AU767010B2 - Isoforms of human calcium sensing receptor - Google Patents

Description

WO 00/06601 PCT/US99/17116 Isoforms of Human Calcium Sensing Receptor 4 FIELD OF THE INVENTION The present invention relates to isoforms of a human calcium sensing receptor, and to the genes encoding these isoforms. The invention further relates to methods of screening for agonists or antagonists of the isoforms, particularly with respect to calcium receptor activity, to diagnostic uses of these isoforms and to therapeutic uses of the agonists or antagonists. The invention also relates to gene therapy using the genes encoding the receptor isoforms or molecules capable of down-regulating receptor activity, such as antisense sequences.

BACKGROUND OF THE INVENTION Calcium is an extracellular messenger (Brown et al. (1995) Cell 83:679-682). Serum calcium levels are regulated by 1,25-dihydroxyvitamin D 3 parathyroid hormone, and calcitonin. A calcium sensing receptor (CaSR) has been identified in bovine parathyroid (WO 94/18959; Brown et al. (1993) Nature 366: 575-580). Cloning of the cDNA encoding this receptor (CaSRa) revealed a G-protein-coupled receptor featuring a large extracellular domain, coupled to a seven membrane spanning domain similar to those found in members of the G protein coupled receptor superfamily. This receptor has been shown to play a key role in Ca" homeostatsis through regulation of parathyroid hormone secretion and renal tubular calcium reabsorption. This receptor recognizes calcium and other polyvalent cations and is coupled by changes in phosphoinositide turnover to the release of calcium from intracellular stores. In addition to its abundant expression in parathyroid gland and kidney, full length CaSRa transcripts have also been found in brain, thyroid, intestine, bone marrow and keratinocytes. The complete cDNA sequence encoding the corresponding human form of CaSRa has recently been reported (Freichel et al. (1996) Endocrinology 137:3842-3848). This 3234 base pair nucleotide sequence (SEQ ID NO: 11) encodes a protein having 1078 amino acids (SEQ ID NO: 12). Various forms of the CaSR, particularly from bone marrow cells, are also disclosed by House et al. ((1997) J. Bone Min. Res. 12:1959-1970) and in US patents 5,688,938 and 5,763,569. The presence of calcium receptors in bone, suggests that they are involved in bone remodeling (Quarles (1997) J. Bone Min. Res. 12, 1971-1974).

An alternatively spliced form of CaSRa, has been identified in human medullary thyroid carcinoma and keratinocytes (see Freichel et al. supra). The medullary thyroid carcinoma isoform, designated CaSRb, contains a 307 base pair deletion between nucleotides 186 and 495, corresponding to exon 2. This deletion results in a reading frame shift and premature termination at nucleotide 766.

Translation of CaSRb transcript could yield an extracellular portion of the receptor without the 7 transmembrane anchor and cytosolic tail. Therefore, CaSRb may be a secretory protein, and, although its exact function has yet to be determined, by analogy to other known souble receptors it may play a role in modulating the interaction of native CaSR with its cationic ligands.

A second isoform of CaSRa has also been identified in kerotinocytes (Oda et al. (1997) FASEB J. 11(9): A925; Abstract #395). This form (CaSRc) lacks exon 4, encoding a portion of the extracellular domain of the receptor including a region of acidic amino acids which may mediate calcium binding, and is present in differentiated cells.

However, there is a need in the art to better understand calcium homeostasis. In particular, there is a need in the art to better understand calcium regulation through the CaSR. Isoforms of the wild-type CaSR could be expected to exhibit different pharmacological profiles and signaling properties relative to the wild-type. For example, different isoforms could couple differentially or uniquely to known or unknown signalling pathways, including phosphoinositide turnover, calcium mobilization, protein kinase C activation, cAMP production, and other ion channel activity. Alternatively, variant isoforms may exhibit different cell surface expression, metabolic half-life, or intracellular trafficking, when compared to the wild-type, or behave in a dominant negative or positive fashion, thereby over-riding the functionality of the wild-type receptor expressed in the same tissue.

The present invention addresses the need in the art, as discussed below. Specifically, Applicants present evidence that CaSRb is also found in human kidney. Significantly, Applicants have also identified in human kidney two other alternatively spliced CaSR transcripts (CaSRc and d) with deletions from nucleotides 1378-1608 and 1075 to 1386, respectively.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

SUMMARY OF THE INVENTION As noted above, the present invention concerns identification of isoforms of a human calcium sensing receptor (CaSR). The present invention reveals the presence of mutiple alternatively spliced transcripts of CaSR in human kidney. Two such isoforms identified, CaSRc and d arise from partial deletion of the wild type sequence. The nucleotide 3 deletions in CaSRc and d have no effect on the reading frame. Thus, CaSRc and d will yield receptor proteins with about 90-100 amino acid stretchs deleted from the extracellular domain. These deletions cover a respective extracellular sequence rich in acidic residues (approximately The location and the charge characteristics of the deleted sequences in CaSRc and d suggest that these two CaSR splice variants may exhibit different cation sensing property from the wild type receptor.

The invention therefore relates to an isolated nucleic acid encoding an isoform of a human calcium sensing receptor, wherein the isolated nucleic acid comprises a deletion of nucleotides when compared to the wild-type form of the receptor comprising a DNA sequence as depicted in SEQ ID NO:11, wherein the deletion comprises: a) nucleotides 1075-1386 of SEQ ID NO:11; b) nucleotides 1378-1608 of SEQ ID NO:11; and c) nucleotides 1075-1608 of SEQ ID NO:1 1.

In another embodiment, the isolated nucleic acid of the present invention has at least one property selected from: it can be amplified by polymerase chain reaction (PCR) using an oligonucleotide primer derived from SEQ ID NO:7, SEQ ID NO:9 or SEQ ID 20 NO:11; i it hybridizes under stringent conditions with a nucleic acid having a S' nucleotide sequence as depicted in SEQ ID NO:7 or SEQ ID NO:9; and it encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, and allelic variants thereof.

Preferably, the nucleic acid the invention can be amplified with at least one oligonucleotide primer selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6.

In still another preferred embodiment of the invention, the isolated nucleic acid encodes a CaSR isoform comprising an amino acid sequence as depicted in 30 SEQ ID NO:8 (CaSRc) or SEQ ID NO:10 (CaSRd), or allelic variants thereof.

Preferably, the isolated nucleic acid comprises a nucleotide sequence as depicted in SEQ ID NO:7 or SEQ ID NO:9, or allelic variants thereof.

As can be readily appreciated by one of ordinary skill in the art, one effective way to prepare a nucleic acid of the invention, particularly a cDNA, is to amplify the nucleic acid from a cDNA library comprising a coding sequence for a CasR isofom using PCR.

Various PCR primers, corresponding to any desired segment from SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 can be used in accordance with the invention. In specific embodiments, infra, PCR primers having the sequences depicted in SEQ ID NOS: 1-6 were used to amplify and isolate nucleic acids of the invention. Alternatively, a nucleic acid of the invention can be isolated or identified with an oligonucleotide probe, of at least 10 bases, which hybridizes under stringent conditions to a nucleotide having the sequence or the complementary sequence depicted in SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11. In a specific aspect, the oligonucleotide can be used in a method for detecting genomic DNA (Southern analysis) or expression of mRNA (Northern analysis) encoding a CaSR isoform in a cell. In either case, the method comprises contacting a sample from the cell with the oligonucleotide which is detectable, by labeling with a radioisotope or a chromophore or fluorophore, and detecting hybridization of the oligonucleotide with genomic DNA or mRNA in the sample, wherein detection of hybridization of the oligonucleotide with genomic DNA indicates the presence of a gene encoding a CaSR isoform in the genome, and detection of hybridization with mRNA indicates expression of mRNA encoding a CaSR isoform. It is also possible to e WO 00/06601 PCT/US99/17116 with mRNA indicates expression of mRNA encoding a CaSR isoform. It is also possible to use quantitative methods, to detect the number of CaSR genes in the genome, or to detect an increase or decrease in the level of expression of mRNA.

An oligonucleotide of the invention can also be an antisense oligonucleotide, one that binds to mRNA encoding a CaSR isoform and prevents its translation in the cell. Such an antisense molecule can be encoded by a vector expressed in the cell, or can be a synthetic oligonucleotide, preferably one that includes non-phosphoester bonds so that it is resistant to intracellular nucleases.

In another aspect, the invention provides a vector comprising nucleic acids encoding an isoform of a human calcium sensing receptor. Preferably, the nucleic acid is operatively associated with an expression control sequence permitting expression of the receptor in an expression competent host cell.

The vector may be an RNA molecule, a plasmid DNA molecule, or a viral vector. The viral vector may be a retrovirus, adenovirus, adeno-associated virus, herpes virus, or vaccinia virus.

In still another aspect, the invention is directed to host cells transfected with a vector comprising nucleic acids encoding an isoform of a human calcium sensing receptor. The host cell may be a bacterial cell, a yeast cell, or a mammalian cell. The host cells of the invention can be used to produce a CaSR isoform recombinantly. This method comprises culturing the host cell in culture medium under conditions permitting expression of the isoform. Therefore, another aspect of this invention is a method for expressing an isoform of human calcium sensing receptor comprising: culturing the host cell in culture medium under conditions permitting expression of the recombinant receptor; and identifying cells expressing the receptor on their surface.

The invention also provides an isolated isoform of a human calcium sensing receptor, wherein the isoform comprises about 974 to about 1001 amino acids and has a deletion of at least about 77 amino acids when compared to the wild-type form of the receptor as depicted in SEQ ID NO:12. The deletion may be from the extracellular domain of the receptor, such as from about amino acids 358- 462, about amino acids 460-536 or about amino acids 358-536 of SEQ ID NO:12. In particular, a CaSR isoform of the invention comprises an amino acid sequence as depicted in SEQ ID NO:8 or SEQ ID NO: 10, or allelic variants thereof.

The present invention advantageously provides methods of screening for molecules that modulate the activity of CaSR isoforms, and thus calcium levels. In particular, the invention provides a method of screening for agonists or antagonists of a CaSR isoform activity, the method comprising incubating a test sample with a CaSR isoform, measuring CaSR isoform activity and comparing the activity to that in the absence of the test sample. Any of the screening methods in the art can be used, particularly high throughput screening. In a specific embodiment, the method comprises screening compounds for their WO 00/06601 PCT/US99/17116 ability to influence intracellular calcium concentration. The compounds may be tested alone, in conjunction with an elevation in extracellular calcium concentration, or in the presence of other agonists or antagonists of CaSR isoform activity. Intracellular calcium can be measured with a fluorescent indicator, such as fura-2. Screening methods of the invention permit the identification of CaSR agonists (calcimimetics) or antagonists (calcilytics).

In yet a further embodiment, the present invention provides pharmaceutical compositions and methods for the treatment of a patient suffering from a disease or disorder associated with abnormal calcium levels, such as in the plasma, by the administration of a therapeutically effective amount of a compound capable of modulating the activity of a CaSR isoform. The compound may be specific for a CaSR isoform. Such diseases include, for example, hyperparathyroidism (primary and secondary) and osteoporosis. Other diseases include Paget's disease, hypercalcemia malignancy, and hypertension. The compound may be a calcimimetic or calcilytic identified using the screening assays of the invention.

Alternatively, the disease or disorder is treated using gene therapy.

In one embodiment, the cells of a patient have been transfected with a vector encoding a CaSR isoform under conditions permitting expression of the isoform.

Alternatively, where desired, the invention provides a method of inhibiting CaSR activity in a patient's cell comprising decreasing the level of CaSR in the cell. The level of CaSR protein can be decreased by introducing a CaSR antisense nucleic acid into the cell, which antisense nucleic acid hybridizes under intracellular conditions to a CaSR mRNA. Alternatively, the level of CaSR protein can be decreased by introducing a single chain Fv antibody (scFv) that specifically binds a CaSR isoform, or nucleic acid encoding and intracellular antibody against the isoform, into the cell at a level sufficient to bind to and inactivate the CaSR.

Yet another object of the invention is to provide for high level expression of CaSR isoforms, either by fermentation of transfected or transduced cells to recover purified protein, or in vivo in cells for further testing in vitro or for regulation of calcium homeostasis in vivo, for gene therapy.

A particular object of the invention is to provide for screening of small molecule modulators, e.g., agonists and antagonists, of CaSR activity, particularly of specific CaSR isoforms.

These and other objects are addressed by this invention, which is explained in greater detail in the attached drawings and the following Detailed Description and Examples.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1. Diagram of the structures of the wild-type calcium sensing receptor (CaSRa), splice variant CaSRd (lacking amino acids encoded by nucleotides 1075-1386), and splice variant CaSRc (lacking amino acids encoded by nucleotides 1378-1608).

WO 00/06601 PCT/US99/17116 DETAILED DESCRIPTION OF THE INVENTION This invention is based, in part, on the identification of isoforms of a human calcium sensing* receptor, termed herein CaSR.

The invention accordingly relates to nucleic acids encoding CaSR isoforms, to the purified protein, to cells which express nucleic acids encoding isoforms of CaSR, in particular splicing variants, and to their use in screening for small molecules or natural products, which agonize or antagonize the activity of the CaSR.

The invention can also be used for the treatment of diseases or disorders associated with an abnormal level of calcium, including gene therapy applications (both coding and antisense molecules can be of use).

In addition, anti-CaSR antibodies can be used in diagnostic and purification applications.

These and other aspects of the invention, particularly isolation of CaSR genes, expression of CaSR protein, generation of anti-CaSR antibodies, screening assays for agonists or antagonists of CaSR activity and delivery of CaSR encoding vectors, in particular for gene therapy applications, are discussed in detail in the following sections. Section headers are provided merely for the reader's convenience, and are not to be deemed limiting in any respect.

Genes Encoding Calcium Sensor Receptor Isoforms The present invention contemplates isolation of genes encoding isoforms of a human calcium sensing receptor. As used herein, the term "gene" refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, Sambrook, Fritsch Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II Glover ed. 1985); Oligonucleotide Synthesis Gait ed. 1984); Nucleic Acid Hybridization Hames S.J. Higgins eds. (1985)]; Transcription And Translation Hames S.J. Higgins, eds. (1984)]; Animal Cell Culture Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. Current Protocols in Molecular Biology, John Wiley Sons, Inc. (1994).

Therefore, if appearing herein, the following terms shall have the definitions set out below.

A "cloning vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element plasmid, chromosome, virus) that functions as an autonomous unit of DNA WO 00/06601 PCT/US99/17116 replication in vivo, capable of replication under its own control. Cloning vectors may be capable of replication in one cell type, and expression in another ("shuttle vector"). A "cassette" refers to a segment of DNA that can be inserted into a vector at specific restriction sites. The segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.

A cell has been "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. A cell has been "transformed" by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change. The transforming DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

A "nucleic acid molecule" refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester anologs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 550, can be used, 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, 40% formamide, with 5x or 6x SCC. High stringency hybridization conditions correspond to the highest Tm, 50% formamide, 5x or 6x SCC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater WO 00/06601 PCT/US99/17116 the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). Preferably a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about nucleotides; and more preferably the length is at least about 20 nucleotides.

In a specific embodiment, the term "standard hybridization conditions" refers to a T, of55 0 C, and utilizes conditions as set forth above. In a preferred embodiment, the Tm is 60 0 C; in a more preferred embodiment, the Tm is 65 0

C.

As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 18 nucleotides, that is specifically hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding CaSR, or an isoform thereof. Oligonucleotides can be labeled, with 32

P-

nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid encoding a CaSR, or an isoform thereof. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning CaSR isoforms, or to detect the presence of nucleic acids encoding CaSR isoforms. In a further embodiment, an oligonucleotide of the invention can form a triple helix with a CaSR DNA molecule. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. In this case, the nucleic acid is "operatively associated" with an expression control sequence permitting expression of the protein in an expression competent host cell Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

WO 00/06601 PCT/US99/17116 A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream direction) coding sequence. For purposes of definirg the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.

As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamilies the immunoglobulin superfamily) and homologous proteins from different species myosin light chain, etc.) (Reeck et al., 1987, Cell 50:667). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity.

Accordingly, the term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and not a common evolutionary origin.

In a specific embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when at least about 50% (preferably at least about 75%, and most preferably at least about 90 or of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, Maniatis et al., supra; DNA Cloning, Vols. I II, supra; Nucleic Acid Hybridization, supra.

Similarly, in a particular embodiment, two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 30% of the amino acids are identical, or greater than about are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program.

The term "corresponding to" is used herein to refer similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.

WO 00/06601 PCT/US99/17116 A nucleic acid or amino acid sequence alignment may include spaces. Thus, the term "corresponding to" refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.

A gene encoding a CaSR isoform, whether genomic DNA or cDNA, can be isolated from a human cDNA or genomic library. Methods for obtaining genes encoding CaSR isoforms are well known in the art, as described above (see, Sambrook et al., 1989, supra).

Accordingly, any human cell potentially can serve as the nucleic acid source for the molecular cloning of a CaSR isoform gene. The DNA may be obtained by standard procedures known in the art from cloned DNA a DNA "library"), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein brain, thyroid and kidney cDNA), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al., 1989, supra; Glover, D.M. 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II). Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. In specific embodiments, isoforms CaSRc and CaSRd were isolated from a human kidney cell library. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.

Once the DNA fragments are generated, identification of the specific DNA fragment containing gene encoding a CaSR isoform may be accomplished in a number of ways. For example, DNA fragments may be screened by nucleic acid hybridization to a labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. As noted above, the greater the degree of homology, the more stringent hybridization conditions can be used. In a specific embodiment, Northern hybridization conditions are used to identify mRNA splicing variants of a CaSR gene.

Further selection can be carried out on the basis of the properties of the gene, if the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, or partial amino acid sequence of a CaSR isoform as disclosed herein. Thus, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, has similar or identical electrophoretic migration, isoelectric focusing or nonequilibrium pH gel electrophoresis behavior, proteolytic digestion maps, or antigenic properties as known for CaSR. In a specific embodiment, the isoform is recognized by a polyclonal antibody that does not recognize wild-type CaSR.

The present invention relates to genes cDNAs) encoding allelic variants, splicing variants, analogs, and derivatives of CaSR isoforms of the invention that have the same or homologous functional activity as the isoforms. The production and use of derivatives and analogs related to CaSR isoforms are WO 00/06601 PCT/US99/17116 within the scope of the present invention. In a specific embodiment, the derivative or analog is functionally active, capable of exhibiting one or more functional activities associated with an ibform of the invention. In particular, such an analog can bind calcium. Alternatively, an allelic variant can comprise a mutation that results an inability to bind calcium.

