Purified and isolated protein zero related (PZR) and therapeutic and screening methods using same

Abstract

Isolated and purified proteins and nucleic acids including a novel member of the immunoglobulin super-family characterized as having SHP-2 binding activity and cell signaling activity and called protein zero related or PZR, and cDNA encoding the same. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also disclosed, along with methods of producing each. Isolated and purified antibodies to PZR, and methods of producing the same, are also disclosed. PZR is characterized as having SHP-2 binding activity and cell signaling activity and thus, therapeutic methods involving these activities are also disclosed.

1 - 4 . (canceled)  
     
     
         5 . An isolated and purified antibody capable of preferentially binding to the polypeptide of claim  1 .  
     
     
         6 . The antibody of  claim 5 , further characterized as a monoclonal antibody or as a polyclonal antibody.  
     
     
         7 . The antibody of  claim 6 , wherein the antibody comprises a monoclonal antibody which preferentially binds to an ectodomain fragment of the polypeptide of claim  1 .  
     
     
         8 . The antibody of  claim 6 , wherein the antibody is humanized.  
     
     
         9 . The antibody of  claim 6 , in a pharmaceutically acceptable diluent or excipient.  
     
     
         10 . A hybridoma cell line which produces a monoclonal antibody of  claim 6 .  
     
     
         11 - 21 . (canceled)  
     
     
         22 . A method of producing an antibody immunoreactive with a PZR polypeptide, the method comprising steps of: 
 (a) transfecting a recombinant host cell with the a nucleic acid molecule of claim  11 , which encodes a PZR polypeptide;    (b) culturing the host cell under conditions sufficient for expression of the polypeptide;    (c) recovering the polypeptide; and    (d) preparing the antibody to the polypeptide.    
     
     
         23 . The method of  claim 22 , wherein the polypeptide comprises an amino acid sequence as set forth in any of SEQ ID NOs:2, 4, 6, 8, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 48.  
     
     
         24 . The method of  claim 22 , wherein the nucleic acid molecule comprises a nucleic acid sequence as set forth in any of SEQ ID NOs:1, 3, 5, 7, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47.  
     
     
         25 . An antibody produced by the method of  claim 25 .  
     
     
         26 . A method of detecting a PZR polypeptide, the method comprising: 
 (a) immunoreacting the polypeptide with an antibody prepared according the method of  claim 22  to form an antibody-polypeptide conjugate; and    (b) detecting the conjugate.    
     
     
         27 . A method of detecting in a sample an nucleic acid molecule that encodes the PZR polypeptide encoded by the nucleic acid of claim  11 , the method comprising: 
 (a) contacting the sample under hybridizing conditions with the nucleic acid molecule of claim  11  to form a duplex; and    (b) detecting the presence of the duplex.    
     
     
         28 . The method of  claim 27 , wherein the nucleic acid molecule to be detected comprise a DNA molecule or an RNA molecule.  
     
     
         29 . An assay kit for detecting the presence of a PZR polypeptide in a biological sample, the kit comprising a first container containing a first antibody capable of immunoreacting with a PZR polypeptide of claim  1 , wherein the first antibody is present in an amount sufficient to perform at least one assay.  
     
     
         30 . The assay kit of  claim 29 , further comprising a second container containing a second antibody that immunoreacts with the first antibody.  
     
     
         31 . The assay kit of  claim 30 , wherein the first antibody and the second antibody comprise monoclonal antibodies.  
     
     
         32 . The assay kit of  claim 30 , wherein the first antibody is affixed to a solid support.  
     
     
         33 . The assay kit of  claim 30 , wherein the first and second antibodies each comprise an indicator.  
     
     
         34 . The assay kit of  claim 33 , wherein the indicator is a radioactive label or an enzyme.  
     
     
         35 . An assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a PZR polypeptide, the kit comprising a first container containing a PZR polypeptide that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.  
     
     
         36 . (canceled)  
     
     
         37 . A transgenic non-human animal having incorporated into its genome a nucleic acid molecule of claim  11 , the nucleic acid molecule being present in the genome in a copy number effective to confer expression in the animal of a PZR polypeptide.  
     
     
         38 . The transgenic non-human animal of  claim 37 , wherein the animal is a mouse.  
     
     
         39 . A method of screening candidate substances for an ability to modulate PZR biological activity, the method comprising: 
 (a) establishing replicate test and control samples that comprise a biologically active PZR polypeptide;    (b) administering a candidate substance to test sample but not the control sample;    (c) measuring the biological activity of PZR in the test and the control samples; and    (d) determining that the candidate substance modulates PZR biological activity if the biological activity of PZR measured for the test sample is greater or less than the biological activity of PZR level measured for the control sample.    
     
