its isoforms, by techniques known to those skilled in the art,
Monoclonal antibodies specific to the cytoplasmic region are produced. Next, for use in the method of the present invention, a nucleic acid encoding an intracellular binding protein or a vector containing the protein and capable of being expressed in a host cell is prepared. Vectors suitable for delivering nucleic acids encoding intracellular binding proteins to cells containing CaSR include:
This corresponds to the above-discussed studies on the delivery of antisense nucleic acids. In a preferred embodiment of the second embodiment, the nucleic acid sequence encoding the CaSR intracellular binding protein further comprises a sequence encoding a localization signal that directs the intracellular binding protein to the intracellular location of CaSR, and / or The cell membrane is provided with a sequence that allows insertion of an intracellular binding protein. The localization signal or insertion sequence is
It is present everywhere on the intracellular binding protein as long as it does not interfere with binding to CaSR or its isoform. Examples of localization signals are disclosed in WO 94/02610. Preferably, the localization signal directs the intracellular binding protein to the cell membrane. The following examples can serve as a reference for understanding the present invention, but these are given as examples of the present invention and do not limit the present invention.

[0098]

【Example】

Materials and Methods General Materials and Methods: Bacterial strain: E. coli TG1, strain genotype supE, hsdD5, thi, D (lac-proAB), F '[tr
a D36 may be used as a means for amplification and isolation of plasmid using ^{pro A + B + lacI q lacZDM15} ]. The bacteria may be cultured in the following medium: LB medium:-NaCl (5 g / l) (Difco)-Bacto-tryptone (10 g / l) (Difco)-Yeast extract (5 g / l) (Difco) The medium is solidified by adding 20 g / l agar (Difco). Ampicillin (100 μg / ml) allows for the selection of bacteria that have received a plasmid carrying the gene conferring resistance to this antibiotic as a marker.

The plasmid Bluescript series vector (Stratagene) may be used. These vectors allow for cloning exactly as performed in the pMTL series (Chambers et al., Gene 1988, 68, pp139-149). Furthermore, the vector pCDNA3 (Invitrogen) and the derivative vectors pSG42 and pCNW8) allow the expression of the protein in animal cells under the control of the CMV promoter)
Also available. In addition, the vector pCRII or pCR2.1 (Invit
rogen) is also available. The genetic engineering techniques used to clone cDNA and insert the cDNA into these plasmids are conventional protocols (Maniatis T. et al., "Molecular Cloning,
a Laboratory Manual ", Cold Spring Harbor Laboratory, Cold Spring Harbor
, NY, 1982; Ausubel FM et al. (Ed.), "Current Protocols in Molecular Bi
ology ", John Wiley & Sons, New York, 1987).

Preparation of Plasmids Large quantities of DNA may be prepared using Promega's rapid DNA preparation kit according to the manufacturer's instructions. Small amounts of DNA may be prepared according to the following method: The bacteria containing the plasmid are cultured in 2 ml of LB medium on a shaker for at least 4 hours with shaking. These were centrifuged in an Eppendorf tube at 14,000 rpm for 2 minutes, and the concentrate was treated with solution I (50 mM glucose, 25 mM Tris-HCl buffer pH 8, 10 mM EDTA
Resuspend in pH 8) and dissolve in 200 μl of solution II (0.2 M NaOH, 1% SDS). Next, 150 μl of this solution was added to solution III (3M potassium acetate, 11.5% (v / v) glacial acetic acid).
Neutralize with. After stirring the tube until a precipitate on cotton is obtained, 150 μl of a phenol / chloroform mixture (water saturated 50% phenol and 50% chloroform) are added and the whole mixture is stirred for 30 seconds. After centrifugation at 14,000 rpm for 2 minutes, D
The aqueous phase containing NA is recovered. The DNA is then precipitated by adding 0.5 volumes of isopropanol, then centrifuged at 14,000 rpm for 5 minutes, air-dried and finally 20 μl TE
-Dissolve in RNase (10 mM Tris-HCl containing 50 μg / ml RNase and 1 mM EDTA).