Derivatives can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Preferably, derivatives are made that have enhanced or increased functional activity relative to native CaSR isoforms.

Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a gene encoding a CaSR isoform, including an amino acid sequence that contains a single amino acid variant, may be used in the practice of the present invention.

These include but are not limited to allelic genes, homologous genes from other species, and nucleotide sequences comprising all or portions of CaSR isoform genes which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change. Likewise, the derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a CaSR isoform including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point.

Particularly preferred substitutions are: Lys for Arg and vice versa such that a positive charge may be maintained; Glu for Asp and vice versa such that a negative charge may be maintained; Ser for Thr such that a free -OH can be maintained; and Gin for Asn such that a free CONH 2 can be maintained.

Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site His can act as an acid or base WO 00/06601 PCT/US99/17116 and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces b-turns in the protein's structure. The genes encoding CaSR isoforms, derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding an CaSR isoform, derivative or analog, care should be taken to ensure that the modified gene remains within the same translational reading frame as the CaSR gene (SEQ ID NO: 11), uninterrupted by translational stop signals, in the gene region where the desired activity is encoded.

Additionally, the CaSR isoform-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Preferably, such mutations enhance the functional activity of the mutated gene product. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, et al., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc. Natl. Acad. Sci.

U.S.A. 83:710), use of TAB® linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applicationsfor DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used.

Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.

Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, E. coli, and WO 00/06601 PCT/US99/17116 facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, cart be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2m plasmid.

Expression of CaSR Isoforms The nucleotide sequence coding for CaSR isoforms, or derivatives or analogs thereof, including a chimeric protein, can be inserted into an appropriate expression vector, a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a "promoter." Thus, the nucleic acid encoding a CaSR isoform of the invention is operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.

An expression vector also preferably includes a replication origin.

The necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding a CaSR, a CaSR isoform and/or its flanking regions.

Potential host-vector systems include but are not limited to mammalian cell systems infected with virus vaccinia virus, adenovirus, etc.); insect cell systems infected with virus baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

A recombinant CaSR isoform of the invention, derivative, or analog thereof, may be expressed chromosomally, after integration of the coding sequence by recombination. In this regard, any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).

The cell into which the recombinant vector comprising the nucleic acid encoding a CaSR isoform is cultured in an appropriate cell culture medium under conditions that provide for expression of protein by the cell.

Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).

Expression of a gene may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be WO 00/06601 PCT/US99/17116 used to control gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the b-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and the animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Omitz et al., 1986, Cold Spring Harbor Symp.

Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.

5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Expression vectors containing a nucleic acid encoding a CaSR isoform of the invention can be identified by five general approaches: PCR amplification of the desired plasmid DNA or specific mRNA, nucleic acid hybridization, presence or absence of selection marker gene functions, (d) analyses with appropriate restriction endonucleases, and expression of inserted sequences. In the first approach, the nucleic acids can be amplified by PCR to provide for detection of the amplified product. In the second approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions b-galactosidase activity, WO 00/06601 PCT/US99/17116 thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. In another example, if the nticleic acid encoding a CaSR isoform is inserted within the "selection marker" gene sequence of the vector, recombinants containing the nucleic acid insert can be identified by the absence of the gene function. In the fourth approach, recombinant expression vectors are identified by digestion with appropriate restriction enzymes. In the fifth approach, recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant, provided that the expressed protein assumes a functionally active conformation.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of and known bacterial plasmids, E. coli plasmids col El, pCRI, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, the numerous derivatives of phage 1, NM989, and other phage DNA, M 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2m plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

For example, in a baculovirus expression systems, both non-fusion transfer vectors, such as but not limited to pVL941 (BamH1 cloning site; Summers), pVL1393 (BamH1, Smal, XbaI, EcoRI, Nol, XmaIII, BglII, and Pstl cloning site; Invitrogen), pVL1392 (BglII, Pstl, Not, XmaIII, EcoRI,XbaI, SmaI, and BamHI cloning site; Summers and Invitrogen), and pBlueBacII (BamHl, BglIl, Pstl, Ncol, and HindIll cloning site, with blue/white recombinant screening possible; Invitrogen), and fusion transfer vectors, such as but not limited to pAc700 (BamHl and KpnI cloning site, in which the BamHl recognition site begins with the initiation codon; Summers), pAc701 and pAc702 (same as pAc700, with different reading frames), pAc360 (BamHl cloning site 36 base pairs downstream of a polyhedrin initiation codon; Invitrogen(195)), and pBlueBacHisA, B, C (three different reading frames, with BamH 1, BglIl, PstI, Ncol, and HindlII cloning site, an N-terminal peptide for ProBond purification, and blue/white recombinant screening of plaques; Invitrogen (220)) can be used.

Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase (DHFR) promoter, any expression vector with a DHFR expression vector, or a DHFR/methotrexate co-amplification vector, such as pED (Pstl, Sall, SbaI, Smal, and EcoRl cloning site, with the vector expressing both the cloned gene and DHFR; see Kaufman, Current Protocols in Molecular Biology, 16.12 (1991). Alternatively, a glutamine synthetase/methionine sulfoximine co-amplification vector, such as pEE14 (HindII, Xbal, Smal, Sbal, WO 00/06601 PCT/US99/17116 EcoRI, and BclI cloning site, in which the vector expresses glutamine synthase and the cloned gene; Celltech). In another embodiment, a vector that directs episomal expression under control of Epsteth Barr Virus (EBV) can be used, such as pREP4 (BamH Sfil, Xhol, Notl, NheI, HindIll, Nhel, Pvull, and KpnI cloning site, constitutive Rous Sarcoma Virus Long Terminal Repeat (RSV-LTR) promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamH 1, Sfil, Xhol, Notl, Nhel, HindIIl, Nhel, Pvull, and KpnI cloning site, constitutive human cytomegalovirus (hCMV) immediate early gene, hygromycin selectable marker; Invitrogen), pMEP4 (Kpnl, PvuI, NheI, HindIII, Notl, Xhol, Sfil, BamH I cloning site, inducible methallothionein IIa gene promoter, hygromycin selectable marker: Invitrogen), pREP8 (BamH1, Xhol, Notl, HindIll, Nhel, and Kpnl cloning site, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, Xhol, Sfil, and BamHI cloning site, RSV-LTR promoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selectable marker, Nterminal peptide purifiable via ProBond resin and cleaved by enterokinase; Invitrogen). Selectable mammalian expression vectors for use in the invention include pRc/CMV (HindllI, BstXI, Notl, Sbal, and Apal cloning site, G418 selection; Invitrogen), pRc/RSV (Hindll, SpeI, BstXI, Notl, XbaI cloning site, G418 selection; Invitrogen), and others. Vaccinia virus mammalian expression vectors (see, Kaufman, 1991, supra) for use according to the invention include but are not limited to pSCI 1 (Smal cloning site, TK- and b-gal selection), pMJ601 (Sall, Smal, Afll, Narl, BspMIl, BamHI, Apal, NheI, Sacll, KpnI, and HindlI cloning site; TK- and b-gal selection), and pTKgptF S (EcoRl, Pstl, Sall, AccI, HindII, SbaI, BamHI, and Hpa cloning site, TK or XPRT selection).

Yeast expression systems can also be used according to the invention to express isoforms of a CaSR. For example, the non-fusion pYES2 vector (XbaI, Sphl, Shol, Notl, GstXI, EcoRI, BstXI, BamH1, Sad, Kpnl, and Hindll cloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, Notl, BstXI, EcoRI, BamHI, Sad, KpnI, and HindIII cloning site, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the invention.

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and WO 00/06601 PCT/US99/17116 modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. Expression in yeast can produce a biologically active product. Expression in eukaryotic cells can increase the likelihood of "native" folding.

Moreover, expression in mammalian cells can provide a tool for reconstituting, or constituting, CaSR activity. Furthermore, different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.

Vectors are introduced into the desired host cells by methods known in the art, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, Wu et al., 1992, J.

Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).

Soluble forms of the protein can be obtained by collecting culture fluid, or solubilizing inclusion bodies, by treatment with detergent, and if desired sonication or other mechanical processes, as described above. The solubilized or soluble protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gel electrophoresis, chromatography ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.

Antibodies to CaSR Isoforms The invention provides an antibody which specifically binds a CaSR isoform. Such antibodies can be used diagnostically, to detect the presence and optionally the quantity of the isoform in cells. Antibodies of the invention, particularly single chain Fv antibodies (scFv) can also be used therapeutically, to suppress CaSR activity (see below). In a specific embodiment, the antibody recognizes an epitope which is not present in the wild type receptor, CaSRa. In another specific embodiment, exemplified infra, the antibody is polyclonal. Monoclonal antibodies, and antibody fragments (in addition to scFv antibodies) are also contemplated by this invention. Using the antibody of the invention, one can specifically detect expression of a CaSR isoform in a cell by contacting a sample from the cell with the antibody under conditions permitting binding of the antibody to the protein in the sample, and detecting binding of the antibody to a protein in the sample, wherein detection of binding of the antibody to the protein indicates expression of a CaSR isoform in the cell. Using quantitative immunoassay or Western blotting methods, it is possible to quantitate the amount of CaSR, and particularly to detect increases or decreases in the amount of CaSR relative to the cell at an earlier time, or to normal cells.

According to the invention, a human CaSR isoform produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as WO 00/06601 PCT/US99/17116 an antigen or immunogen to generate antibodies that recognize the polypeptide. A molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids. An antigenic portion of a molecule can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization. A molecule that is antigenic need not be itself immunogenic, capable of eliciting an immune response without a carrier. Preferably, the antigenic polypeptide comprises an epitope and/or a sequence not present in the wild-type CaSRa, and elicits antibodies which bind to a CaSR isoform.

Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. The antibodies of the invention may be cross reactive, they may recognize CaSR isoforms from different species. Polyclonal antibodies have greater likelihood of cross reactivity. Alternatively, an antibody of the invention may be specific for a single isoform of CaSR.

In a preferred embodiment, the antibodies are capable of specifically recognizing an isoform of CaSR, and are not capable of recognizing the wild-type CaSR.

Various procedures known in the art may be used for the production of polyclonal antibodies. For the production of antibody, various host animals can be immunized by injection with the CaSR isoform, or a derivative fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, a polypeptide or fragment thereof can be conjugated to an immunogenic carrier, bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies against the isoform, or fragment, analog, or derivative thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein [Nature 256:495-497 (1975)], as well as the trioma technique, the human B-cell hybridoma technique [Kozbor et al., Immunology Today 4:72 1983); Cote et al., Proc. Natl. Acad. Sci.

U.S.A. 80:2026-2030 (1983)], and the EBV-hybridoma technique to produce human monoclonal antibodies [Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)]. In an additional embodiment of the invention, monoclonal antibodies can be produced in germfree animals [International Patent Publication No. WO 89/12690, published 28 December 1989]. In fact, according to the invention, techniques developed for the production of "chimeric antibodies" [Morrison et WO 00/06601 PCT/US99/17116 al., J. Bacteriol. 159:870 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)] by splicing the genes from a mouse antibody molecule specific for a CaSR isoform together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention. Such human or humanized chimeric antibodies are preferred for use in therapy of human diseases or disorders (described infra), since the human or humanized antibodies are much less likely than xenogenic antibodies to induce an immune response, in particular an allergic response, themselves.

According to the invention, techniques described for the production of single chain Fv (scFv) antibodies Patent Nos. 5,476,786 and 5,132,405 to Huston; U.S. Patent 4,946,778] can be adapted to produce CaSR isoform-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries [Huse et al., Science 246:1275-1281 (1989)] to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for a CaSR isoform.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the fragment which can be. produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of a CaSR isoform, one may assay generated hybridomas for a product which binds to an isoform containing such epitope. For selection of an antibody specific to an isoform from a particular species of animal, one can select on the basis of positive binding with a CaSR isoform expressed by or isolated from cells of that species of animal, but which does not bind the wild-type CaSR.

The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the CaSR isoform, for Western blotting, imaging theisoform in situ, measuring levels WO 00/06601 PCT/US99/17116 thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art.

In a specific embodiment, antibodies that agonize or antagonize the activity of CaSR, and, in particular, are specific for a CaSR isoform, can be generated. Such antibodies can be tested using the assays described infra for identifying ligands. In particular, such antibodies can be scFv antibodies expressed intracellularly.

Screening Assays Identification and isolation of a gene encoding isoforms of a CaSR of the invention provides for expression of these isoforms in quantities greater than can be isolated from natural sources, or in indicator cells that are specially engineered to indicate CaSR activity after transfection or transformation of the cells. Accordingly, in addition to rational design ofagonists and antagonists based on the structures of CaSR isoforms, the present invention contemplates an alternative method for identifying specific ligands of CaSR isoforms using various screening assays known in the art.

Any screening technique known in the art can be used to screen for CaSR agonists or antagonists.

The present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and agonize (calcimimetics) or antagonize (calcilytics) CaSR activity in vivo. For example, natural products libraries can be screened using assays of the invention for molecules that agonize or antagonize CaSR activity. The present invention provides both the means and methodology for identifying compounds capable of modulating CaSR activity, including the specific modulation of CaSR isoforms. Screening assays for calcimimetics and calcilytics are discussed in WO 94/18959 and US Pat. No. 5,763,569, the entire contents of which are incorporated herein by reference.

In a preferred screening assay compounds are assayed for their ability to influence intracellular calcium concentration. The compounds may be tested alone, in conjunction with an elevation in extracellular calcium concentration, or in the presence of other agonists or antagonists of CaSR isoform activity. Agonists include neomycin, di- and tri-valent cations (gadolinium, calcium, magnesium, strontium, barium, lanthanum), polyamines and other known calcimimetics. Intracellular calcium is measured with the fluorescent indicator, fura-2 (from Molecular Probes). For example, HEK-293 cells transfected with a nucleic acid encoding a CaSR isoform is loaded in buffer containing 0.5uM fura-2, HEPES, pH 7.35, 0.1% BSA, 0.5mM CaCIl, 0.5mM MgCl2, 6.7mM KCI, 3mM glucose and 142mM NaCI for 45 min at 37 0 C. The cells are washed and resuspended to about 2 x 10 6 cells/ml in the loading buffer without fura-2. For intracellular calcium measurement, cells were placed in a quartz cuvette equilibrated at 37C. Fluorescence is detected using excitation monochrometers centered at 340 and 380 nm and emission light collected at 505 nm.

WO 00/06601 PCT/US99/17116 Knowledge of the primary sequence of CaSR isoforms, and the similarity of their sequences with proteins of known function, can provide an initial clue as the inhibitors or antagonists of the protein* Identification and screening of antagonists is further facilitated by determining structural features of the protein, using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce large libraries. Using the "phage method" [Scott and Smith, 1990, Science 249:386-390 (1990); Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)], very large libraries can be constructed (106-108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method [Geysen et al., Molecular Immunology 23:709-715 (1986); Geysen et al. J. Immunologic Method 102:259-274 (1987)] and the method of Fodor et al. [Science 251:767-773 (1991)] are examples. Furka et al. [14th International Congress of Biochemistry, Volume 5, Abstract FR:013 (1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991)], Houghton Patent No. 4,631,211, issued December 1986] and Rutter et al. Patent No. 5,010,175, issued April 23, 1991] describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries [Needels et al., Proc. Natl. Acad. Sci. USA 90:10700-4 (1993); Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993); Lam et al., International Patent Publication No. WO 92/00252; Kocis et al., International Patent Publication No. WO 9428028, each of which is incorporated herein by reference in its entirety], and the like can be used to screen for CaSR ligands according to the present invention.

The screening can be performed with recombinant cells that express a CaSR isoform, or alternatively, using purified protein, produced recombinantly, as described above. For example, the ability of a labeled, soluble CaSR isoform that includes the extracellular calcium binding portion of the molecule, can be used to screen libraries, as described in the foregoing references.

In one embodiment, a CaSR isoform may be directly labeled. In another embodiment, a labeled secondary reagent may be used to detect binding of an isoform to a molecule of interest, a molecule attached to a solid phase support. Binding may be detected by in situ formation of a chromophore by an enzyme label. Suitable enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase. In a further embodiment, a two color assay, using two chromogenic substrates with two enzyme labels on different acceptor molecules of interest, may be used. Cross-reactive and singly-reactive ligands may be identified with a two-color assay.

Other labels for use in the invention include colored latex beads, magnetic beads, fluorescent labels fluorescene isothiocyanate (FITC), phycoerythrin Texas red rhodamine, free or chelated lanthanide series salts, especially Eu 3 to name a few fluorophores), chemiluminescent WO 00/06601 PCT/US99/17116 molecules, radio-isotopes, or magnetic resonance imaging labels. Two color assays may be performed with two or more colored latex beads, or fluorophores that emit at different wavelengths. Labeled may be detected visually or by mechanical/optical means. Mechanical/optical means include fluorescence activated sorting, analogous to FACS, and micromanipulator removal means.

As exemplified herein, the level of the CaSR isoform can be evaluated by metabolic labeling of the proteins. As the metabolic labeling occurs during in vitro incubation of the tissue biopsy in the presence of culture medium supplemented with 3 S]-methionine, the level of each of the markers detected may be affected by the in vitro conditions. In addition to metabolic (or biosynthetic) labeling with 3 5

S]-

methionine, the invention further contemplates labeling with 4 C]-amino acids and 3 H]-amino acids (with the tritium substituted at non-labile positions). Thus, a sample or library of compounds can be directly analyzed after labeling of the proteins therein, by colorimetric staining using silver, gold, coomassie blue, or amido-schwartz, to mention a few techniques; isotopic labeling, with ["P]-orthophosphate, [125], 131 fluorescent or chemiluminescent tags; and immunological detection with labeled antibody or specific binding partner of a marker.

Pharmaceutical Compositions and Therapy Diseases or disorders associated with calcium homeostasis, and therefore, the CaSR, are known in the art. Such diseases are related to the functional responses of cells to calcium, such as parathyroid hormone secretion from parathyroid cells, calcitonin secretion by C-cells and bone resorption by osteoclasts. An example is hyperparathyroidism, which results in elevated levels of parathyroid hormone in the plasma. Therefore, a method of decreasing plasma parathyroid hormone levels is a way of treating hyperparathyroidism. Alternatively, increased levels of plasma calcitonin are associated with inhibition of bone resorption. Inhibition of bone resorption offers a way to treat osteoporosis, for example. The present invention provides both the means and methodology for identifying compounds capable of modulating CaSR activity, including the specific modulation of CaSR isoforms, and of using these compounds for the treatment of diseases or disorders associated with abnormal calcium levels.

