WO2000039277A2 - Ndsp nucleic acids and polypeptides - Google Patents
Ndsp nucleic acids and polypeptides Download PDFInfo
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- WO2000039277A2 WO2000039277A2 PCT/US1999/030744 US9930744W WO0039277A2 WO 2000039277 A2 WO2000039277 A2 WO 2000039277A2 US 9930744 W US9930744 W US 9930744W WO 0039277 A2 WO0039277 A2 WO 0039277A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- Prostate cancer is the most commonly diagnosed cancer and the second most common cause of death from cancer in American men.
- Prostate cancer cells often initially rely on androgen (e.g., testosterone) for their growth and maintenance. Therefore, androgen withdrawal, by castration or through the use of an anti-androgenic drug, is a common treatment for prostate cancer.
- prostate cancer patients develop androgen-independent prostate cancer so that androgen withdrawal treatment is no longer effective.
- the complex process of prostate tumor growth and development involves multiple gene products. Therefore, it is important to identify genes involved in tumor development, growth, and androgen dependence, particularly those genes and gene products that can serve as targets for the diagnosis, prevention, and treatment of prostate cancer.
- the present invention is based, at least in part, on the discovery of a cDNA molecule encoding a novel protein called Nuclear Dual Specificity Phosphatase-Like Protein (NDSP) .
- NDSP Nuclear Dual Specificity Phosphatase-Like Protein
- This protein, fragments, derivatives, and variants thereof are collectively referred to as “polypeptides of the invention” or “proteins of the invention.”
- Nucleic acid molecules encoding polypeptides of the invention are collectively referred to as “nucleic acids of the invention.”
- the NDSP was initially identified as a gene that is expressed at a higher level in prostate cancer cells exposed to androgen than in prostate cancer cells exposed to an anti-androgen, casodex.
- NDSP is not expressed in LN3 LNCaP cells, and androgen independent prostate cancer cell line, or C R22R prostate cancer xenografts, which are also androgen independent. Because NDSP is not expressed in certain androgen independent prostate cancers, but is expressed in an androgen dependent prostate cancer, it can serve as a diagnostic marker.
- NDSP expression at the mRNA or protein level
- activity in a sample of prostate cancer cells determine whether the prostate cancer is androgen dependent or androgen independent. If the prostate cancer is androgen dependent, it can be treated with androgen withdrawal therapy (e.g., by castration or treatment with an anti-androgen, e.g., casodex) .
- NDSP can serve as a target in drug screening.
- a test compound increases NDSP expression or activity in an androgen independent prostate cancer (e.g., a androgen independent prostate cancer cell line)
- the compound may be a therapeutic compound that can be used to confer androgen dependence on an androgen independent prostate cancer.
- This screen can be used to identify both compounds that influence NDSP expression directly and compounds that influence expression or activity of protein in pathways responsible for androgen independent growth of prostate cancers .
- Compound that increase NDSP expression can be used in combination with androgen withdrawal therapy to treat an androgen independent prostate cancer.
- gene expression can be measured at the mRNA or protein level.
- expression can be measured indirectly by .measuring the activity of the protein encoded by the identified gene.
- the present invention provides a method for detecting the presence of NDSP activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of NDSP activity such that the presence of NDSP activity is detected in the biological sample.
- the invention provides a method for identifying a compound for the treatment of prostate cancer (e.g., an androgen independent prostate cancer) by identifying a compound that binds to or modulates the activity of NDSP.
- prostate cancer e.g., an androgen independent prostate cancer
- such methods entail measuring a biological activity of NDSP in the presence and absence of a test compound and identifying those compounds which alter the activity of NDSP.
- the invention also features methods for identifying a compound which modulates the expression of NDSP (at the mRNA or protein level) by measuring the expression of a NDSP nucleic acid or protein in the presence and absence of a compound.
- Differential expression refers to both quantitative, as well as qualitative, differences in the expression pattern of a gene in tumor cells treated with a particular compound (e.g., casodex) and untreated tumor cells.
- a differentially expressed gene can be a target gene.
- a target gene is a differentially expressed gene involved in a disorder (e.g. , prostate cancer) such that modulation of the level of target gene expression or of target gene product activity can act to prevent and/or ameliorate symptoms of the disorder (e.g., androgen- dependent or androgen-independent prostate cancer) .
- Compounds that modulate the expression of the target gene or the activity of the target gene product can be used in the treatment or prevention of the disorder.
- An androgen dependent prostate cancer cell is a cell that requires androgen for continued cell division.
- and androgen independent prostate cancer cell is a cell that can continue to divide in the absence of androgen .
- NDSP has some homology to several phosphatases , including dual specificity phosphatases.
- a number of phosphatases contribute to the regulation of cell growth and signal transduction by dephosphorylating one or more proteins that activate signal transduction when phosphorylated. Thus, phosphatases commonly act to inhibit signal transduction.
- phosphatases appear to act as tumor suppressors.
- tyrosine phosphatase PTP ⁇ chromosome 3p21
- PTEN chromosome 10q23
- PPP2P1 / S a subunit of serine/threonine phosphatase 2A (PP2A) (chromosome llq22- 24) is deleted in a significant proportion of lung cancers and colon cancers.
- NDSP may act as a negative regulator of a signalling pathway that is required for growth of androgen independent prostate cancer. If so, lack of NDSP expresion may contribute to androgen independent growth, and compounds which increase NDSP expression or activity may be useful for the treatment of androgen independent prostate cancer.
- NDSP acts to increase rather than decrease signalling.
- NDSP appears to lack the catalytic domain that is characteristic of many dual specificity phosphatases.
- the vast majority of dual specificity phosphatases include the active site sequence HCXXGXXR(S/T) .
- Studies have shown that the cysteine and the arginine in this sequence are required for activity.
- Two dual specificity phosphatases which lack one or both of these critical residues murine STYX (HGXXGXXRS) and human Sbfl (GLXXGXXIT) , are catalytically inactive.
- NDSP is highly homologous to Sbfl in the region surrounding the active site.
- NDSP has a pseudo active site (CLXXGXXIT) that is nearly identical to that of Sbfl.
- NDSP may not possess phosphatase activity.
- Such catalytically inactive proteins may act to antagonize the activity of endogenous phosphatases.