Enzymatic Amplification of DNA by Polymerase Chain Reaction (PCR) The PCR reaction is double-stranded DNA, dNTP (0.2 mM), PCR buffer (10 mM Tris-HCl pH 8.5).
, 1 mM MgCl ₂ , 5 mM KCl, gelatin 0.01%)), 0.5 μg of each oligonucleotide, and 2.5 IU of Ampli Taq DNA polymerase (Perkin Elmer) with or without formamide (5%) And in a final volume of 100 μl. The mixture is covered with two drops of paraffin oil to reduce evaporation of the sample. The equipment used may be a Crocodile II from Appligene. Unless otherwise specified, denaturation is performed at 90 ° C. for denaturation of the helix and the temperature for hybridization of the oligonucleotide to this denatured (single-stranded) DNA is 5-10 higher than the temperature for separation of the oligonucleotide. ° C lower and at a temperature of 72 ° C for enzymatic extension. The fragment obtained by PCR is used for cloning and, once cloned, the fragment is symmetrically re-sequenced to ensure that there are no mutations that may have occurred during amplification. Oligonucleotides may be synthesized chemically using a β-cyanoethyl protecting group according to the phosphoramidite method. After synthesis, the protecting groups are removed by treatment with ammonia, and the oligonucleotide can be purified and concentrated by two precipitations with butanol. DNA concentration may be determined by measuring the optical density at 260 nm.

Ligation Ligation was performed with 100-200 ng of vector, 0.5-2 μg of insert, 40 IU of T4
DNA ligase enzyme (Biolabs) and ligation buffer (50 mM Tris-HC)
l pH 7.8; 10 mM MgCl ₂ ; 10 mM DTT; 1 mM ATP) at + 14 ° C overnight. A negative control was performed by ligation of the vector without insert. If necessary, prior to ligation, filling of the overhanging 5 'end was performed with the Klenow fragment of E. coli DNA polymerase I (Biolabs) according to the supplier's instructions. 3 '
Protruding end disruptions were performed in the presence of T4 phage DNA polymerase (Biolabs) according to the manufacturer's recommendations.

Bacterial Transformation The entire ligation volume (10 μl) was transferred to Chung et al. (1988, Proc. Natl. Acad.
Sci. 86: 2172-2175) may be used for the transformation of bacteria made competent. The bacteria are cultured in LB liquid medium with shaking in an incubator at 37 ° C. for several hours until an OD 0.6 at 600 nm is obtained. The medium is then centrifuged at 6,000 rpm for 10 minutes. 1/10 of the medium volume at the start of the bacterial concentrate
Volume of TSB (LB medium + 100 g / l PEG 4000, 5% DMSO, 10 mM MgCl ₂ , 10
Dissolve in mM MgSO ₄ ). After incubation at 4 ° C. for 30-60 minutes, 200 μl of the bacteria are contacted with the ligation product on ice for 15 minutes. After adding 200 μl of LB [medium], the bacteria were incubated at 37 ° C. for 30 minutes, then LB
+ Spread on ampicillin medium.

DNA Separation and Extraction DNA separation was performed by electrophoresis as a function of size. To do this, different gels are used, depending on the size of the fragments to be separated: TBE buffer (90 mM T.sub.M) for separating large DNA fragments (greater than 500 bp).
ris base; 90 mM boric acid; 2 mM EDTA) 1% agarose gel (Gibco BRL; NuSie in TBE buffer to separate small DNA fragments (less than 500 bp)
ve agarose gel (FMC Bioproducts). Electrophoresis on an agarose gel or polyacrylamide gel is performed in a TBE buffer and performed in the presence of a molecular weight marker (1 Kb ladder, Gibco BRL). The DNA was mixed with 1/10 of the charge amount of blue (200 g / l Ficoll, 0.5 g / l bromophenol blue, 50 mM EDTA) and gelled. After electrophoresis at 100 V and staining with ethidium bromide (0.5 mg / ml gel), the band is observed under a UV lamp.