Therefore, the present invention provides pharmaceutical compositions and methods for the treatment of a patient suffering from a disease or disorder associated with abnormal calcium levels, such as in the plasma, by the administration of a therapeutically effective amount of a compound capable of modulating the activity of a CaSR isoform. The term "patient" includes both human and other mammals.

"Pharmaceutical composition" refers to a composition comprising the compound and at least one component selected from the group comprising pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, WO 00/06601 PCT/US99/17116 antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms. Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monosterate and gelatin. Examples of suitable carriers, diluents, solvents or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Examples ofexcipients include lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate phosphate. Examples of disintegrating agents include starch, alginic acids and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.

"Pharmaceutically acceptable" means it is, within the scope of sound medical judgement, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.

"Pharmaceuticallly acceptable dosage forms" refers to dosage forms of the compound of the invention, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.

Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition.

"Pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates, malonates, salicylates, propionates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methane-sulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and quinateslaurylsulphonate salts, and the like. (See, for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 66: p.

1 19 (1977) which is incorporated herein by reference.) Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus WO 00/06601 PCT/US99/17116 formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The* sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, omithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, lysine and arginine, and dicyclohexylamine, and the like.

"Solid dosage form" means the dosage form of the compound of the invention is solid form, for example capsules, tablets, pills, powders, dragees or granules. In such solid dosage forms, the compound of the invention is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid, binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia, humectants, as for example, glycerol, disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, solution retarders, as for example paraffin, absorption accelerators, as for example, quaternary ammonium compounds, wetting agents, as for example, cetyl alcohol and glycerol monostearate, adsorbents, as for example, kaolin and bentonite, lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, opacifying agents, buffering agents, and agents which release the compound(s) of the invention in a certain part of the intestinal tract in a delayed manner.

The choice of vehicle and the content of active substance in the vehicle are generally determined in accordance with the solubility and chemical properties of the active compound, the particular mode of administration and the provisions to be observed in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets. To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.

WO 00/06601 PCTIUS99/17116 The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the emulsifying wax, and the way together with the oil and fat make up the emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.

If desired, the aqueous phase of the cream base may include, for example, a least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulphoxide and related analogs.

The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Solid compositions of may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.

The pharmaceutical compositions can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, inhalational, rectal, nasal, buccal, sublingual, vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intraperitoneal. It will be appreciated that the preferred route may vary with for example the condition of the recipient.

The formulations can be prepared in unit dosage form by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

WO 00/06601 PCT/US99/17116 "Formulations suitable for oral administration" may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a* powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tables may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compounds moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Solid compositions for rectal administration include suppositories formulated in accordance with known methods and containing at least one compound of the invention.

If desired, and for more effective distribution, the compounds can be microencapsulated in, or attached to, a slow release or targeted delivery systems such as a biocompatible, biodegradable polymer matrices poly(d,l-lactide co-glycolide)), liposomes, and microspheres and subcutaneously or intramuscularly injected by a technique called subcutaneous or intramuscular depot to provide continuous slow release of the compound(s) for a period of 2 weeks or longer. The compounds may be sterilized, for example, by filtration through a bacteria retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.

Actual dosage levels of active ingredient in the compositions of the invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration. The selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.

Total daily dose of the compounds of this invention administered to a host in single or divided doses may be in amounts, for example, of from about 0.001 to about 100 mg/kg body weight daily and preferably 0.01 to 10 mg/kg/day. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.

The amount of each component administered is determined by the attending clinicians taking into consideration the etiology and severity of the disease, the patient's condition and age, the potency of each component and other factors.

WO 00/06601 PCT/US99/17116 The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials with elastomeric stoppers, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Gene Therapy and Transgenic Vectors The present invention also relates to gene therapy of diseases or disorders associated with abnormal levels of calcium. As discussed above, a "vector" is any means for the transfer of a nucleic acid according to the invention into a host cell. Preferred vectors are viral vectors, such as retroviruses, herpes viruses, adenoviruses, and adeno-associated viruses. Thus, a gene encoding a CaSR, a CaSR isoform, a polypeptide domain fragment thereof, or a nucleic acid encoding a CaSR antisense sequence is introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both.

Expression vectors of the invention can be used, as pointed out above, both to transfect cells for screening or biological testing of modulators of CaSR activity, or for delivery of a CaSR nucleic acid, as described above, or CaSR antisense gene in vivo or ex vivo for gene therapy, to increase or decrease the level of CaSR activity. A vector that expresses an anti-CaSR scFv can also be introduced using the techniques discussed below.

Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNAbased vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art [see, Miller and Rosman, BioTechniques 7:980-990 (1992)]. Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsulating the viral particles.

DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus \iO r0/0660c r-T/S9TCQ /171 I16 VT1 UUUU A 128 (AAV), vaccinia virus, and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not replication competent after introduction into a cell, and thus does not lead to a productive viral infection. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt et al., Molec. Cell. Neurosci. 2:320-330 (1991)], defective herpes virus vector lacking a glyco-protein L gene [Patent Publication RD 371005 or other defective herpes virus vectors [International Patent Publication No. WO 94/21807, published September 29, 1994; International Patent Publication No. WO 92/05263, published April 2, 1994]; an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. Clin. Invest. 90:626-630 (1992); see also La Salle et al., Science 259:988-990 (1993)]; and a defective adeno-associated virus vector [Samulski et al., J. Virol.

61:3096-3101 (1987); Samulski et al., J. Virol. 63:3822-3828 (1989); Lebkowski et al., Mol. Cell. Biol.

8:3988-3996 (1988)].

Preferably, for in vivo administration, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-y (IFN-y), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors [see, Wilson, Nature Medicine (1995)]. In addition, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.

Naturally, the invention contemplates delivery of a vector that will express a therapeutically effective amount of a CaSR, or an antisense thereto, for gene therapy applications. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to reduce by at least about percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host.

Any vector, viral or non-viral, of the invention will preferably be introduced in vivo in a pharmaceutically acceptable vehicle or carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.

Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum.

WO nn/n/0r1 DPrT/TQo/o1 '71 1 *vt w. "V Z -29 animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the lie.

Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

Adenovirus vectors In a preferred embodiment, the vector is an adenovirus vector. Adenoviruses are eukarvotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types.

Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see W094/26914). Those adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (example: Mavl, Beard et al., Virology (1990) 81), ovine, porcine, avian, and simian (example: SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus Manhattan or A26/61 strain (ATCC VR-800), for example).

Preferably, the replication defective adenoviral vectors of the invention comprise the ITRs, an encapsidation sequence and the nucleic acid of interest. Still more preferably, at least the El region of the adenoviral vector is non-functional. The deletion in the El region preferably extends from nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus (Pvull-BglII fragment) or 382 to 3446 (Hinfll-Sau3A fragment). Other regions may also be modified, in particular the E3 region (W095/02697), the E2 region (W094/28938), the E4 region (W094/28152, W094/12649 and W095/02697), or in any of the late genes In a preferred embodiment, the adenoviral vector has a deletion in the El region (Ad Examples of El-deleted adenoviruses are disclosed in EP 185.573, the contents of which are incorporated herein by reference. In another preferred embodiment, the adenoviral vector has a deletion in the El and E4 regions (Ad Examples of EI/E4-deleted adenoviruses are disclosed in W095/02697 and W096/22378, the contents of which are incorporated herein by reference. In still another preferred embodiment, the adenoviral vector has a deletion in the El region into which the E4 region and the nucleic acid sequence are inserted (see FR94 13355, the contents of which are incorporated herein by reference).

The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid which carries, inter alia. the DNA sequence of interest. The homologous recombination is effected following cotransfection of the adenovirus and plasmid into an appropriate cell line. The cell line which is employed should preferably be transformable by the said elements, and (ii) contain the sequences which are able to complement the part of the genome of the ILI/ f'n f PDrT/I TCOQ/1711 f 1J UUUIUIJlP 30 replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination.

Examples of cell lines which may be used are the human embryonic kidney cell line 293 (Graham et al., J.

Gen. Virol. 36 (1977) 59) which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines which are able to complement the El and E4 functions, as described in applications W094/26914 and W095/02697. Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.

Adeno-associated virus vectors The adeno-associated viruses (AAV) are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterised. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions which carry the encapsulation functions: the left-hand part of the genome, which contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, which contains the cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488 528). These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the said gene of interest in vitro (into cultured cells) or in vivo, (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsulation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus). The AAV recombinants which are produced are then purified by standard techniques.

The invention also relates, therefore, to an AAV-derived recombinant virus whose genome encompasses a sequence encoding a nucleic acid encoding a CaSR flanked by the AAV ITRs. The invention also relates to a plasmid encompassing a sequence encoding a nucleic acid encoding a CaSR flanked by two ITRs from an AAV. Such a plasmid can be used as it is for transferring the nucleic acid sequence, with the plasmid, where appropriate, being incorporated into a liposomal vector (pseudo-virus).

Retrovirus vectors In another embodiment the gene can be introduced in a retroviral vector, as described in WO nn/ AAAAI PCfT/TCQQ/ o171 1; TV t~ VUWUUU /31 T/TO711 Anderson et al., U.S. Patent No. 5,399,346: Mann et al., 1983, Cell 33:153; Temin et al.. U.S. Patent No.

4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., 1988, J. Virol. 62:1120; Temin et al., U.S. Patent No. 5,124,263; EP 453242, EP178220; Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689; International Patent Publication No. WO 95/07358, published March 16, 1995, by Dougherty et al.; and Kuo et al., 1993, Blood 82:845. The retroviruses are integrating viruses which infect dividing cells. The retrovirus genome includes two LTRs, an encapsulation sequence and three coding regions (gag, pol and env). In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest.

These vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukaemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Defective retroviral vectors are disclosed in W095/02697.

In general, in order to construct recombinant retroviruses containing a nucleic acid sequence, a plasmid is constructed which contains the LTRs, the encapsulation sequence and the coding sequence.

This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid. In general, the packaging cell lines are thus able to express the gag, pol and env genes. Such packaging cell lines have been described in the prior art, in particular the cell line PA317 (US4,861,719); the PsiCRIP cell line (W090/02806) and the GP+envAm-12 cell line (W089/07150). In addition, the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsulation sequences which may include a part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639). Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

Retroviral vectors can be constructed to function as infections particles or to undergo a single round of transfection. In the former case, the virus is modified to retain all of its genes except for those responsible for oncogenic transformation properties, and to express the heterologous gene. Non-infectious viral vectors are prepared to destroy the viral packaging signal, but retain the structural genes required to package the co-introduced virus engineered to contain the heterologous gene and the packaging signals.

Thus, the viral particles that are produced are not capable of producing additional virus.

Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.

Non-viral vectors Alternatively, the vector can be introduced in vivo by lipofection. For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker wn nn/n01 PrT/I I.SQ9/17116 32 [Feigner, et. al., Proc. Nail. Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey, et al., Proc. Natl. Acad.

Sci U.S.A. 85:8027-8031 (1988); Ulmer et al., Science 259:1745-1748 (1993)]. The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes [Feigner and Ringold, Science 337:387-388 (1989)]. Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications W095/18863 and W096/17823, and in U.S. Patent No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting [see Mackey, et. al., supra]. Targeted peptides, hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide International Patent Publication W095/21931), peptides derived from DNA binding proteins International Patent Publication W096/25508), or a cationic polymer International Patent Publication W095/21931).

It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter [see, Wu et al., J. Biol. Chem.

267:963-967 (1992); Wu and Wu,J. Biol. Chem. 263:14621-14624 (1988); Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990; Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991)]. Receptor-mediated DNA delivery approaches can also be sued [Curiel et al.. Hum.

Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)].

This invention provides several embodiments for specifically inhibiting CaSR activity in a patient suffering from a disease or disorder associated with abnormal calcium levels.

As a first embodiment, CaSR expression is inhibited by nucleic acids comprising a sequence complementary to the sequence encoding CaSR, or isoform thereof, and down-regulating or blocking its expression. A preferred embodiment comprises an antisense polynucleotide molecule. Preparation and use of antisense polynucleotides, DNA encoding antisense RNA molecules and use of oligo and genetic antisense is disclosed in WO 92/15680. the entire contents of which are incorporated herein by reference.

Antisense nucleic acids of the invention are preferably RNA capable of specifically hybridizing with all or part of the sequence selected from the group consisting of SEQ ID No. 7, SEQ ID No. 9, and SEQ ID No. 11 or the corresponding messenger RNA. The antisense sequence of the present invention may be derived from DNA sequences whose expression in the cell produces RNA WO 00/06601 PCTUS99/17116 complementary to all or part of the CaSR. These antisense sequences can be prepared by expression of all or part of the sequence selected from the group consisting of SEQ ID No. 7, SEQ ID No. 9,and SEQ ID No. 11 in the opposite orientation (EP 140 308). Any length of the antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of the CasR, or isoform thereof. Preferably, the antisense sequence is at least nucleotides in length.

In another aspect of this preferred embodiment the nucleic acid encodes antisense RNA molecules. In this embodiment, the nucleic acid is operably linked to signals enabling expression of the nucleic acid sequence, and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the antisense nucleic acid once the vector is introduced into the cell.

Examples of suitable vectors includes plasmids, adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses as described above.

Suitable expression signals include transcriptional promoter and termination sequences.

Among the promoter sequences useful for practice of this invention are tetracycline-regulated transcriptional modulators and CMV, SV-40, Ela, MLP, and LTR promoters. Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, US 5,168,062 and 5,385,839, the entire contents of which are incorporated herein by reference. The nucleic acid constructs of this invention are capable of down-regulating or blocking expression of a CaSR, or isoform thereof, and are delivered, in a preferred aspect of the invention, locally to cells capable of regulating calcium levels in a patient.

A second embodiment of the present invention's method of specifically inhibiting human CaSR activity, or an isoform thereof, at selected sites, comprises inhibiting CaSR function by expression of a nucleic acid sequence encoding an intracellular binding protein capable of selectively interacting with the CaSR, or isoform thereof, within a transfected cell. WO 94/29446 and WO 94/02610, the entire contents of which are incorporated herein by reference, disclose cellular transfection with genes encoding an intracellular binding protein. An intracellular binding protein includes any protein capable of selectively interacting, or binding, with a CaSR, or isoform thereof, in the cell in which it is expressed and of neutralizing the function of bound CaSR. Preferably, the intracellular binding protein is an antibody or a fragment of an antibody. More preferably, the antibody or fragment thereof binds the cytoplasmic domain of the CaSR. Most preferably, the intracellular binding protein is a single chain antibody capable of inhibiting cellular calcium sensing.

WO 94/02610 discloses preparation of antibodies and identification of the nucleic acid encoding a particular antibody. Using the CaSR or an isoform thereof, a monoclonal antibody specific for the cytoplasmic domain is prepared by according to techniques known to those skilled in the art. A vector comprising the nucleic acid encoding an intracellular binding protein, or a portion thereof, and capable of expression in a host cell is subsequently prepared for use in the method of this invention. Suitable vectors and methods of delivering nucleic acids encoding intracellular binding proteins to cells containing a CaSR include those discussed above for delivery of antisense nucleic WO 00/06601 PCT/US99/17116 acids.

In a preferred aspect of this second embodiment, the nucleic acid sequence encoding a daSR intracellular binding protein additionally comprises a sequence encoding a localization signal for targeting the intracellular binding protein to the cellular location of CaSR and/or a sequence enabling insertion of the intracellular binding protein in the plasma membrane. The localization signal or insertion sequence can be located anywhere on the intracellular binding protein, so long as it does not interfere with binding to the CaSR or isoform thereof. Examples of localization signals are disclosed in WO 94/02610. Preferably, the localization signal targets the intracellular binding protein to the plasma membrane.

The present invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention.

EXAMPLES

Material and Methods General Materials and Methods: Bacterial strain. The strain TG1 of Escherichia coli of the genotype supE, hsdD5, thi, D(lacproAB), F'[tra D36 pro A B lacI lacZDM15] may be used as a means to amplify and isolate the recombinant plasmids utilized.

It may be cultivated on: LB medium: -NaCI (5 g/l) (Difco) -Bacto-tryptone (10 g/1) (Difco) -Yeast extract (5 g/1) (Difco) This medium is rendered solid by the addition de 20 g/l of agar (Difco). Ampicillin (100 jg/ml) permits selection of the bacteria that have received the plasmids that carry the gene imparting resistance to this antibiotic as a marker.

Plasmids.

Bluescript series vectors (Stratagene), may used. These vectors permit cloning to be performed just like the pMTL series (Chambers et al.; Gene 1988, 68, pp 139-149).

In addition, the vectors pCDNA3 (Invitrogen) and derivative vectors (pSG42 and pCNW8), which permit the expression of proteins in mammal cells under the control of the CMV promoter, may be used.

Also, the vectors pCRII or pCR2.1 (Invitrogen), which permit cloning of PCR fragments, may be used.

WO 00nn/n PCT/IIS 99117116 v3 The genetic engineering techniques used to clone and insert cDNAs into these plasmids employ routine protocols (Maniatis T. et al., "Molecular Cloning, a Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982; Ausubel F.M. et al. "Current Protocols in Molecular Biology," John Wiley Sons, New York, 1987).

Preparation of theplasmidDNA. Large quantities of DNA may be prepared using Promega's rapid DNA preparation kit in accordance with the manufacturer's instructions. Small quantities of DNA may be prepared in the following manner: bacteria containing the plasmid are cultivated for at least 4 hours in 2 ml of LB medium in a shaker with agitation. They are then centrifuged for 2 minutes at 14,000 rpm in Eppendorf tubes, then the concentrate is put back in suspension in 100 pl of solution I (50 mM of glucose, 25 mM of Tris-HCI pH 8 buffer, 10 mM of EDTA pH lysed with 200 pl of solution II (0.2 M of NaOH, 1% SDS). The lysis solution is then neutralized with 150 pl of solution III (3 M of potassium acetate, 11.5% glacial acetic acid). After agitation of the tubes until a flocculent precipitate is obtained, 150 1l of a mixture of phenol/chloroform (50% phenol and 50% chloroform saturated in water) is added, and the entire mixture is agitated for 30 seconds. The aqueous phase containing the DNA is recovered after centrifugation for 2 minutes at 14,000 rpm. The DNA is then precipitated via the addition of 0.5 volume of isopropanol, then centrifuged for 5 minutes at 14,000 rpm and air-dried in order to finally be dissolved in 20 pl of TE-RNAse (solution of 10 mM of Tris-HCI and I mM of EDTA with 50 pg/ml of RNAse).