     
         40 . The method of  claim 39 , wherein the PZR polypeptide biological activity is selected from the group consisting of modulation of cell migration, modulation of cell proliferation, modulation of density induced growth arrest, and combinations thereof.  
     
     
         41 . The method of  claim 39 , wherein the test and control samples further comprises cells that express the PZR polypeptide.  
     
     
         42 . A recombinant cell line suitable for use in the assay of  claim 41 .  
     
     
         43 . The method of  claim 39 , wherein the method is carried out in a cell-free system, the method further comprising: 
 (a) establishing a control system comprising a PZR polypeptide and a ligand wherein the PZR polypeptide is capable of binding to the ligand;    (b) establishing a test system comprising the PZR polypeptide, the ligand, and a candidate compound;    (c) measuring the binding affinity of the PZR polypeptide and the ligand in the control and the test systems; and    (d) determining that the candidate compound modulates PZR polypeptide biological activity in a cell-free system if the binding affinity measured for the test system is less than or greater than the binding affinity measured for the control system.    
     
     
         44 . The method of  claim 43 , wherein the ligand comprises an antibody which preferentially binds the PZR polypeptide.  
     
     
         45 . The method of  claim 44 , wherein the ligand is a monoclonal antibody.  
     
     
         46 . A method for identifying a compound that modulates tyrosine kinase or tyrosine phosphatase biological activity, the method comprising: 
 (a) contacting a cell lysate of a target cell that has been exposed to a candidate substance with an anchoring molecule that is specific for a PZR protein substrate which has been dephosphorylated as a result of signal transduction in the target cell;    (b) detecting phosphotyrosine residues on the PZR protein substrate that is bound to the anchoring molecule;    (c) determining that the candidate substance modulates tyrosine kinase or tyrosine phosphatase biological activity if the amount of phosphotyrosine residues on the bound PZR protein substrate derived from the target cell lysate is greater or less than an amount of phosphotyrosine residues on a bound PZR protein substrate derived from a control target cell.    
     
     
         47 . The method of  claim 46 , wherein the anchoring molecules are selected from the group consisting of monoclonal antibodies, polyclonal antibodies, and combinations thereof.  
     
     
         48 . The method of  claim 47 , wherein the tyrosine phosphatase comprises SHP-2.  
     
     
         49 . A method for determining the presence or amount of a protein tyrosine phosphatase enzymatic activity in a sample, the method comprising: 
 (a) incubating a sample suspected of containing a protein tyrosine phosphatase with a phosphorylated PZR peptide under conditions and for a time sufficient to permit dephosphorylation of the phosphorylated PZR peptide by a protein tyrosine phosphatase in the sample, wherein the phosphorus atom of the phosphorylated PZR peptide is detectably labeled;    (b) separating non-dephosphorylated PZR peptide from free detectably labeled phosphorus released by dephosphorylation of the phosphorylated PZR peptide; and    (c) detecting the presence or amount of detectable labeled phosphorus released by dephosphorylation of the phosphorylated PZR peptide to thereby determine the presence or amount of protein tyrosine phosphatase enzymatic activity in the sample.    
     
     
         50 . The method of  claim 49 , wherein the detectably labeled phosphorus comprises radioactive phosphorus.  
     
     
         51 . A method for determining the presence or amount of a protein tyrosine kinase enzymatic activity in a sample, the method comprising: 
 (a) incubating a sample suspected of containing a protein tyrosine kinase with a dephosphorylated PZR peptide and a predetermined amount of detectably labeled phosphorus atom under conditions and for a time sufficient to permit phosphorylation of the dephosphorylated PZR peptide by a protein tyrosine kinase in the sample;    (b) separating phosphorylated PZR peptide from free detectably labeled phosphorus not incorporated onto the phosphorylated PZR peptide; and    (c) detecting the presence or amount of detectably labeled phosphorus incorporated onto the phosphorylated PZR peptide to thereby determine the presence or amount of protein tyrosine kinase enzymatic activity in the sample.    
     
     
         52 . The method of  claim 51 , wherein the detectably labeled phosphorus comprises radioactive phosphorus.  
     
     
         53 . A method of modulating cell signaling in a warm-blooded vertebrate, the method comprising administering to the warm-blooded vertebrate a PZR biological. activity-modulating amount of a PZR biological activity-modulating composition; and modulating cell signaling in the warm-blooded vertebrate through the modulating of PZR biological activity with the composition.  
     