Extraction of DNA from agarose gel bands is performed by electroelution as follows: A piece of gel containing DNA fragments is cut out with a scalpel, put into a dialysis tube containing 100-500 μl of TBE, and clamped with two clamps. close. The whole mixture is placed in an electrophoresis tank and an electric field of 100 V is applied. After the DNA is removed from the gel, it is purified by two phenol / chloroform extractions followed by two chloroform extractions, followed by precipitation in the presence of 0.3 M sodium acetate and 2.5 volumes of absolute alcohol. After centrifugation (14,000rpm
5 minutes), dry the DNA concentrate and dissolve in 20 μl of water.

Fluorescent Sequencing of Plasmid DNA Sequencing was performed according to the method of Sanger, using four different markers with different fluorescent markers.
This may be done using deoxyribonucleotides. These dideoxyribonuclei
Incorporation of one of the reotides by Taq polymerase in the DNA to be sequenced
Causes replication to stop. This reaction produces DNA of various sizes, all of which are
The 3 'end is terminated by one of the four dideoxyribonucleotides
. 1 μg of plasmid and 4 pmoles of pramer were obtained from Applied Biosystems PRISM ^C Add to 9.5 μl of “Premix” supplied under the trademark. Final volume is 2
Perform 25 cycles of PCR using 0 μl (denaturing phase at 96 ° C for 30 seconds,
(Implification phase and extension phase at 60 ° C for 4 minutes). After amplification
The resulting fragment is purified on an exclusion column (Clontech's Chromaspin-30), followed by Spe
Dry in ed Vac. All of the dried material was reconstituted with 24 μl EDTA (50 mM) and 120 μl
Dissolve in 5 μl of a mixture made from ion formamide. Change at 96 ° C for 3 minutes
After the development, 3-5 μl is loaded on an electrophoresis gel. Various DNA fragments depend on their size
Laser reading on ABI 370 DNA sequencer (Applied Biosystems)
One after the other, where different fluorophores are detected.

Example 1 PCR Amplification of a CaSR Splicing Variant First strand cDNA isolated from total RNA from normal adult human tissue was purchased from Invitrogen. Subsequently, the RNA was treated with DNase (RNase-free) to remove genomic DNA contamination. Priming 10 microgram (μg) RNA with oligo dT primer, MMLV
Reverse transcription was performed with reverse transcriptase. The reaction was stopped by incubation at 65 ° C for 10 minutes. This cDNA was mixed with 40 μl of RT buffer (1 × RT buffer: 50 mM Tri
s-HCl pH 8.3, 75 mM KCl, 3 mM MgCl ₂ , 10 mM DTT).

The following oligonucleotide primers were used to identify CaSR isoforms. Primer sequence Wild-type CasR position 1-AS 5'-CATGGGTCAATTCACTGTCAT-3 '3383-3403 (SEQ ID NO: 1) 3-AS 5'-GCCAGATCACACAGATGACAA-3' 2207-2227 (SEQ ID NO: 2) 4-AS 5'-GGCATAGACGTTGTAATACCC- 3 '1525-1545 (SEQ ID NO: 3) 5-AS 5'-TGTGGACAGACTTCCTGGGAT-3' 1013-1033 (SEQ ID NO: 4) 5-S 5'-ATCCCAGGAAGTCTGTCCACA-3 '1013-1033 (SEQ ID NO: 5) 7-S 5 '-ACTCCTAGCTGTCTCATCCCT-3' -44- -24 (SEQ ID NO: 6)

The splicing mutant CaSRc was amplified on AmpliTaq Gold from Perkin Elmer. The final reaction mixture is 100 μl reaction, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl ₂ , 0
.001 (w / v) gelatin, 0.8 mM dATP, 0.8 mM dCTP, 0.8 mM dGTP, 0.8 mM dTTP, 2.5
Unit AmpliTaq Gold, 0.4 uM Primer 5-S, and 2 μl of Invitrogen human kidney
Consists of cDNA. Reaction conditions are as follows: maintained at 95 ° C for 9 minutes; 94 ° C-30
40 cycles of 60 seconds at 60 ° C for 1 minute; maintained at 60 ° C for 10 minutes. CaSRb was amplified using the same conditions as CaSRc, except that 0.4 uM primer 5-AS and 0.4 uM primer 7-S were used instead of primers 3-AS and 5-S.