Enzyme amplification ofDNA by Polymerase Chain Reaction (PCR). PCR reactions may be carried out in a final volume of 100 pl in the presence of the double stranded DNA, dNTP (0.2 mM), PCR buffer (10 mM of Tris-HCL pH 8.5, 1 mM of MgCl2, 5 mM of KCI, gelatin 0.5 pg of each of the oligonucleotides. and 2.5 IU of Ampli Taq DNA polymerase (Perkin Elmer) with or without formamide The mixture iss covered with 2 drops of paraffin oil to limit evaporation of the sample. The equipment used may be Appligene's "Crocodile II." Unless otherwise specified, denaturation is effected at a temperature of 90 0 C for denaturation of the helix, a temperature for hybridization of the oligonucleotides to the denatured (single-stranded) DNA that iss 5 to 10 degrees lower than the temperature for the separation of the oligonucleotides, and a temperature of 72 0 C for elongation by the enzyme. The fragments obtained by PCR, which are used for cloning, are systematically resequenced once they were cloned, so as to verify the absence of any mutations that might have occurred during the amplification.

The oligodeoxynucleotides may be chemically synthesized according to the phosphoramidite method by utilizing f-cyanoethyl protector groups. After synthesis, the protector groups are eliminated by treatment with ammonia, and two precipitations with butanol permit purification and concentration of the oligodeoxynucleotides. The DNA concentration may be determined by measuring the optical density at 260 nm.

IIW nn Lnl "r TCnn/1 '711 vwvv UU/lPUU 36 A Ligations. Ligation reactions may be carried out at +14 0 C for one night in a final volume of ID pl in the presence of 100 to 200 ng of vector, 0.5 to 2 pg of insert, 40 IU of enzyme T4 DNA ligase (Biolabs), and a ligation buffer (50 mM of Tris-HCI pH 7.8; 10 mM of MgCl2; 10 mM of DTT; 1 mM of ATP). The negative control is formed by the ligation of the vector in the absence of the insert.

The filling of the prominent 5' ends is carried out, as needed, before ligation via the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications. The destruction of the prominent 3' ends is accomplished in the presence of DNA Polymerase of the T4 phage (Biolabs) used according to the manufacturer's recommendations.

Transformation of bacteria. The entire ligation volume (10 pl) may be used to transform bacteria, which may be rendered competent by the method of Chung et al. (1988, Proc. Natl. Acad. Sci. 86:2172- 2175). The bacteria are placed in culture in a liquid LB medium for several hours in an incubator with agitation at 37 0 C until an OD of 0.6 was obtained at 600 nm. The medium is then centrifuged at 6.000 rpm for 10 mn. The bacteria are rendered competent by dissolving the bacterial concentrate in a volume of TSB (LB medium 100 g/l of PEG 4000, 5% DMSO, 10 mM of MgCl2, 10 mM of MgSO4) corresponding to 1/10 of the volume of the medium of the initial culture. After incubation at 4 0 C for 30 to 60 minutes, 200 pl of bacteria are placed in contact with the ligation products for 15 minutes on ice. After the addition of 200 pl of LB [medium], the bacteria are incubated for 30 mn at 37 0 C, then spread out on an LB ampicillin medium.

Separation and extraction of the DNA. The separation of the DNA is performed by electrophoresis as a function of their size. In order to do this, different gels are used depending on the size of the fragments to be separated: agarose gel (Gibco BRL) in a TBE buffer (90 mM of Tris base; 90 mM of borate: 2 mM of EDTA) to separate large DNA fragments (greater than 500 bp); NuSieve agarose gel (FMC Bioproducts) in a TBE buffer to separate small fragments (less than 500 bp).

Migration on agarose gel or on polyacrylamide gel is carried out in a TBE buffer and in the presence of a molecular weight marker (1 Kb ladder. Gibco BRL). The DNA was mixed with 1/10 of the deposit volume of blue (200 g/l of Ficoll, 0.5 g/l of bromophenol blue, 50 mM of EDTA) before being deposited on the gel. After migration at 100 Volts and staining with ethidium bromide (concentration pg/ml of gel), the bands are viewed under a UV lamp.

Extraction of the DNA from the band of an agarose gel is carried out by means of electroelution as follows: the piece of gel containing the DNA fragment s cut out with a scalpel and placed in a dialysis tube closed with two clamps and containing 100 to 500 pl of TBE. The entire mixture is placed in an electrophoresis tank. where it is subjected to an electrical field of 100 Volts. After being removed from the gel, the DNA is then purified by means of two extractions with phenol/chloroform followed by two wn nn/nn1 pCrT/ TiQ Q/1 71 1 37 extractions with chloroform, then precipitated in the presence of 0.3 M of sodium acetate and 2.5 volume of absolute alcohol. After centrifugation (5 mn at 14.000 rpm), the DNA concentrate is dried and then dissolved in 20 pl of water.

Fluorescent sequencing ofplasmid DNA. The sequencing may be carried out according to Sanger's method using 4 dideoxyribonucleotides possessing a different fluorescent marker. The incorporation of one of these dideoxyribonucleotides causes a halt in the replication by the polymerase Taq of the DNA to be sequenced. This reaction yields DNA fragments of various sizes, all of which are terminated at 3' by one of the 4 dideoxyribonucleotides. One pg of a plasmid and 4 picomoles of a primer are added to 9.5 pl of a "premix" supplied by Applied Biosystems under the trademark PRISM The final volume is 20 pl in order to perform a PCR for 25 cycles, broken down into a denaturation phase at 96 0 C for 30 seconds, a hybridization phase at 50 0 C for 15 seconds, and an elongation phase at 60 0 C for 4 minutes. DNA fragments obtained after amplification are purified on an exclusion column (Chromaspinfrom Clontech) and are then dried in a Speed Vac. All of the dried material is dissolved in 5 pl of a mixture made up of 24 pl of EDTA (50 mM) and 120 pl of deionized formamide. After denaturation at 96 0 C for 3 minutes, 3 to 5 pl are deposited on an electrophoresis gel. The different DNA fragments are separated according to their size and then successively passed in front of a laser reader of the ABI 370 DNA sequencer (Applied Biosystems), where the different fluorescent chromophores are detected.

EXAMPLE 1: PCR amplification of CaSR splice variants First-strand cDNA. isolated from total RNA from normal human adult tissue, was purchased from Invitrogen. The RNA was subsequently treated with DNase (RNase-free) to eliminate genomic DNA contamination. Ten micrograms (Oug) of the RNA is primed with an Oligo (dT) primer and reverse transcribed with MMLV reverse transcriptase. The reaction is stopped by incubating at 65 0

C

for 10 minutes. The cDNA is in 40ul of RT buffer. (lxRT Buffer: 50mM Tris HCI, pH 8.3, 75 mM KCI, 3mM MgC12, 10mM DTT).

WO 00/06601 PCT/US99/17116 The following oligonucleotide primers were used to identify CaSR isoforms: 4 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'-TGTGGACAGACTTCCTGGGAT-3' 1013- 1033 (SEQ ID NO:4) 5'-ATCCCAGGAAGTCTGTCCACA-3' 1013- 1033 (SEQ ID 7-S 5'-ACTCCTAGCTGTCTCATCCCT-3' -44 -24 (SEQ ID NO:6) Splice variant CaSRc was amplified with Perkin Elmer's AmpliTaq Gold. The final reaction mix consisted of 10mM Tris-HCI, pH 8.3, 50mM KCI, 1.5mM MgC12, 0.001 gelatin, 0.8mM dATP, 0.8mM dCTP, 0.8mM dGTP, 0.8mM dTTP, 2.5 Units AmpliTaq Gold, 0.4uM Primer 3-AS, 0.4uM Primer 5-S, and 2ul Invitrogen human kidney cDNA 100ul reaction. Reaction conditions are: hold at 95 0 C for 9 minutes; 40 cycles of 94 0 C 30 seconds, 60 0 C 1 minute; and hold at 60 0 C for minutes.

CaSRb was amplified using the same conditions as CaSRc, except that 0.4uM Primer and 0.4uM Primer 7-S were used in place of Primers 3-AS and Splice variant CaSRd was amplified using the same components as CaSRc with the following exceptions: 0.4uM Primer 1-AS and 0.4uM Primer 7-S were used in place of Primers 3-AS and and 3ul instead of 2ul of Invitrogen human kidney cDNA 100ul reaction was used. Reaction conditions were: hold at 95 0 C for 9 minutes; 20 cycles of 94 0 C 30 seconds, 64°C 3 minutes; cycles of 94 0 C 30 seconds, 64 0 C 3 minutes (increment 10 seconds cycle); hold at 60 0 C for minutes. 50ul of this reaction mix was then electrophoresed on a 1% agarose gel and the bands in the 3.4kb region were excised and extracted using Qiagen's Gel Extraction kit and eluted in l ul of this extract was used as a template for the next reaction which contained the same components as the previous reaction with the following exceptions: 0.4uM Primer 4-AS was used instead of Primer 1-AS, and the template was replaced. Reaction conditions for the first and second WO 00/06601 PCT/US99/17116 amplifications were identical.

All PCR products were cloned into pCR2.1 according to Invitrogen's protocols. Sequencifng was performed with an automated DNA sequencer.

EXAMPLE 2: Expression of CaSRb and CaSRc in human kidney The strategy of searching for CaSR splice variants involved the use of primer pairs to scan different parts of of the sensor cDNA. Human kidney first strand cDNA was used as template and was amplified with either primer pair 3AS/5S or 5AS/7S. Electrophoretic resolution of the PCR mixture obtained with primer pair 3AS/5S revealed the presence of a product with the expected size 1.2kb) of the wild type CaSR and one with lower molecular mass of approximately 1.0kb. The primer pair 5AS/7S also yielded two visible PCR products with estimated size of 1.0 and 0.7kb. The product corresponded to the expected size of the wild type CaSR. The PCR products from both primer pairs were ligated into pCR 2.1 and multiple clones were selected for analysis by restriction digestion with EcoRl to release the insert. Clones bearing the putative wild type CaSR insert and those with smaller insert size were sequenced. The results of these experiments confirmed the presence of the correct CaSR sequence in the putative wild type clones. On the other hand, clones with shorter insert were found to contain either a deletion from nucleotide 186-495 or from nucleotide 1378-1608. The 186-495 deleted CaSR corresponds to CaSRb described originally in medullary thyroid carcinoma. The 1378-1608 deleted CaSR is designated as CaSRc (Figure Unlike CaSRb, the deletion in CaSRc does not cause a shift in reading frame.

Example 3: Expression of CaSRd in human kidney Following an initial PCR enrichment of human kidney first strand cDNA with primer pair 1AS/7S, products in the 3.0 3.4 kb range were gel purified and were amplified further with primer pair 4AS/7S. The PCR products were isolated by TA cloning and sequenced. One of these clones was found to contain a deletion from nucleotide 1075-1386 (Figure The 1075-1386 deleted CaSR is designated CaSRd. No change in reading frame was detected in this alternatively spliced CaSR transcript.

Example 4: Stable expression ofisoform CaSRd in HEK-293 cells Full length CaSRd was cloned into the mammalian expression vector pCEP4 (from Invitrogen). The CaSRd DNA used for transfection was prepared using the Qiagen plasmid 12- 9-03:15:40 ;WATERMARK PATENT :61 3 98196010 3/ 3 39a preparation kit. LipofectAMINE (Life Technologies, Inc.) was used as a carrier for transfection. Transfection of HEK-293 cells with CaSRd DNA was performed according to the general protocol described in the LipofectAMINE transfection kit.

The CaSRd DNA and lipofectamine complex (1 ml) was overlayed onto HEK-293 cells (90% confluent) in 6 well plates. After 5 hr. at 37 0 C, 1 ml of DMEM containing 20% fetal bovine serum, penicillin and streptomycin was added to each well. After incubating at 37 0 C for 16 hours, the media was replaced with 2 ml of DMEM containing 10% bovine serum albumin, penicillin and streptomycin and the cultures were incubated further for 8 hours at 37°C. CaSRd transfectants were isolated by selection in the presence of hygromycin following limited dilution.

The cells in each well were trypsinized, and cultures were diluted in 100 ml of DMEM containing 10% fetal bovine serum, 200 ug/ml of hygromycin, penicillin and streptomycin. 1 ml aliquots of the diluted cultures were added to o* *o *ol COMS ID No: SMBI-00414639 Received by IP Australia: Time 15:52 Date 2003-09-12 each well of several 24 well tissue culture plates. After 4 weeks in culture, wells containing a single colony were identified and each cell clone was expanded into a T75 flask. The expression, of CaSRd in each cell clone was monitored by Northern analysis. A clone, 21/2 with the highest expression level was used for functional analysis as described below.

The function of isoform CaSRd was assayed by its ability to increase intracellular concentration in response to elevation in extracellular calcium concentrations and other agonists. The wild type receptor has been shown to increase intracellular calcium concentration when extracellular calcium concentrations were raised. Intracellular calcium was measured with the fluorescent indicator, fura-2 (from Molecular Probes). HEK-293 cells transfected with CaSRd was loaded in buffer containing fura-2, 20mM HEPES, pH 7.35, 0.1% BSA, 0.5mM CaC12, 0.5mM MgC 2 6.7mM KCI, 3mM glucose and 142mM NaCI for 45 min at 37 0 C. The cells were washed and resuspended to 2 x 106 cells/ml in the loading buffer without fura-2. For intracellular calcium measurement, cells were placed in a quartz cuvette equilibrated at 37 0 C. Excitation monochrometers were centered at 340 and 380 nm with emission light collected at 505 nm. Different CaSR agonists were added to different final concentrations to activate the CaSR. Usually a final concentration of 10mM external CaCl 2 is sufficient to activate the wild type receptor maximally. Results indicated that CaSRd did not respond to agonists such as Ca Mg" and neomycin but did respond to gadolinium (Table In the presence of a CaSR potentiating compound, NPS568 (WO 94/18959, Fox et al., (1993) J. Bone Min. Res. 8: S181; Abstract #260) CaSRd responded to calcium, magnesium and neomycin.

Table I Effects of extracellular Ca", NPS568 and gadolinium on intracellular calcium level in CaSRd expressing HEK-293 cells 25 Increase in fura-2 fluorescence (cps x 106) Expt 1 Expt 2 Ca 2 0.2 0 20mM Ca 2 1.5 1.7 2uM NPS568 10uM NPS568 0.2 0.1 100uM gadolinium The present invention is not to be limited in scope by the specific embodiments described herein.

Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such 41 modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

"Comprises/comprising" when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof." 2 go• o* *o o *oo EDITORIAL NOTE APPLICATION NUMBER 52389/99 The following Sequence Listing pages 1 to 32 are part of the description. The claims pages follow on pages 42 to WO 00/06601 PCTIUS99/17116 1 SEQUENCE LISTING <110> Yu, Kin Tak Thrower, Larry W.

Labaudiniere, Richard F.