- overexpression of Sbfl can lead to transformation of NIH 3T3 cells (Cui et al . , (1998) Nature Genetics 18:331- 337) . It is thought that Sbfl overexpression interferes with the interaction between a dual-specificity phosphatase and SET (Suvar3-9, Enhancer-of-zeste, Trithorax) domain containing protein.
- SET domains occur in many proteins that contribute to epigenetic mechanisms of gene regulation through the propagation of chromatin states that are restrictive or permissive for transcription.
- ⁇ DSP cannot act as a phosphatase, it may interfere with a phosphatase which would otherwise inactivate a signalling pathway that acts to promote growth of prostate cancer cells. If so, a compound which binds to ⁇ DSP and prevents ⁇ DSP from interfering with the activity of a phosphatase could be useful in the treatment of prostate cancer.
- ⁇ DSP maps to chromosomal location llpl5.4-11.15.1, a region associated with several cancers. Accordingly, ⁇ DSP may play a role in prostate cancer and other cancers .
- the invention features nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of SEQ ID NO:l or 3 , or the nucleotide sequence of the cDNA insert of the clone deposited with the American Type Culture Collection, Manassass, VA (ATCC) as Accession Number (the "cDNA of ATCC ”) .
- the invention features nucleic acid molecules which include a fragment of at least 15 (25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1200) nucleotides of the nucleotide sequence of SEQ ID NO:l or 3 or the nucleotide sequence of the cDNA of ATCC .
- the invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence encoded by the cDNA of ATCC .
- the nucleic acid molecules have the nucleotide sequence of SEQ ID NO:l or
- nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 2, the fragment including at least 9, (10, 12, 15, 20, 25, 30, 50, 100, 150, 300, or 400) contiguous amino acids of SEQ ID NO:2, or the polypeptide encoded by the cDNA of ATCC .
- the invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence encoded by the cDNA of
- nucleic acid molecules such as allelic variants are present in cDNA prepared from a mammalian prostate cancer cell (e.g., a prostate cancer cell) .
- isolated polypeptides or proteins having an amino acid sequence that is at least about 65%, preferably 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2.
- isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65%, preferably 75%, 85%, or 95% identical to a nucleic acid sequence encoding SEQ ID NO: 2; and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l or 3 or the non-coding strand of the cDNA of
- polypeptides which are a naturally occurring allelic variant of a polypeptide that includes the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence encoded by the cDNA of
- polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule having the sequence of SEQ ID NO: 1 or 3 under stringent conditions.
- the invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO :1 or 3 or the cDNA of ATCC .
- the nucleic acid molecules are at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1200) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 1 or
- the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention.
- Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention.
- the invention provides host cells containing such a vector.
- the invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a polypeptide of the invention is produced.
- Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention. Preferred proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide.
- an activity, a biological activity, and a functional activity of a polypeptide of the invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on a responsive cell as determined in vivo, or in vi tro, according to standard techniques.
- activities can be a direct activity, such as an association with or an enzymatic activity on a second protein or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with a second protein.
- a polypeptide of the invention has an amino acid sequence sufficiently identical to an identified domain of a polypeptide of the invention.
- the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.
- amino acid or nucleotide sequences which contain a common structural domain having about 65% identity, preferably 75% identity, more preferably 85%, 95%, or 98% identity are defined herein as sufficiently identical.
- the isolated polypeptide and lacks both a transmembrane and a cytoplasmic domain.
- polypeptide lacks both a transmembrane domain and a cytoplasmic domain and is soluble under physiological conditions.
- the polypeptides of the present invention, or biologically active portions thereof can be operably linked to a heterologous amino acid sequence to form a fusion protein.
- the invention further features antibodies that specifically bind a polypeptide of the invention such as monoclonal or polyclonal antibodies.
- the polypeptides of the invention or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
- the present invention provides methods for detecting the presence of the activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity such that the presence of activity is detected in the biological sample.
- Figure 1 depicts the cDNA sequence (SEQ ID NO:l) of a human NDSP clone .
- Figure 2 depicts the open reading frame of SEQ ID NO:l (SEQ ID NO : 3 ) and the predicted amino acid sequence (SEQ ID NO: 2) of human NDSP.
- SEQ ID NO: 3 the predicted amino acid sequence of human NDSP.
- SEQ ID NO: 4 the putative pseudo active site of NDSP is underlined.
- Figure 3 is a hydropathy plot of NDSP. Relative hydrophobicity is shown above the dotted line, and relative hydrophilicity is shown below the dotted line.
- Figure 4 is an alignment of amino acids 706 to 810 of NDSP with a pleckstrin homology domain consensus sequence (SEQ ID NO: 5) .
- the present invention is based, at least in part, on the discovery of a cDNA molecule encoding Nuclear Dual Specificity Phosphatase-Like protein (NDSP) , a protein whose expression in WT LNCaP cells is approximately 3- fold lower in the presence of casodex, an anti-androgen, than in the presence of testosterone.
- NDSP Nuclear Dual Specificity Phosphatase-Like protein
- the NDSP cDNA of SEQ ID NO:l ( Figure 1) has a 2436 nucleotide open reading frame (SEQ ID NO:3; Figure 2) encoding an 812 amino acid protein (SEQ ID NO: 2; Figure 3) .
- Figure 1 has a 2436 nucleotide open reading frame (SEQ ID NO:3; Figure 2) encoding an 812 amino acid protein (SEQ ID NO: 2; Figure 3) .
- nucleic acid molecules that encode a polypeptide of the invention or a biologically active portion thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
- nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
- the nucleic acid molecule can be single- stranded or double-stranded, but preferably is double- stranded DNA.
- an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
- an “isolated” nucleic acid molecule is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
- the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
- an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material , or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
- a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l or 3 or the cDNA of ATCC, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NO:l or 3 or the cDNA of ATCC as a hybridization probe, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al . , eds., Molecular Cloning: A Laboratory Manual , 2nd ed .
- a nucleic acid molecule of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
- the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
- oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
- an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence of SEQ ID NO:l or 3 or the cDNA of ATCC , or a portion thereof.
- a nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.
- a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full length polypeptide of the invention for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a polypeptide of the invention.
- the nucleotide sequence determined from the cloning one gene allows for the generation of probes and primers designed for use in identifying and/or cloning homologues in other cell types, e.g., from other tissues, as well as homologues from other mammals.