The splicing mutant CaSRd was amplified using the same components except for the following:
During the reaction of l, add 0.4 uM primer 1-AS and 0.4 uM primer 7-S to primer 3-AS
And 5-S were used, and 3 μl of Invitrogen human kidney cDNA was used instead of 2 μl. Reaction conditions were as follows: maintained at 95 ° C for 9 minutes;
20 cycles of 3 minutes; 94 ° C for 30 seconds, 64 ° C for 3 minutes (10 seconds / cycle increase)
For 20 cycles; maintained at 60 ° C. for 10 minutes. Next, 50 μl of the reaction mixture was electrophoresed on a 1% agarose gel, a band of 3.0 to 3.4 kb region was cut out, extracted using a gel extraction kit of QIAGEN, and eluted in 50 μl of ddH ₂ O. 1 μl of this extract
Used as template for the next reaction containing the same components as the previous reaction except for the following: 0.4 uM Primer 4-AS was used instead of Primer 1-AS and the template was replaced. The first and second reaction conditions were identical. All PCR products were cloned into PCR 2.1 according to Invitrogen protocol. Sequencing was performed with an automatic DNA sequencer.

Example 2: Expression of CaSRb and CaSRc in human kidney A strategy for searching for CaSR splice variants involves scanning different pairs of sensor cDNAs using primer pairs. Using human kidney first strand cDNA as a template,
Amplification was performed with either primer pair 3AS / 5S or 5AS / 7S. Primer pair 3AS /
Electrophoretic resolution of the PCR mixture obtained by 5S reveals that there is a product of the expected size (1.2 kb) of wild-type CaSR and a lower molecular weight product of about 1.0 kb. Became. Primer pair 5AS / 7S also produced two macroscopic PCR products of approximate size of 1.0 kb and 0.7 kb. The 1.0 kb product corresponds to the expected size of wild-type CaSR. The PCR products from both primer pairs bound to pCR2.1 and multiple clones were selected for analysis by restriction digestion with EcoR1 to release the insert. Clones with putative wild-type CaSR inserts and smaller sized clones were sequenced. These experimental results confirmed that the putative wild-type clone had a valid CaSR sequence. On the other hand, clones with shorter inserts were found to contain a deletion from nucleotides 186-495 or a deletion from nucleotides 1378-1608. The 186-495 deletion CaSR corresponds to CaSRb originally described in medullary thyroid carcinoma. 1378-1608 deleted CaSR is Ca
It is called SRc (Fig. 1). Unlike CaSRb, a deletion in CaSRc does not shift the reading frame.

Example 3 Expression of CaSRd in Human Kidney Following initial PCR enrichment of human kidney first strand cDNA with primer pair 1AS / 7S, 3
Products ranging from 0.0 to 3.4 kb were gel purified and further amplified with primer pair 4AS / 7S. The PCR product was isolated by TA cloning and sequenced. One of these clones was found to contain a deletion from nucleotides 1075-1386 (FIG. 1). The 1075-1386 deleted CaSR is called CaSRd. No alteration in reading frame was detected in this alternatively spliced CaSR transcript.

Example 4: Stable expression of isoform CaSRd in HEK-293 cells Full-length CaSRd was cloned into the mammalian expression vector pCEP4 (Invitrogen). A Qiagen plasmid production kit was used to produce CaSRd DNA for transfection. Lipofect AMINE (Life Technologies, Inc.) was used as a carrier for transfection. HEK-293 cells were transfected with CaSRd DNA by the general method described in the Lipofect AMINE transfection kit. CaSRd DNA and Lipofectamine complex (1 ml) were spread on HEK-293 cells (90% confluent) in 6-well plates. After 5 hours at 37 ° C., 1 ml of DMEM containing 20% fetal calf serum, penicillin and streptomycin was added to each well. After incubation at 37 ° C. for 16 hours, the culture was replaced with 2 ml of DMEM containing 10% fetal bovine serum, penicillin and streptomycin, and the culture was incubated at 37 ° C. for a further 8 hours. The CaSRd transfectants were isolated by dilution in the limit followed by selection in the presence of hygromycin. Trypsinize cells in each well, 10%
The culture was diluted with 100 ml of DMEM containing fetal calf serum, 200 μg / ml hygromycin, penicillin and streptomycin. A 1 ml aliquot of the diluted culture was added to each well of several 24-well tissue culture plates. After 4 weeks of culture, wells containing a single colony were identified and each cell clone was expanded in a T75 flask. The expression of CaSRd in each cell clone was monitored by Northern analysis. The clone with the highest expression level, 21/2, was used for the following functional analysis.