<120> ISOFORMS OF HUMAN CALCIUM SENSING RECEPTOR <130> A2996-US <140> <141> <160> 12 <170> PatentIn Ver. <210> 1 <211> 21 <212> DNA <213> Homo sapiens <400> 1 catgggtcaa ttcactgtca t 21 <210> 2 <211> 21 <212> DNA <213> Homo sapiens <400> 2 gccagatcac acagatgaca a 21 <210> 3 <211> 21 <212> DNA <213> Homo sapiens <400> 3 ggcatagacg ttgtaatacc c 21 <210> 4 <211> 21 <212> DNA <213> Homo sapiens WO 00/06601 WO 0/0601PCT[US99/17116 <400> 4 tgtggacaga <210> <211> 21 <212> DNA <213> Homo <400> atcccaggaa <210> 6 <211> 21 <212> DNA <213> Homo <400> 6 actcctagct cttcctggga t sapiens gtctgtccac a sapiens gtctcatccc t <210> 7 <211> 3003 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(3003) <400> 7 atg gca ttt tat agc 48 Met Ala Phe Tyr Ser 1 5 ace tet gec tac ggg 96 Thr Ser Ala Tyr Gly atc ctt ggg ggg ctc 144 Ile Leu Gly Gly Leu caa gat ctc aaa tca 192 Gin Asp Leu Lys Ser tgc Cys cca Pro ttt Phe agg Arg tge Cys gac Asp ct Pro ccg Pro tgg Trp cag Gin at t Ile 40 gag Glu gte Val cga Arg 25 cat His tet Ser ctc Leu 10 gcc Ala ttt Phe gtg Val ttg Leu caa Gin gga Gly gaa Glu gca Ala aag Lys gta Val tgt Cys ctc Leu aag Lys gea Ala ate Ile acc Thr ggg Gly get Ala agg Arg tgg Trp gac Asp aaa Lys tat Tyr cac His att Ile gat Asp aat Asn WO 00/06601 PCT/US99/17116 3 55 ttc cgt ggg ttt cgc tgg tta cag gct atg ata ttt gcc ata gag gag 240 Phe Arg Gly Phe Arg Trp Leu Gin Ala Met Ile Phe Ala Ile Giu Glu 70 75 ata aac agc ago cca gc ctt ctt ccc aao ttg acg otg gga tao agg 288 Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr Leu Giy Tyr Arg 90 ata ttt gao act tgc aac acc gtt tct aag gcc ttg gaa gcc acc ctg 336 Ile Phe Asp Thr Cys Asn Thr Vai Ser Lys Ala Leu Giu Aia Thr Leu 100 105 110 agt ttt gtt got caa aac aaa att gat tot ttg aac ctt gat gag tto 384 Ser Phe Val Aia Gin Asn Lys Ile Asp Ser Leu Asn Leu Asp Giu Phe 115 120 125 tgc aac tgo toa gag cac att ccc tot acg att got gtg gtg gga gca 432 Cys Asn Cys Ser Giu His Ile Pro Ser Thr Ile Ala Vai Vai Giy Ala 130 135 140 act ggc toa ggc gto too aog gca gtg gca aat ctg otg ggg otc tto 480 Thr Giy Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe 145 150 155 160 tao att coo cag gto agt tat goc too too ago aga otc otc ago aao 528 Tyr Ile Pro Gin Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn 165 170 175 aag aat caa ttc aag tot ttc otc oga aco ato ccc aat gat gag cac 576 Lys Asn Gin Phe Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Giu His 180 185 190 oag gc act gc atg goa gao ato ato gag tat tto cgc tgg aac tgg 624 Gin Ala Thr Ala Met Ala Asp Ile Ile Giu Tyr Phe Arg Trp Asn Trp 195 200 205 gtg ggc aca att gca got gat gao gao tat ggg cgg cog ggg att gag 672 Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu 210 215 220 aaa ttc oga gag gaa got gag gaa agg gat ato tgo ato gao ttc agt WO 00/06601 PCT/US99/17116 4 720 Lys Phe Arg Giu Giu Ala Giu Giu Arg Asp Ile Cys Ile Asp Phe Ser 225 230 235 240 gaa etc ate tcc cag tac tet gat gag gaa gag ate cag cat gtg gta 768 Glu Leu Ile Ser Gin Tyr Ser Asp Giu Giu Glu Ile Gin His Vai Vai 245 250 255 gag gtg att caa aat tcc acg gce aaa gtc atc gtg gtt ttc tcc agt 816 Glu Val Ile Gin Asn Ser Thr Aia Lys Val Ile Vai Val Phe Ser Ser 260 265 270 ggc eca gat ctt gag ccc ctc ate aag gag att gtc cgg cgc aat atc 864 Gly Pro Asp Leu Giu Pro Leu Ile Lys Giu Ile Vai Arg Arg Asn Ile 275 280 285 acg gge aag ate tgg ctg gee age gag gee tgg gee agc tee tee etg 912 Thr Giy Lys Ile Trp Leu Ala Ser Giu Aia Trp Aia Ser Ser Ser Leu 290 295 300 ate gee atg cet eag tac ttc cac gtg gtt ggc gge acc att gga ttc 960 Ile Ala Met Pro Gin Tyr Phe His Vai Val Gly Gly Thr Ile Giy Phe 305 310 315 320 get etg aag get ggg eag ate eca gge tte egg gaa tte ctg aag aag 1008 Ala Leu Lys Ala Gly Gin Ile Pro Gly Phe Arg Giu Phe Leu Lys Lys 325 330 335 gtc cat ccc agg aag tet gtc cac aat ggt ttt gee aag gag ttt tgg 1056 Val His Pro Arg Lys Ser Val His Asn Gly Phe Ala Lys Giu Phe Trp 340 345 350 gaa gaa aea ttt aae tgc eac etc caa gaa ggt gea aaa gga cct tta 1104 Glu Giu Thr Phe Asn Cys His Leu Gin Giu Gly Ala Lys Giy Pro Leu 355 360 365 cct gtg gac ace ttt etg aga ggt cac gaa gaa agt gge gae agg ttt 1152 Pro Val Asp Thr Phe Leu Arg Giy His Giu Glu Ser Giy Asp Arg Phe 370 375 380 age aae age teg aca gee tte ega ccc etc tgt aca ggg gat gag aae 1200 Ser Asn Ser Ser Thr Ala Phe Arg Pro Leu Cys Thr Giy Asp Giu Asn 385 390 395 400 WO 00/06601 PCT/US99/17116 ate age agt gtc gag acc cct tac ata gat tac acg cat tta egg ata 1248 Ile Ser Ser Val Glu Thr Pro Tyr Ile Asp Tyr Thr His Leu Arg Ile 405 410 415 tec tac aat gtg tac tta gca gtc tac tec att gcc cac gcc ttg caa 1296 Ser Tyr Asn Val Tyr Leu Ala Val Tyr Ser Ile Ala His Ala Leu Gin 420 425 430 gat ata tat acc tgc tta cct ggg aga ggg etc ttc ace aat ggc tec 1344 Asp Ile Tyr Thr Cys Leu Pro Gly Arg Gly Leu Phe Thr Asn Gly Ser 435 440 445 tgt gca gac ate aag aaa gtt gag gcg tgg cag gtg ccc ttc tec aac 1392 Cys Ala Asp Ile Lys Lys Val Glu Ala Trp Gin Val Pro Phe Ser Asn 450 455 460 tgc age cga gac tgc ctg gca ggg ace agg aaa ggg ate att gag ggg 1440 Cys Ser Arg Asp Cys Leu Ala Gly Thr Arg Lys Gly Ile Ile Glu Gly 465 470 475 480 gag ccc ace tgc tgc ttt gag tgt gtg gag tgt cct gat ggg gag tat 1488 Glu Pro Thr Cys Cys Phe Glu Cys Val Glu Cys Pro Asp Gly Glu Tyr 485 490 495 agt gat gag aca gat gcc agt gcc tgt aac aag tgc cca gat gac ttc 1536 Ser Asp Glu Thr Asp Ala Ser Ala Cys Asn Lys Cys Pro Asp Asp Phe 500 505 510 tgg tec aat gag aac cac ace tec tgc att gcc aag gag ate gag ttt 1584 Trp Ser Asn Glu Asn His Thr Ser Cys Ile Ala Lys Glu Ile Glu Phe 515 520 525 ctg tcg tgg acg gag ccc ttt ggg ate gca etc ace etc ttt gee gtg 1632 Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu Thr Leu Phe Ala Val 530 535 540 ctg ggc att ttc ctg aca gcc ttt gtg ctg ggt gtg ttt ate aag ttc 1680 Leu Gly Ile Phe Leu Thr Ala Phe Val Leu Gly Val Phe Ile Lys Phe 545 550 555 560 cgc aac aca ccc att gtc aag gcc ace aac cga gag etc tec tac etc 1728 WO 00/06601 WO 0006601PCT[US99/17116 Arg Asn ctc ctc 1776 Leu Leu ggg gag 1824 Giy Giu atc agc 1872 Ile Ser 610 gtc ctc 1920 Val Leu 625 tgg tgg 1968 Trp Trp atg cag 2016 Met Gin agc tac 2064 Ser Tyr cac gag 2112 His Glu 690 ctg ctg 2160 Leu Leu 705 ccg gag 2208 Thr ttc Phe ccc Pro 595 ttc Phe ctg Leu ggg Giy at t Ile cgc Arg 675 ggc Gly gct Ala aac Pro tcc Ser 580 cag Gin gtg Vali gtg Vai ctc Leu gtc Vai 660 aac Asn t cc Ser gcc Aia ttc Ile 565 ctg Leu gac Asp ctc Leu ttt Phe aac Asn 645 at c Ile cag Gin ctc Leu atc Ile aat Val Lys ctc tgc Leu Cys tgg acg Trp Thr tgc atc Cys Ile 615 gag gcc Giu Ala 630 ctg cag Leu Gin tgt gtg Cys Val gag ctg Glu Leu atg gcc Met Ala 695 tgc ttc Cys Phe 710 gaa gcc Ala tgc Cys tgc Cys 600 tca Ser aag Lys ttc Phe at c Ile gag Giu 680 ctg Leu ttc Phe aag Thr Asn 570 ttc tcc Phe Ser 585 cgc ctg Arg Leu tgc atc Cys Ile atc ccc Ile Pro ctg ctg Leu Leu 650 tgg ctc Trp Leu 665 gat gag Asp Giu ggc ttc Gly Phe ttt gc Phe Ala ttc atc Arg Giu Leu Ser Tyr Leu 575 age Ser ege Arg ctg Leu ace Thr 635 gtt Val tac Tyr atc Ile ctg Leu ttc Phe 715 tee Ser cag Gin gtg Val1 620 agc Ser ttc Phe ace Thr atc Ile atc Ile 700 aag Lys ctg Leu c cg Pro 605 aaa Lys ttc Phe etc Leu gcg Aia ttc Phe 685 ggC Gly t cc Ser ttc Phe 590 gcc Ala ace Thr cac His tgc Cys ccc Pro 670 ate Ile tac Tyr egg Arg ttc Phe ttt Phe aac Asn ege Arg acc Thr 655 ccc Pro acg Thr acc Thr aag Lys at c Ile Gly cgt Arg aag Lys 640 ttc Phe tca Ser tgc Cys tgc Cys etg Leu 720 acc ttc agc atg ctc atc Pro Giu Asn Phe Asn Giu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile 725 730 735 WO 00/06601 tte tte ate 2256 7 gte tgg ate tee tte att eca gce PCT/US99/17116 tat gee agc ace tat Tyr Ala Ser Thr Tyr Phe Phe Ile Val Trp Ile 740 gge aag ttt gtc tct gcc 2304 Gly Lys Phe Val Ser Ala 755 ttt ggc ttg etg geg tge 2352 Phe Gly Leu Leu Ala Cys 770 tte aag cca tcc cgc aae 2400 Phe Lys Pro Ser Arg Asn 785 790 get cac get tte aag gtg 2448 Ala His Ala Phe Lys Val 805 gte tee cge aag egg tee 2496 Val Ser Arg Lys Arg Ser 820 cct tee tee tee ate age 2544 Pro Ser Ser Ser Ile Ser 835 eag ccc gag agg eag aag 2592 Gin Pro Giu Arg Gin Lys 850 gag eag eag eag eag cce 2640 Glu Gin Gin Gin Gin Pro 865 870 eag cag cce aga tge aag 2688 Gin Gin Pro Arg Cys Lys 885 ace tte tea etg age ttt 2736 Thr Phe Ser Leu Ser Phe Ser Phe Ile Pro 745 gta gag gtg att Val Giu Val Ile 760 ate tte tte aae Ile Phe Phe Asn 775 ace ate gag gag Thr Ile Giu Glu get gee egg gee Ala Ala Arg Ala 810 age age ctt gga Ser Ser Leu Gly 825 age aag age aae Ser Lys Ser Asn 840 cag eag eag ceg Gin Gin Gin Pro 855 etg ace etc cca Leu Thr Leu Pro cag aag gte ate Gin Lys Val Ile 890 gat gag cet eag Asp Giu Pro Gin Ala gee ate etg Ala Ile Leu 765 aag ate tac Lys Ile Tyr 780 gtg cgt tge Vai Arg Cys 795 acg etg ege Thr Leu Arg gge tee acg Gly Ser Thr age gaa gae Ser Giu Asp 845 etg gee eta Leu Ala Leu 860 750 gca gee age Ala ate Ile age Ser ege Arg gga Gly 830 cca Pro ace Thr Ala Ser att ete Ile Leu ace gca Thr Ala 800 age aae Ser Asn 815 tee acc Ser Thr tte cca Phe Pro cag eaa Gin Gin eag eag eaa ega tet cag Gin Gin Gin Arg 875 ttt gge age gge Phe Giy Ser Gly aag aac gee atg Lys Asn Aia Met Ser Gin 880 acg gte Thr Val 895 gee cae Ala His WO 00/06601 PCT/US99/17116 8 900 905 910 agg aat tct acg cac cag aac tcc ctg gag gcc cag aaa agc agc gat 2784 Arg Asn Ser Thr Hius Gin Asn Ser Leu Glu Ala Gin Lys Ser Ser Asp 915 920 925 acg ctg acc cga cac cag cca tta ctc ccg ctg cag tgc ggg gaa acg 2832 Thr Leu Thr Arg His Gin Pro Leu Leu Pro Leu Gin Cys Gly Glu Thr 930 935 940 gac tta gat ctg acc gtc cag gaa aca ggt ctg caa gga cct gtg ggt 2880 Asp Leu Asp Leu Thr Val Gin Glu Thr Gly Leu Gin Gly Pro Val Gly 945 950 955 960 gga gac cag cgg cca gag gtg gag gac cct gaa gag ttg tcc cca gca 2928 Gly Asp Gin Arg Pro Giu Val Glu Asp Pro Glu Giu Leu Ser Pro Ala 965 970 975 ctt gta gtg tcc agt tca cag agc ttt gtc atc agt ggt gga ggc agc 2976 Leu Val Val Ser Ser Ser Gin Ser Phe Val Ile Ser Gly Gly Gly Ser 980 985 990 act gtt aca gaa aac gta gtg aat tca 3003 Thr Val Thr Giu Asn Val Val Asn Ser 995 1000 <210> 8 <211> 1001 <212> PRT <213> Homo sapiens <400> 8 Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala Leu Thr Trp, His 1 5 10 Thr Ser Ala Tyr Gly Pro Asp Gin Arg Ala Gin Lys Lys Gly Asp Ile 25 Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp 40 Gin Asp Leu Lys Ser Arg Pro Giu Ser Val Glu Cys :Ile Arg Tyr Asn 55 Phe Arg Gly Phe Arg Trp Leu Gin Ala Met Ile Phe Ala Ile Glu Glu 70 75 WO 00/06601 WO 0006601PCT/US99/1 7116 Ile I le Ser Cys Thr 145 Tyr Lys Gin Val Lys 225 Glu Glu Giy Thr Ile 305 Al a Val Glu Asn Phe Phe Asn 130 Gly Ile Asn Ala Gly 210 Phe Leu Val Pro Gly 290 Ala Leu His Glu Ser Asp Val 115 Cys Ser Pro Gin Thr 195 Thr Arg Ile Ile Asp 275 Lys Met Lys Pro Thr Ser Thr 100 Ala Ser Gly Gin Phe 180 Ala Ile Giu Ser Gin 260 Leu Ile Pro Ala Arg 340 Phe Pro Cys Gin Giu Val Vai 165 Lys Met Ala Giu Gin 245 Asn Glu Trp Gin Gly 325 Lys Asn Ala Asn Asn His Ser 150 Ser Ser Ala Ala Ala 230 Tyr Ser Pro Leu Tyr 310 Gin Ser Cys Leu Thr Lys Ile 135 Thr Tyr Phe Asp Asp 215 Glu Ser Thr Leu Al a 295 Phe Ile Val His Leu Vai Ile 120 Pro Ala Ala Leu Ile 200 Asp Giu Asp Ala Ile 280 Se r His Pro His Leu Pro Ser 105 Asp Ser Val1 Ser Arg 185 Ile Asp Arg Giu Lys 265 Lys Glu Vai Gly Asn 345 Gin Asn 90 Lys Ser Thr Ala Ser 170 Thr Giu Tyr Asp Giu 250 Val Giu Ala Vai Phe 330 Gly Glu Leu Al a Leu Ile Asn 155 Ser Ile Tyr Gly Ile 235 Giu Ile Ile Trp Gly 315 Arg Phe Gly Thr Leu Asn Ala 140 Leu Arg Pro Phe Arg 220 Cys Ile Val Val1 Ala 300 Gly Giu Ala Ala Leu Giu Leu 125 Val Leu Leu Asn Arg 205 Pro Ile Gin Val Arg 285 Ser Thr Phe Lys Lys *Gly Ala 110 Asp Val Gly Leu Asp 190 Trp Giy Asp His Phe 270 Arg Ser Ile Leu Glu 350 Gly Tyr Thr Giu Gly Leu Ser 175 Giu Asn Ile Phe Val1 255 Ser Asn Ser Gly Lys 335 Phe Pro Arg Leu Phe Ala Phe 160 Asn His Trp Giu Ser 240 Val Ser Ile Leu Phe 320 Lys Trp Leu WO 00/06601 WO 0006601PCT[US99/17116 Pro Ser 385 Ile Ser Asp Cys Cys 465 Glu Ser Trp Leu Leu 545 Arg Leu Gly Ile Val 625 Val 370 Asn Ser Tyr Ile Al a 450 Ser Pro Asp Ser Ser 530 Gly Asn Leu Giu Ser 610 Leu 355 Asp Ser Ser Asn Tyr 435 Asp Arg Thr Glu Asn 515 Trp Ile Thr Phe Pro 595 Phe Leu Thr Ser Val Val 420 Thr Ile Asp Cys Thr 500 Glu Thr Phe Pro Ser 580 Gin Val Val Phe Thr Giu 405 Tyr Cys Lys Cys