- the probe/primer typically comprises substantially purified oligonucleotide.
- the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti-sense strand of SEQ ID NO: lor 3 or the cDNA of
- Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences encoding the same protein molecule encoded by a selected nucleic acid molecule .
- the probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
- Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
- a nucleic acid fragment encoding a "biologically active portion" of a polypeptide of the invention can be prepared by isolating or preparing a nucleic acid molecule having the sequence of a portion of SEQ ID NO: 3 or a portion of the nucleotide sequence of the cDNA of ATCC which encodes a polypeptide having a biological activity, expressing the encoded portion of the polypeptide protein (e.g., by recombinant expression in vi tro) and assessing the activity of the encoded portion of the polypeptide.
- the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO: 1 or 3 or the cDNA of ATCC due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence of SEQ ID NO:l or
- DNA sequence polymorphisms that lead to changes in the amino acid sequence may exist within a population (e.g., the human population) .
- Such genetic polymorphisms may exist among individuals within a population due to natural allelic variation.
- An allele is one of a group of genes which occur alternatively at a given genetic locus.
- allelic variant refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence.
- the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention.
- Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene.
- Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
- nucleic acid molecules encoding proteins of the invention from other species which have a nucleotide sequence which differs from that of the protein described herein are intended to be within the scope of the invention.
- Nucleic acid molecules corresponding to natural allelic variants and homologues of a cDNA of the invention can be isolated based on their identity to the nucleic acid molecule disclosed herein using a cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
- a cDNA encoding a soluble form of a membrane-bound protein of the invention isolated based on its hybridization to a nucleic acid molecule encoding all or part of the membrane-bound form.
- a cDNA encoding a membrane-bound form can be isolated based on its hybridization to a nucleic acid molecule encoding all or part of the soluble form.
- an isolated nucleic acid molecule of the invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1200) nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NO:l or 3 or the cDNA of ATCC .
- hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%) identical to each other typically remain hybridized to each other.
- stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989) , 6.3.1-6.3.6.
- a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
- an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a nucleic acid molecule having the sequence of SEQ ID NO: 1 or 3, or the cDNA of ATCC corresponds to a naturally-occurring nucleic acid molecule.
- a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein) .
- allelic variants of a nucleic acid molecule of the invention sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues.
- a "non-essential” amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
- amino acid residues that are not conserved or only semi-conserved among homologues of various species may be non-essential for activity and thus would be likely targets for alteration.
- amino acid residues that are conserved among the homologues of various species e.g., murine and human
- amino acid residues that are conserved among the homologues of various species may be essential for activity and thus would not be likely targets for alteration.
- nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from SEQ ID NO: 2 yet retain biological activity.
- the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 45% identical, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2.
- An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l or 3 or the cDNA of
- ATCC such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
- Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
- conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
- a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
- amino acids with basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid, glutamic acid
- uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
- nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
- beta-branched side chains e.g., threonine, valine, isoleucine
- aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
- mutant polypeptide that is a variant of a polypeptide of the invention can be assayed for: (1) the ability to form protein:protein interactions with other proteins that naturally interact with the polypeptide of the invention; (2) the ability to bind a ligand of the polypeptide of the invention; (3) the ability to bind to an intracellular or extracellular target protein of the polypeptide of the invention.
- the mutant polypeptide can be assayed for the ability to modulate cellular proliferation or cellular differentiation.
- the present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a polypeptide of the invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
- the antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame) .
- An antisense nucleic acid molecule can be antisense to all or part of a noncoding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention.
- the noncoding regions (“5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.
- Useful antisense oligonucleotides can hybridize to the portion of NDSP mRNA encoding the pseudo active site
- An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
- An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
- an antisense nucleic acid e.g., an antisense oligonucleotide
- an antisense nucleic acid e.g., an antisense oligonucleotide
- modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil , dihydrouracil , beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2 , 2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2- thiouracil, beta-D-mannosylqueosine,
- the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
- the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in si tu such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a selected polypeptide of the invention to thereby inhibit expression, e.g., by inhibiting transcription and/or translation.
- the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
- An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
- antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
- antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
- the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
- vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
- An antisense nucleic acid molecule of the invention can be an ⁇ -anomeric nucleic acid molecule.
- An of-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual -units, the strands run parallel to each other (Gaultier et al . (1987) Nucleic Acids Res . 15:6625-6641).
- the antisense nucleic acid molecule can also comprise a 2' -o-methylribonucleotide (Inoue et al . (1987) Nucleic Acids Res . 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al . (1987) FEBS Lett . 215:327- 330) .
- Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
- ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)
- a ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of the invention can be designed based upon the nucleotide sequence of a cD ⁇ A disclosed herein.
- Useful ribozymes can cleave the portion of ⁇ DSP mR ⁇ A encoding the pseudo active site (SEQ ID ⁇ O:4) or the pleckstrin homology domain (amino acids 706 to 810 of NDSP ( Figure 4) .
- a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (Cech et al . U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742).
- an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e. g. , Bartel and Szostak (1993) Science 261:1411-1418.
- the invention also encompasses nucleic acid molecules which form triple helical structures.
- expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells.
- nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide e.g., the promoter and/or enhancer
- the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
- the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al . (1996) Bioorganic & Medicinal Chemistry 4(1) : 5-23) .
- peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
- the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
- the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al . (1996), supra ; Perry-O' Keefe et al . (1996) Proc . Natl . Acad . Sci . USA 93: 14670-675.
- PNAs can be used in therapeutic and diagnostic applications.
- PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
- PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996) , supra; or as probes or primers for DNA sequence and hybridization (Hyrup (1996), supra ; Perry-O' Keefe et al. (1996) Proc . Natl . Acad . Sci . USA 93: 14670-675).
- PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
- PNA-DNA chimeras can be generated which may combine the advantageous properties of PNA and DNA.
- Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
- PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) , supra) .
- the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al . (1996) Nucleic Acids Res . 24(17) : 3357-63.
- a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs.
- the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo) , or agents facilitating transport across the cell membrane (see, e . g. , Letsinger et al . (1989) Proc. Natl . Acad. Sci . USA 86:6553-6556; Lemaitre et al . (1987) Proc . Natl . Acad . Sci . USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier ( see, e . g. , PCT Publication No. WO 89/10134) .