The function of isoform CaSRd was assayed by increasing extracellular calcium levels and the ability to increase intracellular levels in response to other agonists. The wild-type receptor is
The intracellular calcium concentration was increased when the extracellular calcium concentration increased. Intracellular calcium was measured using a fluorescent indicator, fura-2 (manufactured by Molecular Probes). HEK-293 cells transfected with CaSRd were treated with 0.5 μM fula-2, 20 mM HEPES, pH 7.35,
_{0.1% BSA, 0.5mM CaCl 2,} 0.5mM MgCl 2, 6.7mM KCl, 3mM glucose and 142 mM NaC
1 for 45 minutes at 37 ° C. The cells were washed and resuspended in a supplement containing no fura-2 to 2 × 10 ⁶ cells / ml. For intracellular calcium measurements, cells were placed in quartz cuvettes equilibrated at 37 ° C. Emission light collected at 505 nm caused the excitation monochromator to center at 340 and 380 nm. Different Ca
SR agonists were added to various final concentrations to activate CaSR. Usually, a final concentration of 10 mM external CaCl ₂ is sufficient to maximize activation of the wild-type receptor. The results show that CaSRd does not respond to agosts such as Ca ⁺⁺ , Mg ⁺⁺ and neomycin, but does respond to gadolinium (Table I). In the presence of a CaSR enhancing compound, NPS568 (WO94 / 18959, Fox et al., (1993) J. Bone Min Res. 8: S181; Ab
(stract # 260) CaSRd responded to calcium, magnesium and neomycin.

Table I Effect of extracellular Ca ⁺⁺ , NPS568 and gadolinium on intracellular calcium concentration in CaSRd expressing HEK-293 cells Increased fula-2 fluorescence (cps × 10 ⁶ ) Expt 1 Expt 2 20 mM Ca ²⁺ 0.2 0 20 mM Ca ²⁺ + 1.5 1.7 2 μM NPS568 10 μM NPS568 0.2 0.1 100 μM Gadolinium 2.0

[0116] The present invention is not limited to 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 accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is further to be understood that all base or amino acid sizes and all molecular weight or molecular mass values for nucleic acids or polypeptides are approximate and are provided for the foregoing. Various publications are cited herein, the contents of which are included in the disclosure herein.

[Sequence list]

[Brief description of the drawings]