Cys 485 Asp Asn Glu Leu Ile 565 Leu Asp Leu Phe Leu Ala 390 Thr Leu Leu Lys Leu 470 Phe Ala His Pro Thr 550 Val Leu Trp Cys Glu 630 Arg 375 Phe Pro Ala Pro Val1 455 Al a Glu Ser Thr Phe 535 Ala Lys Cys Thr Ile 615 Ala 360 Gly Arg Tyr Val Gly 440 Giu Gly Cys Ala Ser 520 Gly Phe Ala Cys Cys 600 Ser Lys His Pro Ile Tyr 425 Arg Ala Thr Val Cys 505 Cys Ile Val Thr Phe 585 Arg Cys Ile Glu Leu Asp 410 Ser Gly Trp Arg Glu 490 Asn Ile Ala Leu Asn 570 Ser Leu Ile Pro Glu Cys 395 Tyr Ile Leu Gin Lys 475 Cys Lys Al a Leu Gly 555 Arg Ser Arg Leu Thr 635 Ser 380 Thr Thr Ala Phe Val 460 Gly Pro Cys Lys Thr 540 Val Giu Ser Gin Val 620 Ser 365 Gly Gly His His Thr 445 Pro Ile Asp Pro Giu 525 Leu Phe Leu Leu Pro 605 Lys Phe Asp Asp Leu Al a 430 Asn Phe Ile Gly Asp 510 Ile Phe Ile Ser Phe 590 Ala Thr His Arg Giu Arg 415 Leu Gly Ser Glu Glu 495 Asp Glu Ala Lys Tyr 575 Phe Phe Asn Arg Phe Asn 400 Ile Gin Ser Asn Gly 480 Tyr Phe Phe Val1 Phe 560 Leu Ile Gly Arg Lys 640 WO 00/06601 PCT[US99/7116 11 Trp Trp Gly Leu Asn Leu Gin Phe Leu Leu Val Phe Leu Cys Thr Phe 645 Met Gin Ile Val Ile Cys Val 660 Ser Tyr Arg Asn Gin Glu Leu 675 His Glu Gly Ser Leu Met Ala 690 695 Leu Leu Ala Ala Ile Cys Phe 705 710 Pro Glu Asn Phe Asn Glu Ala 725 Phe Phe Ile Val Trp Ile Ser 740 Gly Lys Phe Val Ser Ala Val 755 Phe Gly Leu Leu Ala Cys Ile 770 775 Phe Lys Pro Ser Arg Asn Thr 785 790 Ala His Ala Phe Lys Val Ala 805 Val Ser Arg Lys Arg Ser Ser 820 Pro Ser Ser Ser Ile Ser Ser 835 Gin Pro Glu Arg Gin Lys Gin 850 855 Glu Gin Gin Gin Gin Pro Leu 865 870 Gin Gin Pro Arg Cys Lys Gin 885 Thr Phe Ser Leu Ser Phe Asp 900 Arg Asn Ser Thr His Gin Asn 915 Ile Glu 680 Leu Phe Lys Phe Glu 760 Phe Ile Ala Ser Lys 840 Gin Thr Lys Glu Ser 920 650 Trp Leu Tyr Thr Ala 665 Asp Glu Ile Ile Phe 685 Gly Phe Leu Ile Gly 700 Phe Ala Phe Lys Ser 715 Phe Ile Thr Phe Ser 730 Ile Pro Ala Tyr Ala 745 Val Ile Ala Ile Leu 765 Phe Asn Lys Ile Tyr 780 Glu Glu Val Arg Cys 795 Arg Ala Thr Leu Arg 810 Leu Gly Gly Ser Thr 825 Ser Asn Ser Glu Asp 845 Gin Pro Leu Ala Leu 860 Leu Pro Gin Gin Gin 875 Val Ile Phe Gly Ser 890 Pro Gin Lys Asn Ala 905 Leu Glu Ala Gin Lys 925 Pro 655 Pro Ser 670 Ile Thr Cys Tyr Thr Cys Arg Lys Leu 720 Met Leu Ile 735 Ser Thr Tyr 750 Ala Ala Ser Ile Ile Leu Ser Thr Ala 800 Arg Ser Asn Gly 830 Pro Thr Arg Gly Met 910 Ser 815 Ser Thr Phe Pro Gin Gin Ser Gin 880 Thr Val 895 Ala His Ser Asp WO 00/06601 WO 0006601PCT[US99/1 7116 Thr Asp 945 Gly Leu Thr Leu 930 Leu Asp Val1 Val1 Thr Asp Gin Val Thr 995 Arg Leu Arg Ser 980 Giu His Thr Pro 965 Ser Asn Gin Vai 950 Giu Ser Val 12 Pro Leu Leu 935 Gin Glu Thr Vai Giu Asp Gin Ser Phe 985 Val Asn Ser 1000 Pro Leu Gin Cys Gly 940 Gly Leu Gin Gly Pro 955 Pro Giu Giu Leu Ser 970 Val Ile Ser Gly Giy 990 Giu Val Pro 975 Gly Thr Giy 960 Al a Ser <210> 9 <211> 2922 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(2922) <400> 9 atg Met 1 acc Thr at c 144 Ile caa 192 Gin ttc 240 Phe ata gca Ala tct Ser ctt Leu gat Asp cgt Arg aac ttt Phe gcc Ala ggg Giy ctc Leu ggg Gly agc tat Tyr tac Tyr ggg Giy aaa Lys ttt Phe agc agc Ser 5 ggg Gly ctc Leu tca Ser cgc Arg cca tgc Cys cca Pro ttt Phe agg Arg tgg Trp 70 gcc tgc Cys gac Asp cct Pro ccg Pro 55 tta Leu ctt t gg Trp cag Gin att Ile 40 gag Giu cag Gin ctt gtc Val cga Arg 25 cat His tct Ser gct Ala ccc ctc Leu 10 gcc Ala ttt Phe gtg Vai atg Met aac ttg Leu caa Gin gga Giy gaa Giu ata Ile 75 ttg gca Aia aag Lys gta Val tgt Cys ttt Phe acg ctc Leu aag Lys gca Aia atc Ile gcc Ala Ctg acc Thr ggg Giy gct Aia agg Arg ata Ile gga tgg Trp gac Asp aaa Lys tat Tyr gag Giu tac cac His att Ile gat Asp aat Asn gag Giu agg WO 00/06601 288 Ile Asn Ser ata ttt gac 336 Ile Phe Asp agt ttt gtt 384 Ser Phe Vai 115 tgc aac tgc 432 Cys Asn Cys 130 act ggc tca 480 Thr Giy Ser 145 tac att ccc 528 Tyr Ile Pro aag aat caa 576 Lys Asn Gin cag gcc act 624 Gin Aia Thr 195 gtg ggc aca 672 Val Gly Thr 210 aaa ttc cga 720 Lys Phe Arg 225 gaa ctc atc 768 Glu Leu Ile PCT/US99/17116 Ser Pro Ala Leu Leu Pro act tgc aac acc gtt tct Thr Cys Asn Thr Val Ser 100 105 gct caa aac aaa att gat Ala Gin Asn Lys Ile Asp 120 tca gag cac att ccc tct Ser Glu His Ile Pro Ser 135 ggc gtc tcc acg gca gtg Gly Val Ser Thr Ala Val 150 cag gtc agt tat gcc tcc Gin Val Ser Tyr Ala Ser Asn 90 aag Lys tct Ser acg Thr gca Ala tcc Ser 165 ttc aag tct ttc Phe Lys Ser Phe 180 gcc atg gca gac Ala Met Ala Asp att gca gct gat Ile Ala Ala Asp 215 gag gaa gct gag Glu Giu Ala Glu 230 tcc cag tac tct 170 ctc cga acc Leu Arg Thr 185 atc atc gag Ile Ile Glu 200 gac gac tat Asp Asp Tyr gaa agg gat Glu Arg Asp gat gag gaa Leu Thr Leu Giy Tyr Arg gcc ttg gaa gcc acc ctg Ala Leu Giu Ala Thr Leu 110 ttg aac ctt gat gag ttc Leu Asn Leu Asp Giu Phe 125 att gct gtg gtg gga gca Ile Ala Val Val Gly Ala 140 aat ctg ctg ggg ctc ttc Asn Leu Leu Gly Leu Phe 155 160 agc aga ctc ctc agc aac Ser Arg Leu Leu Ser Asn 175 atc ccc aat gat gag cac Ile Pro Asn Asp Giu His 190 tat ttc cgc tgg aac tgg Tyr Phe Arg Trp Asn Trp 205 ggg cgg ccg ggg att gag Gly Arg Pro Gly Ile Glu 220 atc tgc atc gac ttc agt Ile Cys Ile Asp Phe Ser 235 240 gag atc cag cat gtg gta Glu Ile Gin His Vai Val 255 Ser Gin Tyr Ser Asp Giu Glu 245 250 WO 00/06601 WO 0006601PCTIUS99/1 7116 gag gtg 816 Glu Val ggc cca 864 Gly Pro acg ggc 912 Thr Gly 290 atc gcc 960 Ile Ala 305 gct ctg 1008 Ala Leu gtc cat 1056 Val His gaa gaa 1104 Giu Glu atg ggg 1152 Met Gly 370 tat tcc 1200 Tyr Ser 385 ttt aag 1248 Phe Lys ctc ttc 1296 att Ile gat Asp 275 aag Lys atg Met aag Lys ccc Pro aca Thr 355 gag Giu atc Ile gaa Giu atc caa Gin 260 ctt Leu atc Ile cct Pro gct Ala agg Arg 340 ttt Phe cag Gin atc Ile gtc Val aac aat Asn gag Glu tgg Trp cag Gin ggg Gly 325 aag Lys aac Asn gtg Val aac Asn ggg Gly 405 gag tcc Ser ccc Pro ctg Leu tac Tyr 310 cag Gin tct Ser tgc Cys acc Thr tgg Trp 390 tat Tyr gag acg Thr ctc Leu gcc Ala 295 ttc Phe atc Ile gtc Val cac His ttt Phe 375 cac His tac Tyr aaa gcc Ala atc Ile 280 agc Ser cac His cca Pro cac His cta Leu 360 gat Asp ctc Leu aac Asn atc aaa Lys 265 aag Lys gag Glu gtg Val ggc Gly aat Asn 345 cgg Arg gag Giu tcc Ser gt c Val ctg gtc Val gag Giu gcc Ala gt t Val ttc Phe 330 ggt Gly cat His tgt Cys cca Pro tat Tyr 410 t gg atc Ile at t Ile tgg Trp, ggc Gly 315 cgg Arg ttt Phe cta Leu ggt Gly gag Glu 395 gcc Al a agt gtg Val gtc Val gcc Ala 300 ggc Gly gaa Giu gcc Ala aac Asn gac Asp 380 gat Asp aag Lys ggt gtt Val cgg Arg 285 agc Se r acc Thr ttc Phe aag Lys ttt Phe 365 ctg Leu ggc Gly aag Lys ttc ttc Phe 270 cgc Arg tcc Ser att Ile ctg Leu gag Giu 350 aca Thr gtg Val1 tcc Ser gga Gly tcc tcc Ser aat Asn tcc Ser gga Gly aag Lys 335 ttt Phe aac Asn ggg Gly atc Ile gaa Giu 415 agg agt Ser atc Ile ctg Leu ttc Phe 320 aag Lys tgg Trp, aat Asn aac Asn gtg Val 400 aga Arg gag WO 00/06601 Leu Phe Ile Asn 420 gtg ccc ttc tcc 1344 Val Pro Phe Ser 435 ggg atc att gag 1392 Gly Ile Ile Glu 450 cet gat ggg gag 1440 Pro Asp Gly Giu 465 tgc cca gat gac 1488 Cys Pro Asp Asp aag gag ate gag 1536 Lys Glu Ile Giu 500 aec etc ttt gc 1584 Thr Leu Phe Ala 515 gtg ttt atc aag 1632 Val Phe Ile Lys 530 gag ctc tcc tac 1680 Glu Leu Ser Tyr 545 tcec ctg tte ttc 1728 Ser Leu Phe Phe cag ccg gcc ttt 1776 Gin Pro Ala Phe 580 PCT/US99/17116 Giu Glu Lys Ile Leu Trp Ser Gly Phe Ser Arg Giu aac Asn ggg Gly tat Tyr ttc Phe 485 ttt Phe gtg Val tte Phe etc Leu atc Ile 565 ggc Gly tge Cys gag Giu agt Ser 470 tgg Trp ctg Leu ctg Leu ege Arg ctc Leu 550 ggg Giy ate Ile age Ser ccc Pro 455 gat Asp tee Ser t eg Ser gge Gly aae Asn 535 etc Leu gag Glu age Ser cga Arg 440 ace Thr gag Giu aat Asn tgg Trp, att Ile 520 aca Thr ttc Phe ccc Pro ttc Phe 425 gac Asp tgc Cys aca Thr gag Giu aeg Thr 505 tte Phe ccc Pro tee Ser cag Gin gtg Val1 585 tge Cys tgc Cys gat Asp aae Asn 490 gag Glu ctg Leu at t Ile etg Leu gac Asp 570 etc Leu ctg Leu ttt Phe gee Aia 475 cac His ccc Pro aca Thr gte Vai etc Leu 555 tg Trp tgc Cys 430 gca ggg ace Ala Gly Thr 445 gag tgt gtg Giu Cys Val 460 agt gee tgt Ser Ala Cys ace tee tgc Thr Ser Cys ttt ggg ate Phe Gly Ile 510 gee ttt gtg Ala Phe Val 525 aag gee ace Lys Ala Thr 540 tge tge tte Cys Cys Phe aeg tgc ege Thr Cys Arg ate tea tge Ile Ser Cys 590 agg aaa Arg Lys gag tgt Glu Cys aac aag Asn Lys 480 att gee Ile Ala 495 gea etc Ala Leu etg ggt Leu Giy aae ega Asn Arg tee age Ser Ser 560 ctg ege Leu Arg 575 ate ctg Ile Leu WO 00/06601 WO 0006601PCTIUS99/171 16 gtg aaa 1824 Val Lys age ttc 1872 Ser Phe 610 tte ctc 1920 Phe Leu 625 acc gcg 1968 Thr Ala atc ttc 2016 Ile Phe ate ggc 2064 Ile Gly aag tc 2112 Lys Ser 690 tte age 2160 Phe Ser 705 tat gcc 2208 Tyr Ala ate ctg 2256 Ile Leu atc tac 2304 ace Thr 595 cac His tge Cys ccc Pro ate Ile tac Tyr 675 cgg Arg atg Met agc Ser gca Ala ate aac Asn cgc Arg acc Thr ccc Pro acg Thr 660 ace Thr aag Lys ctc Leu ac Thr gc c Ala 740 att cg t Arg aag Lys ttc Phe tca Ser 645 tgc Cys tgc Cys ctg Leu atc Ile tat Tyr 725 agc Ser ctc gte Val tgg Trp atg Met 630 age Ser cac His ctg Leu ccg Pro ttc Phe 710 ggc Gly ttt Phe tte ctc Leu tgg Trp 615 cag Gln tac Tyr gag Glu ctg Leu gag Giu 695 ttc Phe aag Lys ggc Giy aag ctg Leu 600 ggg Gly att Ile cgc Arg gge Gly get Ala 680 aac Asn ate Ile ttt Phe ttg Leu cca gtg Val1 ctc Leu gtc Val1 aac Asn tee Ser 665 gcc Ala ttc Phe gtc Val gte Val ctg Leu 745 tcc ttt Phe aac Asn atc Ile c ag Gin 650 etc Leu atc Ile aat Asn tgg Trp tct Ser 730 gcg Ala cge gag Giu ctg Leu tgt Cys 635 gag Glu atg Met tgc Cys gaa Glu atc Ile 715 gcc Ala tgc Cys aac gee Al a cag Gin 620 gtg Val1 ctg Leu gc Ala ttc Phe gcc Al a 700 tcc Ser gta Val1 atc Ile ac a'ag Lys 605 ttc Phe ate Ile gag Giu ctg Leu ttc Phe 685 aag Lys tte Phe gag Glu ttc Phe atc ate Ile ctg Leu tgg Trp gat Asp ggc Gly 670 ttt Phe tte Phe att Ile gtg Val1 tte Phe 750 gag cee Pro ctg Leu etc Leu gag Glu 655 tte Phe gee Ala ate I le cca Pro att Ile 735 aac Asn gag ace Thr gt t Val tac Tyr 640 ate Ile ctg Leu ttc Phe ace Thr gee Ala 720 gee Ala aag Lys gtg Ile Tyr Ile Ile Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val WO 00/06601 17 755 760 cgt tgc agc acc gca gct cac gct ttc 2352 Arg Cys Ser Thr Ala Ala His Ala Phe 770 775 ctg cgc cgc agc aac gte tcc cgc aag 2400 Leu Arg Arg Ser Asn Val Ser Arg Lys 785 790 tcc acg gga tcc acc cct tcc tce tcc 2448 Ser Thr Gly Ser Thr Pro Ser Ser Ser 805 gaa gac cca ttc cca cag ccc gag agg 2496 Glu Asp Pro Phe Pro Gin Pro Giu Arg 820 825 gcc cta ace cag caa gag cag cag cag 2544 Ala Leu Thr Gin Gin Giu Gin Gin Gin 835 840 cag caa cga tct cag cag cag ccc aga 2592 Gin Gin Arg Ser Gin Gin Gin Pro Arg 850 855 ggc age ggc aeg gtc acc ttc tea etg 2640 Gly Ser Gly Thr Vai Thr Phe Ser Leu 865 870 aae gee atg gee cac agg aat tet acg 2688 Asn Ala Met Ala His Arg Asn Ser Thr 885 cag aaa age age gat aeg ctg ace ega 2736 Gin Lys Ser Ser Asp Thr Leu Thr Arg 900 905 cag tgc ggg gaa aeg gac tta gat ctg 2784 Gin Cys Gly Giu Thr Asp Leu Asp Leu 915 920 caa gga cct gtg ggt gga gac cag egg PCT/US99/17116 aag Lys egg Arg ate Ile 810 cag Gin cag Gin tge Cys age Ser eac His 890 eac His ace Thr cca gtg Vai tee Ser 795 age Ser aag Lys ccc Pro aag Lys ttt Phe 875 cag Gin cag Gin gte Val gag get Ala 780 age Ser age Ser eag Gin etg Leu cag Gin 860 gat Asp aac Asn eca Pro eag Gin gtg 765 gee Ala age Ser aag Lys eag Gin ace Thr 845 aag Lys gag Glu tee Ser tta Leu gaa Glu 925 gag egg Arg ett Leu age Ser eag Gin 830 etc Leu gte Va1 cct Pro ctg Leu etc Leu 910 aca Thr gac gee acg Ala Thr gga gge Gly Gly 800 aae age Asn Ser 815 ccg etg Pro Leu eca cag Pro Gin ate ttt Ile Phe eag aag Gin Lys 880 gag gee Glu Ala 895 ccg ctg Pro Leu ggt ctg Gly Leu cct gaa WO 00/06601 WO 0006601PCT/US99/17116 2832 Gin Gly 930 gag ttg 2880 Giu Leu 945 agt ggt 2922 Ser Gly Pro tcC Ser gga Gly Val1 cca Pro ggc Gly Gly gca Ala agc Ser 965 18 Gly Asp Gin Arg 935 Ctt gta gtg tcc Leu Val Val Ser 950 act gtt aca gaa Thr Val Thr Giu Pro Giu Val 940 agt tca cag Ser Ser Gin 955 aac gta gtg Asn Val Val 970 Giu Asp Pro Giu agc ttt gtc atc Ser Phe Val Ile 960 aat tca Asn Ser <210> <211> 974 <212> PRT <213> Homo sapiens <400> Met i Thr Ile Gin Phe Ile Ile Ser Cys Thr 145 Ala Ser Leu Asp Arg Asn Phe Phe Asn 130 Gly Phe Aia Giy Leu Gly Ser Asp Vai 115 Cys Ser Tyr Tyr Gly Lys Phe Ser Thr 100 Ala Ser Gly Ser 5 Gly Leu Ser Arg Pro Cys Gin Giu Val Cys Pro Phe Arg Trp 70 Ala Asn.