- other appended groups such as peptides (e.g., for targeting host cell receptors in vivo) , or agents facilitating transport across the cell membrane ( see, e . g. , Letsinger et al . (1989) Proc. Natl . Ac
- oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e . g. , Krol et al . (1988) Bio/Techniques 6:958-976) or intercalating agents (see, e . g. , Zon (1988) Pharm.
- the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
- One aspect of the invention pertains to isolated proteins and polypeptides of the invention, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide of the invention.
- the native polypeptide can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
- polypeptides of the invention are produced by recombinant DNA techniques.
- a polypeptide of the invention can be synthesized chemically using standard peptide synthesis techniques .
- an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
- the language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
- protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein”) .
- the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
- culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
- the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
- Biologically active portions of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein (e.g., the amino acid sequence of SEQ ID NO: 2), which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein.
- biologically active portions comprise a domain or motif with at least one activity of the corresponding protein.
- a biologically active portion of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
- other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.
- Preferred polypeptides have the amino acid sequence of SEQ ID NO: 2.
- Other useful proteins are substantially identical (e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to SEQ ID NO: 2 and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
- the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence) .
- the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
- the two sequences are the same length.
- the determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
- a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc . Natl . Acad . Sci . USA 87 : 2264-2268 , modified as in Karlin and Altschul (1993) Proc . Natl . Acad . Sci . USA 90:5873-5877.
- Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al . (1990) J. Mol . Biol . 215:403-410.
- Gapped BLAST can be utilized as described in Altschul et al . (1997) Nucleic Acids Res . 25:3389-3402.
- PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. Id .
- Gapped When utilizing BLAST, Gapped
- BLAST, and PSI-Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
- Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
- ALIGN program version 2.0
- a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
- the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
- a "chimeric protein” or “fusion protein” comprises all or part (preferably biologically active) of a polypeptide of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the same polypeptide of the invention) .
- a heterologous polypeptide i.e., a polypeptide other than the same polypeptide of the invention.
- the term "operably linked” is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other.
- the heterologous polypeptide can be fused to the N-terminus or C-terminus of the polypeptide of the invention.
- One useful fusion protein is a GST fusion protein in which the polypeptide of the invention is fused to the C-terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.
- the fusion protein contains a heterologous signal sequence at its N-terminus.
- the native signal sequence of a polypeptide of the invention can be removed and replaced with a signal sequence from another protein.
- the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al . , eds., John Wiley & Sons, 1992) .
- Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California) .
- useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al . , supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey) .
- the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide of the invention is fused to sequences derived from a member of the immunoglobulin protein family.
- the immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor) , to thereby suppress signal transduction in vivo .
- the immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of ligand/receptor interaction may be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g. promoting or inhibiting) cell survival.
- the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands.
- Chimeric and fusion protein of the invention can be produced by standard recombinant DNA techniques.
- the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
- PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence ( see, e . g. , Ausubel et al . , supra) .
- many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide) .
- a nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention.
- a signal sequence can be used to facilitate secretion and isolation of a protein or polypeptide of the invention.
- Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events.
- Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.
- the signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved.
- the protein can then be readily purified from the extracellular medium by art recognized methods.
- the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
- the present invention also pertains to variants of the polypeptides of the invention. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can be generated by mutagenesis, e.g., discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest.
- Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.
- Variants of a protein of the invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the protein of the invention for agonist or antagonist activity.
- a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
- a variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) .
- methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art ( see, e . g.
- libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants.
- a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
- an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.
- Recursive ensemble mutagenesis (REM) , a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention
- An isolated polypeptide of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation.
- the full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens .
- the antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence of SEQ ID NO: 2 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
- Antibodies which inhibit NDSP activity can bind to the pseudo active site of NDSP (SEQ ID NO: 4) or the pleckstrin homology domain of NDSP (amino acids 706 to 810 of NDSP; ( Figure 4) .
- Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions.
- Figure 3 is a hydrophobicity plot of NDSP. This plot or similar analyses can be used to identify hydrophilic regions.
- An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) .
- An appropriate immunogenic preparation can contain, for example, recombinantly expressed chemically synthesized polypeptide.
- the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
- antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention.
- a molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
- immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
- the invention provides polyclonal and monoclonal antibodies.
- the term "monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
- Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen.
- the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
- ELISA enzyme linked immunosorbent assay
- the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
- antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al . (1983) Immunol .
- Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
- a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest.
- Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene Surf ZAPTM Phage Display Ki t , Catalog No.
- recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
- Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al . (1988) Science 240:1041-1043; Liu et al .
- Fully human antibodies are particularly desirable for therapeutic treatment of human patients.
- Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
- the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
- Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
- the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies.
- Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection. " In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
- a selected non-human monoclonal antibody e.g., a murine antibody
- An antibody directed against a polypeptide of the invention e.g., monoclonal antibody
- an antibody can be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide.
- the antibodies can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
- suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase;
- suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
- suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
- an example of a luminescent material includes luminol ;
- examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 15 I, 131 I, 35 S or 3 H.
- vectors preferably expression vectors, containing a nucleic acid encoding a polypeptide of the invention (or a portion thereof) .
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
- viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
- vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors) . However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) , which serve equivalent functions.
- viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
- the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell .
- the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
- "operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence (s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vi tro transcription/translation system or in a host cell when the vector is introduced into the host cell) .
- regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) . Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) . It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
- the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
- the recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli , insect cells
- the recombinant expression vector can be transcribed and translated in vi tro, for example using T7 promoter regulatory sequences and T7 polymerase .
- Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
- Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
- a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
- enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase .
- Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S- transferase (GST) , maltose E binding protein, or protein A, respectively, to the target recombinant protein.
- GST glutathione S- transferase
- Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al . , (1988) Gene 69:301-315) and pET lid (Studier et al . , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89) .
- Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
- Target gene expression from the pET lid vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl) . This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
- Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al .
- nucleic acid sequences of the invention can be carried out by -standard DNA synthesis techniques.
- the expression vector is a yeast expression vector.
- yeast expression vectors for expression in yeast S . cerivisae include pYepSecl (Baldari et al . (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA) , and pPicZ (Invitrogen Corp, San Diego, CA) .
- the expression vector is a baculovirus expression vector.
- Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol . Cell Biol . 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
- a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al . (1987) EMBO J.
- the expression vector's control functions are often provided by viral regulatory elements.
- viral regulatory elements For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
- suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al . , supra .
- the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid) . Tissue-specific regulatory elements are known in the art.
- tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al . (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol . 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore
- promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the o;-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
- the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention.
- Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
- the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
- a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
- host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- a host cell can be any prokaryotic or eukaryotic cell (e.g., E. coli , insect cells, yeast or mammalian cells) .
- Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
- transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE- dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al . ( supra) , and other laboratory manuals. . For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome.
- a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
- selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
- Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die) .
- a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a polypeptide of the invention. Accordingly, the invention further provides methods for producing a polypeptide of the invention using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
- the host cells of the invention can also be used to produce nonhuman transgenic animals.
- a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequences encoding a polypeptide of the invention have been introduced.
- Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a polypeptide of the invention have been introduced into their genome or homologous recombinant animals in which endogenous encoding a polypeptide of the invention sequences have been altered.
- Such animals are useful for studying the function and/or activity of the polypeptide and for identifying and/or evaluating modulators of polypeptide activity.
- a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
- Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
- a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
- an "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
- a transgenic animal of the invention can be created by introducing nucleic acid encoding a polypeptide of the invention (or a homologue thereof) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
- Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
- a tissue- specific regulatory sequence (s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells.
- transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes .
- a vector which contains at least a portion of a gene encoding a polypeptide of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene.
- the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector) .
- the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein) .
- the altered portion of the gene is flanked at its 5' and -3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell.
- the additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene.
- flanking DNA both at the 5' and 3' ends
- flanking DNA both at the 5' and 3' ends
- the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e . g. , Li et al . (1992) Cell 69:915).
- the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e . g. , Bradley in Teratocarcinomas and Embryonic Stem Cells : A Practi cal
- a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
- Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
- transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene.
- a system is the cre/loxP recombinase system of bacteriophage PI.
- cre/loxP recombinase system of bacteriophage PI.
- a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O' Gorman et al . (1991) Science 251:1351-1355.
- mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
- Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
- Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al . (1997) Nature 385:810- 813 and PCT Publication ⁇ OS . WO 97/07668 and WO 97/07669.
- compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
- the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
- the invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid of the invention. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention and one or more addtional active compounds.
- a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
- routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal , and rectal administration.
- Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene giycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens ; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
- the parenteral preparation can be enclosed
- compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, NJ) or phosphate buffered saline (PBS) .
- the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi .
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene giycol, and liquid polyetheylene giycol, and the like), and suitable mixtures thereof.
- the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the ' required particle size in the case of dispersion and by the use of surfactants .
- Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
- Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
- compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystallme cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
- a binder such as microcrystallme cellulose, gum tragacanth or gelatin
- an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
- a lubricant such as magnesium stearate or Sterotes
- a glidant such as colloidal silicon dioxide
- a sweetening agent such as sucrose
- the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
- a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
- Systemic administration can also be by transmucosal or transdermal means .
- penetrants appropriate to the barrier to be permeated are used in the formulation.
- penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
- Transmucosal administration can be accomplished through the use of nasal sprays or suppositories .
- the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art .
- the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
- suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
- retention enemas for rectal delivery.
- the active compounds are prepared with' carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
- a controlled release formulation including implants and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides , polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
- the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
- Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
- Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
- the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
- the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg) .
- the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain) . A method for lipidation of antibodies is described by Cruikshank et al . ((1997) J " . Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193) .
- the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors .
- Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470) or by stereotactic injection (see, e . g. , Chen et al . (1994) Proc . Natl . Acad . Sci . USA 91:3054-3057).
- the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
- the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
- the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
- nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) detection assays (e.g., chromosomal mapping, tissue typing, forensic biology) ; c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics) ; and d) methods of treatment (e.g., therapeutic and prophylactic).
- detection assays e.g., chromosomal mapping, tissue typing, forensic biology
- predictive medicine e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics
- methods of treatment e.g., therapeutic and prophylactic.
- the isolated nucleic acid molecules of the invention can be used to express proteins (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect mRNA (e.g., in a biological sample) or a genetic lesion, and to modulate an activity of a polypeptide of the invention.
- the polypeptides of the invention can be used to screen drugs or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or aberrant activity compared to the wild type protein.
- the antibodies of the invention can be used to detect and isolate a protein of the and modulate activity of a protein of the invention.
- This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
- the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to polypeptide of the invention or have an inhibitory or stimulatory effect on, for example, expression or activity of a polypeptide of the invention, particularly an inhibitory effect on expression (e.g., expression in a prostate cancer cell in the presence of androgen) .
- modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to polypeptide of the invention or have an inhibitory or stimulatory effect on, for example, expression or activity of a polypeptide of the invention, particularly an inhibitory effect on expression (e.g., expression in a prostate cancer cell in the presence of androgen) .
- the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a polypeptide of the invention or biologically active portion thereof.
- the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
- the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des . 12:145) .
- an assay is a cell-based assay in which a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the polypeptide determined.
- the cell for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
- test compounds can be labeled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
- test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product .
- the assay comprises contacting a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or a biologically active portion thereof as compared to the known compound.
- an assay is a cell-based assay comprising contacting a cell expressing a membrane- bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide or a biologically active portion thereof can be accomplished, for example, by determining the ability of the polypeptide protein to bind to or interact with a target molecule .
- a target molecule is a molecule with which a selected polypeptide (e.g., a polypeptide of the invention binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses the selected protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
- a target molecule can be a polypeptide of the invention or some other polypeptide or protein.
- Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
- the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca + , diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g.
- a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g.
- an assay of the present invention is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the polypeptide or biologically active portion thereof. Binding of the test compound to the polypeptide can be determined either directly or indirectly as described above.
- the assay includes contacting the polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or biologically active portion thereof as compared to the known compound.
- an assay is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished, for example, by determining the ability of the polypeptide to bind to a target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished by determining the ability of the polypeptide of the invention to further modulate the target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
- the cell-free assay comprises contacting a polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the polypeptide to preferentially bind to or modulate the activity of a target molecule.
- the cell-free assays of the present invention are amenable to use of both a soluble form or the membrane- bound form of a polypeptide of the invention.