FIG. 1. Wild-type calcium sensory receptor (CaSRa), splice variant CaSRd (lacking the amino acid encoded at nucleotides 1075-1386), and splice variant C
FIG. 2 is a structural diagram of aSRc (lacking the amino acid encoded by nucleotides 1378-1608).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 45/00 A61P 3/14 4C084 48/00 5/18 4C086 A61P 3/14 9/12 4C087 5/18 19/10 4H045 9/12 35/00 19/10 43/00 111 35/00 C07K 14/705 43/00 111 16/28 C07K 14/705 C12N 1/19 16/28 1/21 C12N 1/19 C12P 21/08 1/21 C12Q 1/02 5/10 G01N 33/15 Z C12P 21/08 33/50 Z C12Q 1/02 C12R 1:91) G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 B // (C12N 5/10 A61K 37/02 C12R 1:91) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT , LU, MC, NL, PT, SE) OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU ZA, ZW (72) Inventor Labodiniere Richard F. United States 19426 Pennsylvania College Way Richard Way 220 (72) Inventor Thrower Rally AW United States Pennsylvania 19468 Loyersford Brook Drive 1204 F-term (reference) 2G045 AA40 DA36 DB07 4B024 AA01 AA11 BA63 CA04 CA20 DA02 DA05 DA12 EA02 EA04 GA13 HA17 4B063 QA01 QA05 QQ21 QQ41 QQ61 QQ89 QR66 QR77 QR80 QX02 4B064 AG01 AG20 AG27 AG31 CA02 CA06 CA10 CA19 CA20 CC01 ACOA ADA DA01 BA25 BB01 BC03 BD50 CA24 CA44 CA46 4C084 AA02 AA06 AA07 AA13 AA17 BA35 BA44 CA18 CA53 CA59 DB32 NA14 ZA422 ZA972 ZB262 ZC062 ZC422 4C086 AA01 AA03 AA04 EA16 MA01 MA04 NA14 ZA42 ZA97 ZB26 ZA97 ZB26 ZA97 ZB26 ZA97 ZB26 AB04 ZC42 4H045 AA10 AA11 AA30 BA10 CA40 DA50 DA75 EA20 EA50 FA71 FA74

Claims (50)