Asn His Ser 150 Cys Asp Pro Pro 55 Leu Leu Thr Lys Ile 135 Thr Trp Gin Ile 40 Giu Gin Leu Val Ile 120 Pro Ala Vai Arg 25 His Ser Al a Pro Ser 105 Asp Ser Val Leu 10 Ala Phe Val Met Asn 90 Lys Ser Thr Ala Leu Gin Gly Giu Ile 75 Leu Al a Leu Ile Asn.

155 Ala Lys Val Cys Phe Thr Leu Asn Ala 140 Leu Leu Lys Ala Ile Ala Leu Glu Leu 125 Val1 Leu Thr Giy Al a Arg Ile Gly Ala 110 Asp Val1 Giy Trp Asp Lys Tyr Glu Tyr Thr Giu Gly Leu His Ile Asp Asn Giu Arg Leu Phe Ala Phe 160 Tyr Ile Pro Gin Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn 165 170 175 WO 00/06601 WO 0006601PCT/US99/171 16 Lys Gin Val Lys 225 Giu Giu Gly Thr Ile 305 Ala Val Giu Met Tyr 385 Phe Leu Vai Giy Asn Aia Giy 210 Phe Leu Vali Pro Giy 290 Aia Leu His Giu Giy 370 Ser Lys Phe Pro Ile Gin Thr 195 Thr Arg Ile Ile Asp 275 Lys Met Lys Pro Thr 355 Giu Ile Giu Ile Phe 435 Ile Phe 180 Aia Ile Giu Ser Gin 260 Leu Ile Pro Ala Arg 340 Phe Gin Ile Val Asn 420 Ser Giu Lys Met Ala Giu Gin 245 Asn Giu Trp Gin Giy 325 Lys Asn Val Asn Gly 405 Giu Asn Gly Ser Ala Ala Aia 230 Tyr Ser Pro Leu Tyr 310 Gin Ser Cys Thr Trp 390 Tyr Giu Cys Giu Phe Asp Asp 215 Giu Ser Thr Leu Ala 295 Phe Ile Val His Phe 375 His Tyr Lys Ser Pro Leu Arg 185 Ile Ile 200 Asp Asp Giu Arg Asp Giu Ala Lys 265 Ile Lys 280 Ser Giu His Vai Pro Gly His Asn 345 Leu Arg 360 Asp Glu Leu Ser Asn Val Ile Leu 425 Arg Asp 440 Thr Cys Thr GiU Tyr Asp Giu 250 Val Giu Ala Vai Phe 330 Giy His Cys Pro Tyr 410 Trp Cys Cys Ile Tyr Giy Ile 235 Giu Ile Ile Trp Gly 315 Arg Phe Leu Gly Glu 395 Al a Ser Leu Phe Pro Phe Arg 220 Cys Ile Val Val Ala 300 Gly Glu Ala Asn Asp 380 Asp Lys Gly Ala Giu Asn Arg 205 Pro Ile Gin Val1 Arg 285 Ser Thr Phe Lys Phe 365 Leu Giy Lys Phe Gly 445 Cys Asp 190 Trp, Gly Asp His Phe 270 Arg Ser Ile Leu Giu 350 Thr Val Ser Gly Ser 430 Thr Val Giu His Asn Trp Ile Glu Phe Ser 240 Val Vai 255 Ser Ser Asn Ile Ser Leu Gly Phe 320 Lys Lys 335 Phe Trp Asn Asn Gly Asn Ile Vai 400 Giu Arg 415 Arg Giu Arg Lys Giu Cys WO 00/06601 450 Pro Asp Gly 465 Cys Pro Asp Lys Glu Ile Thr Leu Phe 515 Val Phe Ile 530 Glu Leu Ser 545 Ser Leu Phe Gin Pro Ala Val Lys Thr 595 Ser Phe His 610 Phe Leu Cys 625 Thr Ala Pro Ile Phe Ile Ile Gly Tyr 675 PCT/US99/17116 Glu Tyr Ser 470 Asp Phe Trp 485 Glu Phe Leu 500 Ala Val Leu Lys Phe Arg Tyr Leu Leu 550 Phe Ile Gly 565 Phe Gly Ile 580 Asn Arg Val Arg Lys Trp Thr Phe Met 630 Pro Ser Ser 645 Thr Cys His 660 Thr Cys Leu 455 Asp Glu Thr Asp Ala 475 Ser Asn Glu Asn His 490 Ser Trp Thr Glu Pro 505 Gly Ile Phe Leu Thr 520 Asn Thr Pro Ile Val 535 Leu Phe Ser Leu Leu 555 Glu Pro Gin Asp Trp 570 Ser Phe Val Leu Cys 585 Leu Leu Val Phe Glu 600 Trp Gly Leu Asn Leu 615 Gin Ile Val Ile Cys 635 Tyr Arg Asn Gin Glu 650 Glu Gly Ser Leu Met 665 Leu Ala Ala Ile Cys 680 Glu Asn Phe Asn Glu 695 Phe Ile Val Trp Ile 715 460 Ser Ala Cys Asn Lys 480 Thr Ser Phe Gly Ala Phe 525 Lys Ala 540 Cys Cys Thr Cys Ile Ser Ala Lys 605 Gin Phe 620 Val Ile Leu Glu Ala Leu Phe Phe 685 Ala Lys 700 Ser Phe Cys Ile Ala 495 Ile Ala Leu 510 Val Leu Gly Thr Asn Arg Phe Ser Ser 560 Arg Leu Arg 575 Cys Ile Leu 590 Ile Pro Thr Leu Leu Val Trp Leu Tyr 640 Asp Glu Ile 655 Gly Phe Leu 670 Phe Ala Phe Phe Ile Thr Ile Pro Ala 720 Lys Phe 705 Ser Arg Lys Leu Pro 690 Ser Met Leu Ile Phe 710 Tyr Ala Ser Thr Tyr Gly 725 Lys Phe Val Ser 730 Ala Val Glu Val Ile Ala 735 WO 00/06601 WO 0/0601PCT[US99/17116.

Ile Ile Arg Leu 785 Ser Giu Ala Gin Giy 865 Asn Gin Gin Gin Giu 945 Ser Leu Tyr cys 770 Arg Thr Asp Leu Gin 850 Ser Ala Lys Cys Gly 930 Leu Gly Al a Ile 755 Ser Arg Giy Pro Thr 835 Arg Gly Met Ser Gly 915 Pro Ser Gly Ala Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys 740 Ile Thr Ser Ser Phe 820 Gin Ser Thr Ala Ser 900 Giu Val Pro Leu Ala Asn Thr 805 Pro Gin Gin Val1 His 885 Asp Thr Gly Ala Phe Ala Vai 790 Pro Gin Giu Gin Thr 870 Arg Thr Asp Gly Leu 950 Lys His 775 Ser Ser Pro Gin Gin 855 Phe Asn Leu Leu Asp 935 Val1 Pro 760 Ala Arg Ser Giu Gin 840 Pro Ser Ser Thr Asp 920 Gin Val1 745 Ser Phe Lys Ser Arg 825 Gin Arg Leu Thr Arg 905 Leu Arg Ser Arg Lys Arg Ile 810 Gin Gin Cys Ser His 890 His Thr Pro Ser 750 Asn Val Ser 795 Ser Lys Pro Lys Phe 875 Gin Gin Val Giu Ser 955 Thr Ala 780 Ser Ser Gin Leu Gin 860 Asp Asn Pro Gin Val 940 Gin Ile 765 Aia Ser Lys Gin Thr 845 Lys Giu Ser Leu Giu 925 Giu Ser Giu Arg Leu Ser Gin 830 Leu Vai Pro Leu Leu 910 Thr Asp Phe Giu Al a Gly Asn 815 Pro Pro Ile Gin Glu 895 Pro Gly Pro Val1 Val1 Thr Gly 800 Ser Leu Gin Phe Lys 880 Al a Leu Leu Glu Ile 960 Gly Ser Thr Val Thr Glu Asn Val Val Asn Ser 965 970 <210> 11 <211> 3234 <212> DNA <213> Homo sapiens <220> <221> CDS WO 00/06601 <222> -(3234) PCTIUS99/17116 <400> 11 atg Met 1 acc Thr at c 144 Ile caa 192 Gin ttc 240 Phe ata 288 Ile ata 336 Ile agt 384 Ser tgc 432 Cys act 480 Thr 145 gca Ala tct Ser ctt Leu gat Asp cgt Arg aac Asn ttt Phe ttt Phe aac Asn 130 ggC ttt Phe gcc Ala ggg Gly ctc Leu ggg Gly agc Ser gac Asp gtt Vai 115 tgc Cys tca tat Tyr tac Tyr ggg Gly aaa Lys ttt Phe agc Ser act Thr 100 gct Ala tca Ser ggc agc Ser 5 ggg Gly ctc Leu tca Ser cgc Arg cca Pro tgc Cys caa Gin gag Giu gtc tgc tgc tgg gtc ctc ttg gca ctc acc tgg cac Cys cca Pro ttt Phe agg Arg t gg Trp 70 gcc Ala aac Asn aac Asn cac His tcc Ser 150 Cys gac Asp cct Pro ccg Pro 55 tta Leu ctt Leu acc Thr aaa Lys at t Ile 135 acg Trp cag Gin att Ile 40 gag Glu cag Gin ctt Leu gtt Val att Ile 120 ccc Pro gca Val cga Arg 25 cat His tct Ser gct Ala ccc Pro tct Ser 105 gat Asp tct Ser gtg Leu 10 gcc Ala ttt Phe gtg Val atg Met aac Asn 90 aag Lys t ct Ser acg Thr gca Leu caa Gin gga Gly gaa Glu ata Ile 75 ttg Leu gcc Ala ttg Leu att Ile aat Asn 155 Ala aag Lys gta Val1 tgt Cys ttt Phe acg Thr ttg Leu aac Asn gct Ala 140 ctg Leu aag Lys gca Ala atc Ile gcc Ala ctg Leu gaa Giu ctt Leu 125 gtg Val1 ctg Thr Trp ggg gac Gly Asp gct aaa Ala Lys agg tat Arg Tyr ata gag Ile Giu gga tac Gly Tyr gcc acc Ala Thr 110 gat gag Asp Giu gtg gga Val Gly ggg ctc His att Ile gat Asp aat Asn gag Giu agg Arg ctg Leu ttc Phe gca Al a ttc Phe 160 Gly Ser Gly Val Thr Ala Val Ala Leu Leu Gly Leu WO 00/06601 WO 0006601PCT[US99/1 7116 tac att 528 Tyr Ile aag aat 576 Lys Asn cag gcc 624 Gin Ala gtg ggc 672 Vai Gly 210 aaa tte 720 Lys Phe 225 gaa ctc 768 Giu Leu gag gtg 816 Giu Vai ggc cca 864 Giy Pro acg ggc 912 Thr Gly 290 atc gcc 960 Ile Ala 305 gct ctg 1008 ccc Pro caa Gin act Thr 195 aca Thr cga Arg atc Ile att Ile gat Asp 275 aag Lys atg Met aag cag Gin ttc Phe 180 gcc Ala att Ile gag Giu tcc Ser caa Gin 260 ctt Leu atc Ile cct Pro gct gtc Vali 165 aag Lys atg Met gca Ala gaa Giu cag Gin 245 aat Asn gag Giu tgg Trp cag Gin ggg agt Ser tet Ser gca Ala gct Ala get Ala 230 tac Tyr tcc Ser ccc Pro ctg Leu tac Tyr 310 cag tat Tyr ttc Phe gac Asp gat Asp 215 gag Giu tct Ser acg Thr ctc Leu gcc Ala 295 ttc Phe atc gcc Aia ctc Leu ate Ile 200 gac Asp gaa Giu gat Asp gcc Al a atc Ilie 280 agc Ser cac His cca tcc Se r cga Arg 185 at c Ile gac Asp agg Arg gag Giu aaa Lys 265 aag Lys gag Giu gtg Val ggc tcc Ser 170 ac Thr gag Giu tat Tyr gat Asp gaa Giu 250 gtc Val gag Giu gcc Ala gtt Val ttc agc Ser atc Ile tat Tyr ggg Gly ate Ile 235 gag Glu atc Ile att Ile tgg Trp gge Gly 315 cgg aga Arg ccc Pro ttc Phe cgg Arg 220 tgc Cys ate Ile gtg Val1 gtc Vai gcc Ala 300 gge Gly gaa etc Leu aat Asn cgc Arg 205 ceg Pro ate Ile cag Gin gtt Val egg Arg 285 agc Se r ace Thr ttc etc Leu gat Asp 190 tgg Trp ggg Giy gae Asp eat His ttc Phe 270 cgc Arg tee Ser att Ile ctg agc Ser 175 gag Giu aac Asn at t Ile ttc Phe gtg Vai 255 tcc Ser aat Asn tcc Ser gga Giy aag aae Asn cac His tgg Trp gag Giu agt Ser 240 gta Vai agt Ser ate Ile etg Leu ttc Phe 320 aag Aia Leu Lys Ala Gly Gin Ile Pro Giy Phe Arg Glu Phe Leu Lys Lys WO 00/06601 WO 0006601PCT/US99/171 16 gtc cat 1056 Val His gaa gaa 1104 Glu Giu cct gtg 1152 Pro Val 370 agc aac 1200 Ser Asn 385 ato agc 1248 Ile Ser tcc tac 1296 Ser Tyr gat ata 1344 Asp Ile tgt gca 1392 Cys Ala 450 cgg cat 1440 Arg His 465 gag tgt 1488 Glu Cys tcc oca coo Pro aca Thr 355 gao Asp ago Ser agt Ser aat Asn tat Tyr 435 gao Asp ota Leu ggt Gly gag agg Arg 340 ttt Phe aco Thr tog Ser gtc Val gtg Val 420 aco Thr at o Ile aao Asn gao Asp gat 325 aag Lys aac Asn ttt Phe aoa Thr gag Glu 405 tao Tyr tgo Cys aag Lys ttt Phe otg Leu 485 ggo tot gto Ser Val tgo cac Cys His otg aga Leu Arg 375 goo tto Ala Phe 390 aoo cot Thr Pro tta gca Leu Ala tta cot Leu Pro aaa gtt Lys Val 455 aoa aac Thr Asn 470 gtg ggg Val Gly too ato cac His oto Leu 360 ggt Gly cga Arg tao Tyr gto Val ggg Gly 440 gag Glu aat Asn aac Asn gtg aat Asn 345 caa Gin cac His coo Pro ata Ile tao Tyr 425 aga Arg gog Ala atg Met tat Tyr ttt 330 ggt Gly gaa Glu gaa Glu oto Leu gat Asp 410 too Ser ggg Gly tgg Trp ggg Gly too Ser 490 aag ttt Phe ggt Gly gaa Glu tgt Cys 395 tao Tyr att Ile oto Leu cag Gin gag Glu 475 ato Ile gaa gc Ala gca Ala agt Ser 380 aca Thr aog Thr gc Al a tto Phe gto Val 460 cag Gin at c Ile gtc aag Lys aaa Lys 365 ggc Gly ggg Gly oat His cac His aoo Thr 445 otg Leu gtg Val aao Asn ggg gag Glu 350 gga Gly gao Asp gat Asp tta Leu goc Aia 430 aa t Asn aag Lys aco Thr tgg Trp tat 335 ttt Phe cot Pro agg Arg gag Giu Arg 415 ttg Leu ggo Gly cac His ttt Phe cac His 495 tao tgg Trp tta Leu ttt Phe aac Asn 400 ata Ile caa Gin too Ser ota Leu gat Asp 480 oto Leu aac WO 00/06601 WO 0006601PCTIUS99/17116 1536 Ser Pro gtc tat 1584 Val Tyr ctg tgg 1632 Leu Trp 530 gac tgc 1680 Asp Cys 545 tgc tgc 1728 Cys Cys aca gat 1776 Thr Asp gag aac 1824 Giu Asn acg gag 1872 Thr Giu 610 ttc ctg 1920 Phe Leu 625 ccc att 1968 Pro Ile tcc ctg 2016 Ser Leu Glu gcc Ala 515 agt Ser ctg Leu ttt Phe gcc Al a cac His 595 ccc Pro aca Thr gtc Val ctc Leu Asp Gly Ser Ile Val 500 aag Lys ggg Gly gca Ala gag Glu agt Ser 580 acc Thr ttt Phe gcc Ala aag Lys tgc Cys 660 aag Lys ttc Phe ggg Gly tgt Cys 565 gcc Ala tcc Ser ggg Gly ttt Phe gcc Ala 645 tgc Cys gga Gly tcc Ser acc Thr 550 gtg Val tgt Cys tgc Cys atc Ile gtg Val 630 acc Thr ttc Phe gaa Glu agg Arg 535 agg Arg gag Glu aac Asn att Ile gca Ala 615 ctg Leu aac Asn tcc Ser aga Arg 520 gag Giu aaa Lys tgt Cys aag Lys gcc Al a 600 ctc Leu ggt Gly cga Arg agc Ser Phe 505 ctc Leu gtg Val ggg Gly cct Pro tgc Cys 585 aag Lys ac Thr gtg Val1 gag Glu tcc Ser 665 Lys ttc Phe ccc Pro atc Ile gat Asp 570 cca Pro gag Glu ctc Leu ttt Phe ctc Leu 650 ctq Leu Glu atc Ile ttc Phe att Ile 555 ggg Gly gat Asp atc Ile ttt Phe atc Ile 635 tcc Ser ttc Phe Val aac Asn t cc Ser 540 gag Giu gag Giu gac Asp gag Giu gcc Ala 620 aag Lys tac Tyr ttc Phe Gly gag Glu 525 aac Asn ggg Gly tat Tyr ttc Phe ttt Phe 605 gtg Val ttc Phe ctc Leu atc Ile Tyr 510 gag Glu tgc Cys gag Giu agt Ser tgg Trp 590 ctg Leu ctg Leu cgc Arg ctc Leu ggg Gly 670 Tyr aaa Lys agc Ser ccc Pro gat Asp 575 tcc Ser t cg Ser ggc Gly aac Asn ctc Leu 655 gag Glu Asn atc Ile cga Arg acc Thr 560 gag Glu aat Asn tgg Trp att Ile aca Thr 640 ttc Phe ccc Pro WO 00/06601 WO 0006601PCT/US99/171 16 cag gac tgg 2064 Gin Asp Trp 675 gtg ctc tgc 2112 Val Leu Cys 690 gtg ttt gag 2160 Val Phe Giu 705 ctc aac ctg 2208 Leu Asn Leu gtc atc tgt 2256 Vai Ile Cys aac cag gag 2304 Asn Gin Glu 755 tcc ctc atg 2352 Ser Leu Met 770 gcc atc tgc 2400 Aia Ile Cys 785 ttc aat gaa 2448 Phe Asn Giu gtc tgg atc 2496 Vai Trp Ile gtc tct gcc 2544 acg Thr atc Ile gcc Ala c ag Gin gtg Val1 740 ctg Leu gcc Ala ttc Phe gc Ala tcc Ser 820 gta tgc Cys tca Ser aag Lys ttc Phe 725 at c Ile gag Giu ctg Leu ttc Phe aag Lys 805 ttc Phe gag cgc Arg tgc Cys atc Ile 710 ctg Leu tgg Trp gat Asp ggc Gly ttt Phe 790 ttc Phe att Ile gtg ctg Leu atc Ile 695 ccc Pro ctg Leu ctc Leu gag Giu ttc Phe 775 gcc Aia atc Ile cca Pro att cgc Arg 680 ctg Leu acc Thr gtt Val tac Tyr atc Ile 760 ctg Leu ttc Phe acc Thr gcc Ala gcc cag Gin gtg Val agc Ser ttc Phe acc Thr 745 atc Ile atc Ile aag Lys ttc Phe tat Tyr 825 atc ccg Pro aaa Lys ttc Phe ctc Leu 730 gcg Ala ttc Phe ggC Gly tcc Ser agc Ser 810 gcc Aia ctg gc Al a ac Thr cac His 715 tgc Cys ccc Pro atc Ile tac Tyr cgg Arg 795 atg Met agc Ser gca ttt Phe aac Asn 700 cgc Arg acc Thr ccc Pro acg Thr acc Thr 780 aag Lys ctc Leu acc Thr gcc ggc Gly 685 cgt Arg aag Lys ttc Phe t ca Ser tgc Cys 765 tgc Cys ctg Leu at c Ile tat Tyr agc atc Ile gtc Val1 t99 Trp atg Met agc Ser 750 cac His ctg Leu ccg Pro ttc Phe ggC Gly 830 ttt agc Ser ctc Leu t gg Trp cag Gin 735 tac Tyr gag Giu ctg Leu gag Giu ttc Phe 815 aag Lys 9gC ttc Phe ctg Leu ggg Giy 720 att Ile cgc Arg ggc Gly gct Ala aac Asn 800 atc Ile ttt Phe ttg WO 00/06601 WO 0006601PCTIUS99/1 7116 Val Ser otg gcg 2592 Leu Ala 850 tcc ogo 2640 Ser Arg 865 ttc aag 2688 Phe Lys aag cgg 2736 Lys Arg tcc ato 2784 Ser Ile agg cag 2832 Arg Gin 930 cag cag 2880 Gin Gin 945 aga tgc 2928 Arg Cys ctg ago 2976 Leu Ser aog cac 3024 Ala 835 tgc Cys aac Asn gtg Val1 too Ser ago Ser 915 aag Lys 000 Pro aag Lys ttt Phe cag Val1 atc Ile ace Thr got Ala ago Ser 900 ago Ser cag Gin otg Leu oag Gin gat Asp 980 aa o Giu tto Phe ato Ile goo Aia 885 ago Ser aag Lys oag Gin aco Thr aag Lys 965 gag Giu too Vai tto Phe gag Giu 870 ogg Arg ott Leu ago Ser oag Gin oto Leu 950 gto Val1 oot Pro otg Ile aa o Asn 855 gag Giu goo Ala gga Gly aao Asn cog Pro 935 oca Pro ato Ile cag Gin gag Ala 840 aag Lys gtg Val1 aog Thr ggo Gly ago Ser 920 otg Leu cag Gin ttt Phe aag Lys gco Ile at o Ile ogt Arg otg Leu too Ser 905 gaa.