     
         54 . The method of  claim 53 , further comprising administering to the warm-blooded vertebrate the PZR polypeptide activity-modulating amount of the PZR biological activity-modulating composition, whereby a PZR polypeptide within the warm-blooded vertebrate is contacted by the composition.  
     
     
         55 . The method of  claim 53 , wherein the PZR biological activity is selected from the group consisting of modulation of cell migration, modulation of cell aggregation, modulation of cell proliferation, modulation of density induced growth arrest, and combinations thereof.  
     
     
         56 . The method of  claim 53 , wherein the composition comprises a PZR polypeptide, or fragment or derivative thereof.  
     
     
         57 . The method of  claim 56 , wherein the PZR polypeptide activity-modulating amount of the PZR polypeptide ranges from about 0.1 to about 300 milligrams per kilogram body weight of the warm-blooded vertebrate.  
     
     
         58 . The method of  claim 57 , wherein the PZR polypeptide activity-modulating amount of the PZR polypeptide ranges from about 0.2 to about 200 milligrams per kilogram body weight of the warm-blooded vertebrate.  
     
     
         59 . The method of  claim 58 , wherein the PZR polypeptide activity-modulating amount of the PZR polypeptide ranges from about 0.5 to about 20 milligrams per kilogram body weight of the warm-blooded vertebrate.  
     
     
         60 . The method of  claim 53 , wherein the composition comprises a monoclonal antibody or fragment thereof which preferentially binds the PZR polypeptide.  
     
     
         61 . The method of  claim 60 , wherein the monoclonal antibody preferentially binds to an ectodomain of the PZR polypeptide.  
     
     
         62 . The method of  claim 60 , wherein the PZR polypeptide activity-modulating amount of the monoclonal antibody ranges from about 0.1 to about 300 milligrams per kilogram body weight of the warm-blooded vertebrate.  
     
     
         63 . The method of  claim 62 , wherein the PZR polypeptide activity-modulating amount of the monoclonal antibody ranges from about 0.2 to about 200 milligrams per kilogram body weight of the warm-blooded vertebrate.  
     
     
         64 . The method of  claim 63 , wherein the PZR polypeptide activity-modulating amount of the monoclonal antibody ranges from about 0.5 to about 20 milligrams per kilogram body weight of the warm-blooded vertebrate.  
     
     
         65 . The method of  claim 60 , wherein the antibody is humanized.  
     
     
         66 . The method of  claim 65 , wherein the humanized antibody preferentially binds an ectodomain of the PZR polypeptide.  
     
     
         67 . The method of  claim 53 , wherein the modulating of PZR biological activity further comprises modulating expression of the PZR polypeptide in the warm-blooded vertebrate.  
     
     
         68 . The method of  claim 67 , wherein the PZR biological activity-modulating composition further comprises a PZR antisense oligonucleotide.  
     
     
         69 . The method of  claim 53 , wherein the administering is selected for the group consisting of intravenous administration, intrasynovial administration, transdermal administration, intramuscular administration, subcutaneous administration and oral administration.  
     
     
         70 . A method for modulating cell signaling in a warm-blooded vertebrate, the method comprising introducing to a cell in the warm-blooded vertebrate a construct comprising a nucleic acid sequence encoding a PZR gene product operatively linked to a promoter, wherein production of the PZR gene product in the cell results in modulation of cell signaling.  
     
     
         71 . The method of  claim 70 , wherein the construct further comprises a vector selected from the group consisting of a plasmid vector or a viral vector.  
     
     
         72 . The method of  claim 70 , wherein the construct further comprises a liposome complex.  
     
     
         73 . The method of  claim 70 , wherein the PZR gene product comprises a protein having an amino acid sequence as set forth in any of SEQ ID NOs:2, 4, 6, 8, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 48.  
     
     
         74 . The method of  claim 70 , wherein the nucleic acid sequence is selected from the group consisting of: 
 (a) a DNA acid sequence as set forth in any of SEQ ID NOs:1, 3, 5, 7, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47, or its complementary strands;    (b) a DNA sequence which hybridizes to a nucleic acid sequence as set forth in any of SEQ ID NOs:1, 3, 5, 7, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47 under wash stringency conditions represented by a wash solution having about 200 mM salt concentration and a wash temperature of at least about 45° C., and which encodes a PZR polypeptide; and    (c) a DNA sequence differing from an isolated nucleic acid molecule of (a) or (b) above due to degeneracy of the genetic code, and which encodes a PZR polypeptide encoded by the isolated nucleic acid molecule of (a) or (b) above.    
     
     
         75 - 79 . (canceled)