[Claims] 1. An isolated nucleic acid encoding an isoform of the human calcium sensory receptor, the nucleic acid comprising from about 2922 to about 3003 nucleotides,
And an isolated nucleic acid lacking about 231 nucleotides when compared to the wild-type receptor set forth in SEQ ID NO: 11. 2. The nucleic acid of claim 1, wherein said deletion is from a region encoding the extracellular domain of said receptor. 3. The deletion comprises about nucleotides 1075-1386 of SEQ ID NO: 11.
The nucleic acid according to claim 2, which is from 4. The method according to claim 1, wherein said deletion is at about nucleotide 1378-1608 of SEQ ID NO: 11.
The nucleic acid according to claim 2, which is from 5. The method according to claim 1, wherein the deletion comprises about nucleotides 1075-1608 of SEQ ID NO: 11.
The nucleic acid according to claim 2, which is from 6. The device according to claim 1, wherein the device has at least one characteristic selected from the following.
3. The nucleic acid according to 1. a) it can be amplified by the polymerase chain reaction (PCR) using oligonucleotide primers derived from SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11; b) Hybridizes with a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9; and c) is derived from SEQ ID NO: 8, SEQ ID NO: 10, and allelic variants thereof. Encodes a polypeptide having an amino acid sequence selected from the group consisting of: 7. The isolated nucleic acid of claim 2, wherein said isoform protein comprises the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10, or an allelic variant thereof. 8. The nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 9,
Or the isolated nucleic acid of claim 2, comprising an allelic variant thereof. 9. The method according to claim 1, wherein the nucleic acid can be amplified by a polymerase chain reaction (PCR) using an oligonucleotide primer selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 6. Isolated nucleic acids. 10. A vector comprising the nucleic acid according to claim 1. 11. The vector according to claim 10, wherein said nucleic acid is operably linked to an expression control sequence enabling expression of said receptor in an expression competent host. 12. The vector according to claim 11, wherein the vector is selected from the group consisting of an RNA molecule, a plasmid DNA molecule, and a viral vector. 13. The vector according to claim 12, which is a plasmid DNA molecule. 14. The vector according to claim 12, which is a viral vector selected from the group consisting of retrovirus, adenovirus, adenoassociated virus, herpes virus, and vaccinia virus. 15. A host cell transfected with the vector according to claim 10. 16. A host cell transfected with the vector according to claim 13. 17. The host cell according to claim 15, wherein the host cell is selected from the group consisting of a bacterial cell, a yeast cell, and a mammalian cell. 18. A method for expressing a human calcium sensory receptor, comprising: a) culturing the host cell of claim 17 in a culture medium under conditions that allow expression of the receptor; and b) identifying cells expressing the receptor on the surface of those cells. . 19. An isolated isoform of the human calcium sensory receptor, said isoform comprising about 974 to about 1001 amino acids and comprising SEQ ID NO:
An isolated isoform protein lacking about 77 amino acids when compared to the wild-type receptor shown in D NO: 12. 20. The isoform of claim 19, wherein said deletion is from an extracellular region of said receptor. 21. The isoform of claim 20, wherein said deletion is from about amino acids 358-462 of SEQ ID NO: 12. 22. The isoform of claim 20, wherein said deletion is from about amino acids 460-536 of SEQ ID NO: 12. 23. The isoform of claim 20, wherein said deletion is from about amino acids 358-536 of SEQ ID NO: 12. 24. The method according to claim 24, wherein the isoform is SEQ ID NO: 8 or SEQ ID NO: 10.
20. The isoform protein according to claim 19, which comprises the amino acid sequence shown in (1) or an allelic variant thereof. 25. A method for screening for an agonist or antagonist of CaSR isoform protein activity, comprising the steps of: incubating a test sample with CaSR isoform protein; measuring CaSR isoform protein activity; Comparing the activity with no activity. 26. The method of claim 25, wherein said CaSR activity is its ability to affect intracellular calcium concentration. 27. The method of claim 25, wherein the test sample is tested alone, with increased extracellular calcium concentration, or in the presence of an agonist or antagonist of CaSR isoform protein activity. 28. The method of claim 26, wherein said intracellular calcium concentration is measured with a fluorescent indicator. 29. The method according to claim 28, wherein the indicator is fura-2. 30. A method of treating a patient suffering from a disease or disorder associated with abnormal calcium levels, comprising administering a therapeutically effective amount of a compound capable of modulating the activity of a CaSR isoform protein. 31. The method according to claim 30, wherein the disease is hyperparathyroidism or osteoporosis. 32. The disease is Paget's disease, hypercalcemic malignancy,
31. The method of claim 30, wherein the disorder is hypertension. 33. The method of claim 30, wherein said compound is an agonist or antagonist of a CaSR isoform protein. 34. A method of increasing or decreasing the level of CaSR activity in a cell, comprising administering to said cell a nucleic acid capable of increasing or decreasing CaSR activity. 35. The method of claim 34, wherein said cells are in a patient suffering from a disease or disorder associated with abnormal calcium levels. 36. The method of claim 35, wherein said disease is hyperparathyroidism or osteoporosis. 37. The disease is Paget's disease, hypercalcemic malignancy,
36. The method of claim 35, wherein the disease is hypertension. 38. The method of claim 35, wherein said nucleic acid is in a vector. 39. The nucleic acid encodes a human calcium sensory receptor isoform protein, the nucleic acid comprises from about 2922 to about 3003 nucleotides, and
39. The method of claim 38, wherein about 231 nucleotides have been deleted when compared to the wild type of the receptor set forth in SEQ ID NO: 11. 40. The method according to claim 38, wherein said vector is selected from the group consisting of a plasmid, a retrovirus, a herpes simplex virus, an adenoassociated virus, an adenovirus, and a vaccinia virus. 41. The method according to claim 27, wherein the nucleic acid encodes an intracellular binding protein capable of binding to CaSR or an isoform thereof. 42. The method according to claim 41, wherein the intracellular binding protein is an antibody or a fragment thereof. 43. The method of claim 42, wherein said antibody or fragment thereof binds to the cytoplasmic domain of CaSR. 44. The method of claim 43, wherein said antibody or fragment thereof is a single chain antibody. 45. The method of claim 41, wherein said nucleic acid further comprises a sequence encoding a localization signal for targeting said intracellular binding protein to a CaSR cellular location. 46. The localization signal is specific for a plasma membrane.
The method described in. 47. The method of claim 34, wherein said nucleic acid encodes an antisense molecule that is complementary to a sequence encoding CaSR and is capable of selectively inhibiting expression of said sequence. 48. The method of claim 47, wherein said antisense molecule is at least about 20 nucleotides in length. 49. An antigenic polypeptide comprising an epitope and / or sequence that is not present in wild-type CaSR, wherein said polypeptide elicits an antibody that binds to a CaSR isoform protein. 50. A protein specifically recognizing the CaSR isoform protein,
Antibodies that do not bind SR.