Giu gc Al a cag Gin ggc Gly aac Asn 985 cag Leu Ala tao ato Tyr Ile tgo ago Cys Ser 875 ogo ogo Arg Arg 890 aog gga Thr Gly gao oca Asp Pro ota aco L eu Thr caa oga Gin Arg 955 ago ggc Ser Gly 970 goc atg Ala Met aaa ago Ala att Ile 860 aco Thr ago Ser too Ser tto Phe cag Gin 940 tot Ser acg Thr gc Ala ago Ser 845 oto Leu gca Ala aac Asn aco Thr oca Pro 925 caa Gin cag Gin gto Val cac His gat Phe tto Phe got Ala gt o Val1 cot Pro 910 oag Gin gag Giu oag Gin aco Thr agg Arg 990 acg Gly aag Lys cac His too Ser 895 too Ser coo Pro cag Gin cag Gin tto Phe 975 aat Asn otg Leu oca Pro got Ala 880 ego Arg too Ser gag Giu cag Gin 000 Pro 960 t oa Ser tot Ser ac Thr His Gin Asn Ser Leu Giu Ala Gin Lys Ser Ser Asp Thr Leu Thr 1005 995 1000 WO 00/06601 WO 0006601PCTJUS99/17116 cga cac cag cca tta ctc cog ctg cag tgc ggg gaa 3072 Arg His Gin Pro Leu Leu Pro Leu Gin Cys Giy Glu 1010 1015 1020 ctg acc gtc cag gaa aca ggt ctg caa gga cct gtg 3120 Leu Thr Vai Gin Giu Thr Gly Leu Gin Gly Pro Val 1025 1030 1035 cgg cca gag gtg gag gac oct gaa gag ttg too oca 3168 Arg Pro Giu Val Giu Asp Pro Giu Giu Leu Ser Pro 1045 1050 tcc agt tca cag agc ttt gtc atc agt. ggt gga ggc 3216 Ser Ser Ser Gin Ser Phe Val Ile Ser Gly Gly Gly 1060 1065 gaa aac gta gtg aat tca 3234 Glu Asn Val Val Asn Ser 1075 acg gac tta gat Thr Asp Leu Asp ggt gga gac cag Gly Gly Asp Gin 1040 gca ctt gta gtg Ala Leu Val Val 1055 ago act gtt aca Ser Thr Val Thr 1070 <210> 12 <211> 1078 <212> PRT <213> Homo sapiens <400> 12 Met Ala Phe Tyr Ser Cys Cys Trp Vai Leu Leu Ala Leu Thr Trp, His 1 5 10 Thr Ser Ala Tyr Gly Pro Asp Gin Arg Ala Gin Lys Lys Gly Asp Ile 25 Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp 40 Gin Asp Leu Lys Ser Arg Pro Giu Ser Val Giu Cys Ile Arg Tyr Asn 55 Phe Arg Gly Phe Arg Trp, Leu Gin Ala Met Ile Phe Ala Ile Glu Giu 70 75 Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg 90 Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Giu Ala Thr Leu 100 105 110 WO 00/06601 PCT/US99/17116 29 Ser Phe Val Ala Gin Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe 115 120 125 Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala 130 135 140 Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe 145 150 155 160 Tyr Ile Pro Gin Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn 165 170 175 Lys Asn Gin Phe Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His 180 185 190 Gin Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe Arg Trp Asn Trp 195 200 205 Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu 210 215 220 Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser 225 230 235 240 Glu Leu Ile Ser Gin Tyr Ser Asp Glu Glu Glu Ile Gin His Val Val 245 250 255 Glu Val Ile Gin Asn Ser Thr Ala Lys Val Ile Val Val Phe Ser Ser 260 265 270 Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val Arg Arg Asn Ile 275 280 285 Thr Gly Lys Ile Trp Leu Ala Ser Glu Ala Trp Ala Ser Ser Ser Leu 290 295 .300 Ile Ala Met Pro Gin Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe 305 310 315 320 Ala Leu Lys Ala Gly Gin Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys 325 330 335 Val His Pro Arg Lys Ser Val His Asn Gly Phe Ala Lys Glu Phe Trp 340 345 350 Glu Glu Thr Phe Asn Cys His Leu Gin Glu Gly Ala Lys Gly Pro Leu 355 360 365 Pro Val Asp Thr Phe Leu Arg Gly His Glu Glu Ser Gly Asp Arg Phe 370 375 380 Ser Asn Ser Ser Thr Ala Phe Arg Pro Leu Cys-Thr Gly Asp Glu Asn 385 390 395 400 WO 00/06601 WO 0006601PCT/US99/171 16 Ile Ser Asp Cys Arg 465 Glu Ser Val Leu Asp 545 Cys Thr Glu Thr Phe 625 Pro Ser Ser Ser Tyr Asn Ile Tyr 435 Ala Asp 450 His Leu Cys Gly Pro Giu Tyr Ala 515 Trp Ser 530 Cys Leu Cys Phe Asp Ala Asn His 595 Giu Pro 610 Leu Thr Ile Val Leu Leu Val1 Val 420 Thr Ile Asn Asp Asp 500 Lys Gly Ala Giu Ser 580 Thr Phe Al a Lys Cys 660 Giu 405 Tyr Cys Lys Phe Leu 485 Gly Lys Phe Gly Cys 565 Ala Ser Gly Phe Ala 645 Cys Thr Leu Leu Lys Thr 470 Vai Ser Giy Ser Thr 550 Val Cys Cys Ile Val 630 Thr Phe Pro Al a Pro Vai 455 Asn Gly Ile Glu Arg 535 Arg Giu Asn Ile Ala 615 Leu Asn Ser Tyr Val Giy 440 Giu Asn Asn Val1 Arg 520 Giu Lys Cys Lys Ala 600 Leu Gly Arg Ser Ile Tyr 425 Arg Ala Met Tyr Phe 505 Leu Val1 Gly Pro Cys S55 Lys Thr Val Giu Ser 665 Asp 410 Ser Gly Trp, Gly Ser 490 Lys Phe Pro Ile Asp 570 Pro Giu Leu Phe Leu 650 Leu Tyr Ile Leu Gin Giu 475 Ile Giu Ile Phe Ile 555 Giy Asp Ile Phe Ile 635 Ser Phe Thr Ala Phe Val 460 Gin Ile Val Asn Ser 540 Giu Giu Asp Giu Ala 620 Lys Tyr Phe His His Thr 445 Leu Vai Asn Gly Giu 525 Asn Gly Tyr Phe Phe 605 Val1 Phe Leu Ile Leu Ala 430 Asn Lys Thr Trp Tyr 510 Glu Cys Giu Ser Trp, 590 Leu Leu Arg Leu Gly 670 Arg 415 Leu Gly His Phe His 495 Tyr Lys Ser Pro Asp 575 Ser Ser Gly Asn Leu 655 Giu Ile Gin Ser Leu Asp 480 Leu Asn Ile Arg Thr 560 Giu Asn Trp Ile Thr 640 Phe Pro Gin Asp Trp Thr Cys Arg Leu Arg Gin Pro Ala Phe Gly Ile Ser Phe WO 00/06601 WO 0006601PCT/US99/171 16 Val1 Val 705 Leu Val Asn Ser Ala 785 Phe Val Val Leu Ser 865 Phe Lys Ser Arg Leu 690 Phe Asn Ile Gin Leu 770 Ile Asn Trp Ser Ala 850 Arg Lys Arg Ile Gin 930 675 Cys Glu Leu Cys Glu 755 Met Cys Glu Ile Ala 835 Cys Asn Vai Ser Ser 915 Lys Ile Ala Gin Val 740 Leu Ala Phe Ala Ser 820 Val Ile Thr Ala Ser 900 Ser Gin Ser Lys Phe 725 Ile Glu Leu Phe Lys 805 Phe Giu Phe Ile Ala 885 Ser Lys Gin Cys Ile 710 Leu Trp Asp Gly Phe 790 Phe Ile Val Phe Giu 870 Arg Leu Ser Gin Ile 695 Pro Leu Leu Glu Phe 775 Ala Ile Pro Ile Asn 855 Glu Ala Gly Asn Pro 935 680 Leu Thr Vai Tyr Ile 760 Leu Phe Thr Ala Al a 840 Lys Val Thr Gly Ser 920 Leu 685 Val Ser Phe Thr 745 Ile Ile Lys Phe Tyr 825 Ile Ile Arg Leu Ser 905 Giu Ala Lys Thr Phe Leu 730 Ala Phe Gly Ser Ser 810 Ala Leu Tyr Cys Arg 890 Thr Asp Leu His 715 Cys Pro Ile Tyr Arg 795 Met Ser Ala Ile Ser 875 Arg Gly Pro Thr Asn Arg 700 Arg Lys Thr Phe Pro Ser Thr Cys 765 Thr Cys 780 Lys Leu Leu Ile Thr Tyr Ala Ser 845 Ile Leu 860 Thr Ala Ser Asn Ser Thr Phe Pro 925 Gin Gin 940 Val Trp Met Ser 750 His Leu Pro Phe Gly 830 Phe Phe Ala Val Pro 910 Gin Giu Leu Trp Gin 735 Tyr Giu Leu Giu Phe Lys Gly Lys His Ser 895 Ser Pro Gin Leu Gly 720 Ile Arg Gly Ala Asn 800 Ile Phe Leu Pro Ala 880 Arg Ser Giu Gin Gin Gin Pro Leu Thr Leu Pro Gin Gin Gin Arg Ser Gin Gin Gin Pro 945 950 955 960 WO 00/06601 Arg Cys Lys Gin Lys Val Ile Phe 965 Leu Ser Phe Asp Giu Pro Gin Lys 980 Thr His Gin Asn Ser Leu Giu Ala 995 1000 Arg His Gin Pro Leu Leu Pro Leu 1010 1015 Leu Thr Vai Gin Giu Thr Gly Leu 1025 1030 Arg Pro Giu Val Glu Asp Pro Giu 1045 Ser Ser Ser Gin Ser Phe Val Ile 1060 Glu Asn Vai Val Asn Ser 1075 PCT/US99/17116.

Giy Ser Gly Thr Val 970 Asn Aia Met Aia His 985 Gin Lys Ser Ser Asp 1005 Gin Cys Giy Giu Thr 1020 Gin Gly Pro Vai Giy 1035 Glu Leu Ser Pro Aia 1050 Ser Giy Giy Giy Ser 1065 Thr Phe Ser 975 Arg Asn Ser 990 Thr Leu Thr Asp Leu Asp Gly Asp Gin 1040 Leu Val Val 1055 Thr Val Thr 1070

Claims (31)

1. An isolated nucleic acid encoding an isoform of a human calcium sensing receptor, wherein the isolated nucleic acid comprises a deletion of nucleotides when compared to the wild-type form of the receptor comprising a DNA sequence as depicted in SEQ ID NO:11, wherein the deletion comprises: a) nucleotides 1075-1386 of SEQ ID NO:11; b) nucleotides 1378-1608 of SEQ ID NO:11; or c) nucleotides 1075-1608 of SEQ ID NO:11.

2. The isolated nucleic acid according to claim 1, having at least one property selected from a) it can be amplified by polymerase chain reaction (PCR) using an oligonucleotide primer derived from SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11; b) it hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence as depicted in SEQ ID NO:7 or SEQ ID NO:9; and c) it encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:8, and SEQ ID

3. An isolated nucleic acid which encodes an isoform of a human calcium sensing receptor which comprises an amino acid sequence as depicted in SEQ ID NO:8 or SEQ ID

4. The isolated nucleic acid of claim 1 comprising a nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:9.

5. The isolated nucleic acid of claim 1 wherein the nucleic acid can be amplified by polymerase chain reaction (PCR) using an oligonucleotide primer selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6. oo

6. A vector comprising the nucleic acid of claim 1.

7. The vector according to claim 6 wherein the nucleic acid is operatively associated with an expression control sequence permitting expression of the receptor in an expression competent host cell.

8. The vector according to claim 7 selected from the group consisting of an RNA molecule, a plasmid DNA molecule, and a viral vector.

9. The vector according to claim 8 which is a plasmid DNA molecule.

The vector according to claim 8 which is a viral vector selected from the group consisting of a retrovirus, adeno-associated virus, herpes virus, and vaccinia virus.

11. A host cell transfected with the vector of claim 6.

12. A host cell transfected with the vector of claim 7.

13. The host cell of claim 11 selected from the group consisting of a bacterial cell, a yeast cell and a mammalian cell.

14. A method for expressing an isoform of human calcium sensing receptor S. comprising: a) culturing the host cell of claim 12 in culture medium under conditions permitting expression of the receptor; and b) identifying cells expressing the receptor on their surface.

An isolated isoform of a human calcium sensing receptor, wherein the isoform comprises a deletion of amino acids when compared to the wild-type form of the receptor comprising an amino acid sequence as depicted in SEQ ID NO:12, wherein the deletion comprises: a) amino acids 358-462 of SEQ ID NO:12; b) amino acids 460-536 of SEQ ID NO:12; or c) amino acids 358-536 of SEQ ID NO:12. 44

16. An isolated isoform of a human calcium sensing receptor which comprises an amino acid sequence as depicted in SEQ ID NO:8 or SEQ ID

17. A method of screening for agonists or antagonists of a CaSR isoform activity, the method comprising incubating a test sample with a CaSR isoform of Claim 15, measuring the CaSR isoform activity and comparing the activity to that in the absence of the test sample.

18. The method according to claim 17, wherein the CaSR activity is its ability to influence intracellular calcium concentration.

19. The method according to claim 17, wherein the test sample is tested alone, in conjunction with an elevation in extracellular calcium concentration, or in the presence of other agonists or antagonists of CaSR isoform activity.

The method according to claim 18, wherein the intracellular calcium concentration is measured with a fluorescent indicator.

21. The method according to claim 20, wherein the indicator is fura-2.

22. A method of increasing or decreasing the level of CaSR activity in a cell, the method comprising administering to the cell a nucleic acid capable of increasing or decreasing activity of a CaSR comprising an amino acid sequence of SEQ ID NO:8 or SEQ ID

23. The method according to claim 22, wherein the cells are in a patient suffering from a disease or disorder associated with abnormal calcium levels.

24. The method according to Claim 23, wherein the disease is hyperparathyroidism or osteoporosis.

The method according to claim 23, wherein the disease is Paget's disease, hypercalcemia malignancy or hypertension.

26. The method according to claim 23, wherein the nucleic acid is in a vector.

27. The method according to claim 26, wherein said vector is selected from the group consisting of plasmids, retroviruses, herpes simplex viruses, adeno- associated viruses, adenoviruses, and vaccinia viruses.

28. The method according to claim 22, wherein the nucleic acid encodes an antibody having an isoform of a CaSR comprising an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:10 as an immunogen.

29. The method according to claim 22, wherein said nucleic acid encodes an antisense molecule complementary to the isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:9 and is capable of selectively inhibiting the expression of said sequence.

The method according to claim 29, wherein said antisense molecule is at least about 20 nucleotides in length.

31. An antibody that specifically recognizes an isoform of a CaSR that comprises an amino acid sequence of SEQ ID NO:8 or SEQ ID NO:10, and does not bind the wild-type CaSR. S: "DATED this 2 nd day of September 2003 AVENTIS PHARMACEUTICALS INC WATERMARK PATENT TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA P19000AU P19000AU00 KJS/KMH/MEH 9 *eo* eo 0•