- non-ionic detergents such as n-octylglucoside
- binding of a test compound to the polypeptide, or interaction of the polypeptide with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
- a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
- glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH) . Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the invention can be determined using standard techniques .
- polypeptide of the invention or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
- Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL) , and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical) .
- antibodies reactive with the polypeptide of the invention or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptidede of the invention trapped in the wells by antibody conjugation.
- Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the polypeptide of the invention or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the polypeptide of the invention or target molecule.
- modulators of expression of a polypeptide of the invention are identified in a method in which a cell is contacted with a candidate compound and the expression of the selected mRNA or protein (i.e., the mRNA or protein corresponding to a polypeptide or nucleic acid of the invention) in the cell is determined.
- the level of expression of the selected mRNA or protein in the presence of the candidate compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate compound.
- the candidate compound can then be identified as a modulator of expression of the polypeptide of the invention based on this comparison.
- the candidate compound when expression of the selected mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the selected mRNA or protein expression.
- the candidate compound when expression of the selected mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the selected mRNA or protein expression.
- the level of the selected mRNA or protein expression in the cells can be determined by methods described herein.
- a polypeptide of the inventions can be used as "bait proteins" in a two-hybrid assay or three hybrid assay
- binding proteins are also likely to be involved in the propagation of signals by the polypeptide of the inventions as, for example, upstream or downstream elements of a signaling pathway involving the polypeptide of the invention.
- This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
- Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing) ; and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
- sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, nucleic acid molecules described herein or fragments thereof, can be used to map the location of the corresponding genes on a chromosome .
- the mapping of the sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease. . Briefly, genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the sequence of a gene of the invention.
- PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler.
- mapping strategies which can similarly be used to map a gene to its chromosome include in si tu hybridization (described in Fan et al . (1990) Proc. Natl . Acad. Sci . USA 87 :6223-27) , pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
- Fluorescence in si tu hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
- FISH Fluorescence in si tu hybridization
- Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
- differences in the D ⁇ A sequences between individuals affected and unaffected with a disease associated with a gene of the invention can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that D ⁇ A sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
- the nucleic acid sequences of the present invention can also be used to identify individuals from minute biological samples.
- the United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
- RFLP restriction fragment length polymorphism
- an individual's genomic D ⁇ A is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
- This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
- the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
- sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
- the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
- Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
- the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
- the nucleic acid sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
- Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals .
- a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
- positive identification of the individual, living or dead can be made from extremely small tissue samples.
- DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime.
- PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids,- e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
- sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual) .
- an "identification marker” i.e. another DNA sequence that is unique to a particular individual
- actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
- Sequences targeted to noncoding regions are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
- polynucleotide reagents include the nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncoding regions having a length of at least 20 or 30 bases.
- nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in si tu hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
- polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in si tu hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.
- the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics , and monitoring clinical trails are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
- diagnostic assays for determining expression of a polypeptide or nucleic acid of the invention and/or activity of a polypeptide of the invention, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant expression or activity of a polypeptide of the invention.
- a biological sample e.g., blood, serum, cells, tissue
- the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, mutations in a gene of the invention can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with aberrant expression or activity of a polypeptide of the invention.
- Another aspect of the invention provides methods for expression of a nucleic acid or polypeptide of the invention or activity of a polypeptide of the invention in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics") .
- Pharmacogenomics allows for the selection of agents
- the genotype of the individual e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent
- Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of a polypeptide of the .invention in clinical trials.
- agents e.g., drugs or other compounds
- An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample.
- a preferred agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention.
- the nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NO:l, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention.
- a preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide of the invention, preferably an antibody with a detectable label.
- Antibodies can be polyclonal, or more preferably, monoclonal.
- an intact antibody, or a fragment thereof can be used.
- labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
- biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic
- vi tro techniques for detection of mRNA include Northern hybridizations and in si tu hybridizations.
- vi tro techniques for detection of A polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs) , Western blots, immunoprecipitations and immunofluorescence .
- vi tro techniques for detection of genomic DNA include Southern hybridizations.
- in vivo techniques for detection of a polypeptide of the invention include introducing into a subject a labeled antibody directed against the polypeptide.
- the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
- the biological sample contains protein molecules from the test subject.
- the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
- a preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject .
- the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting a polypeptide of the invention or mRNA or genomic DNA encoding a polypeptide of the invention, such that the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide is detected in the biological sample, and comparing the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the control sample with the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the test sample.
- the invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid of the invention in a biological sample (a test sample) .
- kits can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of a polypeptide of the invention (e.g., androgen-independent prostate cancer) .
- the kit can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA encoding the polypeptide in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide) .
- Kits may also include instruction for observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide if the amount of the polypeptide or mRNA encoding the polypeptide is above or below a normal level.
- the kit may comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
- a first antibody e.g., attached to a solid support
- a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.
- the kit may comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide of the invention.
- an oligonucleotide e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention
- a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide of the invention.
- the kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
- the kit may also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate) .
- the kit may also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained.
- Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide.
- the methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention.
- the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention.
- the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder.
- test sample refers to a biological sample obtained from a subject of interest.
- a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
- the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention.
- an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
- agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
- agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
- such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the
- the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of a polypeptide of the invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the polypeptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant expression or activity of the polypeptide) .
- the methods of the invention can also be used to detect genetic lesions or mutations in a gene of the invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide of the invention.
- the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding the polypeptide of the invention, or the mis-expression of the gene encoding the polypeptide of the invention.
- such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from the gene; 2) an addition of one or more nucleotides to the gene; 3) a substitution of one or more nucleotides of the gene; 4) a chromosomal rearrangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an aberrant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational modification of the protein encoded by the gene.
- detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e . g. , U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e . g. , Landegran et al . (1988) Science 241:1077-1080; and Nakazawa et al . (1994) Proc . Natl . Acad. Sci .
- PCR polymerase chain reaction
- LCR ligation chain reaction
- This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
- nucleic acid e.g., genomic, mRNA or both
- Alternative amplification methods include: self sustained sequence replication (Guatelli et al . (1990) Proc. Natl . Acad . Sci . USA 87:1874-1878), transcriptional amplification system (Kwoh, et al . (1989) Proc. Natl . Acad. Sci . USA 86:1173-1177), Q-Beta Replicase (Lizardi et al . (1988) Bio /Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers .
- mutations in a selected gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
- sample and control DNA is isolated, amplified (optionally) , digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
- sequence specific ribozymes see, e . g. , U.S. Patent No. 5,498,531
- sequence specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
- genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al . (1996) Human Mutation 7:244-255; Kozal et al . (1996) Nature Medicine 2:753-759).
- genetic mutations can be identified in two- dimensional arrays containing light-generated D ⁇ A probes as described in Cronin et al . , supra . Briefly, a first hybridization array of probes can be used to scan through long stretches of D ⁇ A in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes.
- This step allows the identification of point mutations.
- This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
- Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
- any of a variety of sequencing reactions known in the art can be used to directly sequence the selected gene and detect mutations by comparing the sequence of the sample nucleic acids with the corresponding wild-type (control) sequence.
- Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ( (1977) Proc . Natl . Acad . Sci . USA 74:560) or Sanger ((1977) Proc . Natl . Acad . Sci . USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Bio /Techniques 19:448), including sequencing by mass spectrometry (see, e. g.
- the technique of "mismatch cleavage” entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample.
- the double-stranded duplexes are treated with an agent which cleaves single- stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands .
- RNA/DNA duplexes can be treated with RNase to digest mismatched regions, and DNA/DNA hybrids can be treated with SI nuclease to digest mismatched regions.
- either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e . g. , Cotton et al . (1988) Proc . Natl . Acad . Sci . USA 85:4397; Saleeba et al . (1992) Methods Enzymol . 217:286-295.
- the control DNA or RNA can be labeled for detection.
- the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells.
- DNA mismatch repair enzymes
- the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662) .
- a probe based on a selected sequence is hybridized to a cDNA or other DNA product from a test cell(s) .
- the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e . g. , U.S. Patent No. 5,459,039.
- alterations in electrophoretic mobility will be used to identify mutations in genes.
- SSCP single strand conformation polymorphism
- Single-stranded DNA fragments of sample and control nucleic acids will be denatured and allowed to renature.
- the secondary structure of single- stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
- the DNA fragments may be labeled or detected with labeled probes.
- the sensitivity of the assay may be enhanced by using RNA (rather than DNA) , in which the secondary structure is more sensitive to a change in sequence.
- the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al . (1991) Trends Genet . 7:5) .
- the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al . (1985) Nature 313:495) .
- DGGE denaturing gradient gel electrophoresis
- D ⁇ A will be modified to insure that it does not completely denature, for example by adding a ⁇ GC clamp of approximately 40 bp of high-melting GC-rich D ⁇ A by PCR.
- a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample D ⁇ A (Rosenbaum and Reissner (1987) Biophys . Chem. 265:12753).
- oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al . (1986) Nature 324:163); Saiki et al . (1989) Proc . Natl . Acad. Sci . USA 86:6230).
- allele specific oligonucleotides are hybridized to PCR amplified target D ⁇ A or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target D ⁇ A.
- allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.
- Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al . (1989) Nucleic Acids Res .
- amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc . Natl . Acad . Sci . USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
- the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention.
- any cell type or tissue, preferably peripheral blood leukocytes, in which the polypeptide of the invention is expressed may be utilized in the prognostic assays described herein.
- Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity of the polypeptide.
- the pharmacogenomics i.e., the study of the relationship between an individual ' s genotype and that individual ' s response to a foreign compound or drug
- Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
- the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens.
- effective agents e.g., drugs
- the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention in an individual can be determined to thereby select appropriate agent (s) for therapeutic or prophylactic treatment of the individual .
- Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e . g. , Linder (1997) Clin . Chem. 43(2) :254- 266.
- two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are referred to as "altered drug action.” Genetic conditions transmitted as single factors altering the way the body acts on drugs are referred to as "altered drug metabolism” . These pharmacogenetic conditions can occur either as rare defects or as polymorphisms.
- the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent (s) for therapeutic or prophylactic treatment of the individual.
- pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the exemplary screening assays described herein.
- agents e.g., drugs, compounds
- expression or activity of a polypeptide of the invention and preferably, that of other polypeptides that have been implicated in prostate cancer can be used as a markers.
- genes, including those of the invention, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates activity or expression of a polypeptide of the invention e.g., as identified in a screening assay described herein
- an agent e.g., compound, drug or small molecule
- cells can be isolated and RNA prepared and analyzed for the levels of expression of a gene of the invention and other genes implicated in the disorder.
- the levels of gene expression i.e., a gene expression pattern
- the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene of the invention or other genes.
- the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent.
- this response state may be determined before, and at various points during, treatment of the individual with the agent .
- the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention in the pre-administration sample (optionally, in the presence and absence of an androgen) ; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level the of the polypeptide or nucleic acid of the invention in the post- administration samples (optionally, in the presence and absence of an androgen) ; (v) comparing the level (or androgen inducibility) of the polypeptid
- an agent e
- the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant expression or activity of a polypeptide of the invention (e.g., an androgen-independent prostate cancer) .
- a polypeptide of the invention e.g., an androgen-independent prostate cancer
- the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of a polypeptide of the invention, by administering to the subject an agent which modulates expression or at least one activity of the polypeptide.
- Subjects at risk for a disease which is caused or contributed to by aberrant expression or activity of a polypeptide of the invention can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
- Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
- an agonist or antagonist agent can be used for treating the subject. In the case of prostate cancer, an agonist is used therapeutically.
- the appropriate agent can be determined based on screening assays described herein.
- the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of the polypeptide.
- An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally- occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or other small molecule.
- the agent stimulates one or more of the biological activities of the polypeptide. Examples of such stimulatory agents include the active polypeptide of the invention and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell.
- the agent inhibits one or more of the biological activities of the polypeptide of the invention.
- inhibitory agents include antisense nucleic acid molecules and antibodies. These modulatory methods can be performed in vi tro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject) .
- the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity a polypeptide of the invention.
- the method involves administering an agent (e.g., an agent identified by a screening assay described herein) , or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity.
- an agent e.g., an agent identified by a screening assay described herein
- the method involves administering a polypeptide of the invention or a nucleic acid molecule of the invention as therapy to compensate for reduced or aberrant expression or activity of the polypeptide.
- Stimulation of activity is desirable in situations in which activity or expression is abnormally low downregulated and/or in which increased activity is likely to have a beneficial effect. Conversely, inhibition of activity is desirable in situations in which activity or expression is abnormally high or upregulated and/or in which decreased activity is likely to have a beneficial effect.
- Human NDSP cDNA was identified using a strategy designed to identify genes that are differentially expressed in casodex-treated prostate cancer cells and testosterone-treated cancer cells.
- Wild type LNCaP (WT LNCaP) cells were routinely grown in T162 flasks coated with Matrigel in RPMI-1640 medium supplemented with 10% FBS and 50 nM testosterone.
- LNCaP cells were first incubated in the absence of androgens and then treated with either R1881 or casodex.
- Ten T162 flasks of LNCaP cells were pre-incubated for 24.5 hours in dye-free RPMI-1640 containing 2% charcoal stripped serum.
- five T162 flasks of pretreated cells were treated with testosterone- containing medium (dye-free RPMI-1640, 2% CSS, lOnM testosterone, 0.09% DMSO)
- five T162 flasks of pretreated cells were treated with casodex-containing medium (dye-free RPMI-1640, 2% CSS, 100 ⁇ M casodex, 0.09% DMSO) .
- RNA was prepared from the cell pellets using the RNeasy protocol (Qiagen) . Approximately 260 ⁇ g of total RNA was obtained from each cell pellet. Next, polyA-f RNA was prepared form approximately 240 ⁇ g of each total RNA sample using the Oligotex protocol (Qiagen) , approximately 6 ⁇ g of polyA-f RNA was obtained from each
- RNA sample 240 ⁇ g total RNA sample, and 2 ⁇ g of each polyA ⁇ RNA sample was used for the generation of subtraction libraries using the PCR-select protocol (Clontech; Palo Alto, CA) .
- the PCR products representing partial cDNAs of putatively differentially expressed genes, were subcloned into pCR2.1 (InVitrogen) and transformed into INValphaF 1 cells .
- the cDNA inserts from individual clones of the subtraction libraries were PCR amplified and spotted onto nylon to generated high-density arrays .
- the arrays were probed with first strand cDNA from: WT LNCaP cells treated with 10 nM testosterone, WT LNCaP cells treated with 100 ⁇ M casodex, or WT LNCaP cells treated with stripped serum. Quantitative analysis of radioactive signal at each cDNA spot was performed to identify the relative differential expression of each cDNA among the above cell lines and drug treatments. The clones chosen for further analysis demonstrated higher expression levels in WT LNCaP cells treated with testosterone than in WT LNCaP cells treated with casodex.
- the NDSP cDNA of SEQ ID NO:l ( Figure 1) has a 2436 nucleotide open reading frame (SEQ ID NO:3; Figure 2) encoding an 812 amino acid protein (SEQ ID NO:2; Figure 3) .
- the nucleotide sequence of NDSP was compared to known nucleotide sequences. This search identified both Homo sapiens nuclear dual-specificity phosphatase (Genbank Accession No. U93181; Cui et al . (1998) Nature Genet . 18:331-337) and Mus musculus myotubularin homologous protein 1 (Genbank Accession No. AF073879;
- ⁇ DSP is predicted to have a region with similarity to the pleckstrin homology domain (PF00169) , a domain that is thought to be responsible for recruiting signalling molecules to cellular membranes (Gibson et al .
- ⁇ DSP is also predicted to have potential myristoylation sites (amino acids 9-14, 87-92, 161-166, 170-175, 228-233, 250-255, and 652-657) accordingly, ⁇ DSP may be anchored on the cell membrane.
- Figure 4 is a hydropathy plot of ⁇ DSP. Relative hydrophobicity is shown above the dotted line, and relative hydrophilicity is shown below the dotted line.
- ⁇ DSP maps to chromosomal location llpl5.4-11.15.1, a region associated with several cancers . Chromosomal translocations and loss of heterozygosity.
- a clone ( ) containing a cD ⁇ A encoding ⁇ DSP was deposited with the American Type Culture Collection (ATCC) , Manassass, VA on and assigned Accession
- Example 3 Screening for Compounds that Increase the Expression of NDSP
- Compounds potentially useful for the treatment of prostate cancer or the restoration of androgen dependence to an androgen independent prostate cancer can be identified as follows.
- Androgen-independent prostate cancer cells e.g., LN3 LNCaP cells
- suitable conditions e.g., in T162 flasks coated with Matrigel in RPMI-1640 medium supplemented with 10% FBS and 50 nM testosterone
- the level of NDSP expression is meaured in the presence and absence of the test compound.
- Alterations in expression can be detected by measuring NDSP mRNA using one of the methods described above, e.g., quantitative PCR, or by measuring NDSP protein using one of the methods described above, e.g., Western blotting using a monoclonal anti-NDSP antibody.
- NDSP may interfere with the activity of one or more endogenous phosphatases that act to reduce signalling activity.
- inappropriate NDSP expression may contribute to disorders other than prostate cancer, e.g., other cancers, disorders related to inappropriate cell activation or proliferation.
- compounds which bind to NDSP and prevent NDSP from interferring with phosphatases or compounds reduce NDSP expression may be useful in the treatment of numerous disorders associated with inappropriate signalling.
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Title |
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DATABASE EMBL ON MEDLINE BETHESDA, MD, USA. LAPORTE J. ET AL.: 'Characterization of the Myotubularin Dual Specificity Phosphatase Gene Family from Yeast to Human' Retrieved from medline, accession no. 98409499 Database accession no. Q9Z2D2 & HUM. MOL. GEN. vol. 7, July 1998, pages 1703 - 1713 * |
DATABASE EST, INST. FOR GENOMIC RES. (ROCKVILLE, MD, USA.) ACC. NO. AA307870, 18 April 1997 VENTER J. ET AL.: 'Initial Assessment of Human Gene Diversity and Expression Patterns Based Upon 83 Million Nucleotides of cDNA Sequence' & NATURE vol. 377, no. SUPPL. 6547, 1995, pages 3 - 174 * |
DATABASE EST, NAT. CANCER INST., CANCER GENOME ANATOMY PROJECT, (BETHESDA. MD,USA) ACC. NO. AA87329, 25 March 1998 STRAUSBERG R. * |
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