Chemokine variants and methods of use

Abstract

The present invention provides the nucleotide and amino acid sequence of truncated RANTES (3-68), which has the same amino acid sequence as the wild-type RANTES, but with a Serine/Proline truncation at positions 1 and 2 from the N-terminus, respectively. CD26 is a leukocyte activation marker that possesses dipeptidyl peptidase IV (DPPIV) activity but whose natural substrates and immunological functions had not been previously defined. Several chemokines, including RANTES (regulated on activation, normal T expressed and secreted) are provided, which are substrates for human CD26. The truncated RANTES (3-68) lacked the ability of native RANTES (1-68) to increase the cytosolic calcium concentration in human monocytes, but it still induces this response in macrophages activated with macrophage colony-stimulating factor (M-CSF). RANTES (3-68) retains the ability to stimulate CCR5 receptors and to inhibit the cytopathic effects of HIV-1. The invention provides methods for identifying compounds that affect DPPIV-medicated chemokine cleavage, methods for inhibiting HIV infection and treating individuals having or at risk of having HIV infection, methods for diagnosis and/or prognosis of individuals having a chemokine-associated disorder and methods for accelerating wound healing and angiogenesis, all based on the discovery of DPPIV-mediated cleavage of chemokines.

What is claimed is:  
     
         1 . A substantially pure polypeptide having an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         2 . An isolated polynucleotide which encodes an amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         3 . An isolated polynucleotide selected from the group consisting of: 
 a) SEQ ID NO:1;    b) SEQ ID NO:1, wherein T can also be U;    c) nucleic sequences complementary to SEQ ID NO:1;    d) fragments of a), b), or c) that are at least 15 bases in length and that will hybridize to DNA which encodes SEQ ID NO:2.    
     
     
         4 . An expression vector containing in operable linkage the polynucleotide as in  claim 2 .  
     
     
         5 . A host cell containing the vector of  claim 4 .  
     
     
         6 . The host cell of  claim 5 , wherein the cell is a eukaryotic cell.  
     
     
         7 . A method for identifying a compound which modulates Dipeptidyl peptidase IV (DPPIV)-mediated chemokine processing comprising: 
 a) incubating components comprising the compound, DPPIV and a chemokine under conditions sufficient to allow the components to interact; and    b) determining the N-terminal amino acid sequence of the chemokine before and after incubating in the presence of the compound.    
     
     
         8 . The method of  claim 7 , wherein the modulating is inhibition of DPPIV-mediated chemokine processing.  
     
     
         9 . The method of  claim 7 , whererin the modulating is stimulation of DPPIV-mediated chemokine processing.  
     
     
         10 . The method of  claim 7 , wherein the compound is a peptide.  
     
     
         11 . The method of  claim 7 , wherein the compound is a peptidomimetic.  
     
     
         12 . The method of  claim 7 , wherein the DPPIV is expressed in a cell.  
     
     
         13 . The method of  claim 7 , wherein the chemokine contains a proline or an alanine at position 2 from the N-terminus.  
     
     
         14 . A method of inhibiting membrane fusion between HIV and a target cell or between an HIV-infected cell and a CD4 positive uninfected cell comprising contacting the target or CD4 positive cell with a fusion-inhibiting effective amount of the polypeptide of SEQ ID NO:2.  
     
     
         15 . The method of  claim 14 , wherein the contacting is by in vivo administration to a subject.  
     
     
         16 . The method of  claim 14 , wherein the polypeptide is administered by intravenous, intramuscular or subcutaneous injection.  
     
     
         17 . The method of  claim 14 , wherein the polypeptide is formulated in a pharmaceutically acceptable carrier.  
     
     
         18 . A method of treating a subject having or at risk of having an HIV infection or disorder, comprising administering to the subject, a therapeutically effective amount of a polypeptide of SEQ ID NO:2, wherein the polypeptide inhibits cell-cell fusion in cells infected with HIV.  
     
     
         19 . The method of  claim 18 , wherein the subject is suffering from AIDS or ARC.  
     
     
         20 . The method of  claim 18 , wherein the polypeptide is formulated in a pharmaceutically acceptable carrier.  
     
     
         21 . A method of treating a subject having an HIV-related disorder associated with expression of CCR5 comprising administering to an HIV infected or susceptible cell of the subject, a polypeptide of SEQ ID NO:2 or a nucleic acid sequence encoding the polypeptide of SEQ ID NO:2 or other variant chemokine.  
     
     
         22 . The method of  claim 21 , wherein the polypeptide or nucleic acid is introduced into the cell using a carrier.  
     
     
         23 . The method of  claim 22 , wherein the carrier is a vector.  
     
     
         24 . The method of  claim 21 , wherein the administering is ex vivo.  
     
     
         25 . The method of  claim 21 , wherein the administering is in vivo.  
     
     
         26 . A pharmaceutical composition comprising the polypeptide of SEQ ID NO:2 in a pharmaceutically acceptable carrier.  
     
     
         27 . A pharmaceutical composition comprising CD26 polypeptide in a pharmaceutically acceptable carrier.  
     
     
         28 . A method for producing a variant chemokine having an activity different from the activity of the wild-type chemokine, comprising contacting the wild-type chemokine with an N-terminal processing effective amount of Dipeptidyl peptidase IV (DPPIV), thereby truncating the chemokine and producing a variant chemokine.  
     
     
         29 . The method of  claim 28 , wherein the chemokine contains a proline or an alanine at position 2 from the N-terminus.  
     
     
         30 . The method of  claim 29 , wherein the chemokine is selected from the group consisting of RANTES, MIP-1, IP-10, cotaxin, MDC and MCP-2.  
     
     
         31 . The method of  claim 29 , wherein the chemokine is RANTES.  
     
     
         32 . A method for inhibiting HIV-1 replication in a host cell susceptible to HIV-1 infection, comprising contacting the cell or the host with an effective amount of Dipeptidyl peptidase IV (DPPIV) enzyme such that macrophage-derived chemokine (MDC) is cleaved to produce truncated MDC, thereby providing antiviral activity and inhibiting HIV-1 replication.  
     
     
         33 . A method for inhibiting Dipeptidyl peptidase IV (DPPIV)-mediated chemokine processing comprising contacting DPPIV with an inhibiting effective amount of a compound which inhibits DPPIV expression or activity.  
     
     
         34 . A method for inhibiting an allergic or inflammatory reaction in a subject, comprising administering to the subject an effective amount of Dipeptidyl peptidase IV (DPPIV) enzyme such that a chemokine is cleaved to produce a truncated chemokine, thereby inhibiting an allergic or inflammatory reaction.  
     
     
         35 . The method of  claim 34 , wherein the chemokine is eotaxin.  
     
     
         36 . The method of  claim 34 , wherein the subject is a human.  
     
     
         37 . A method for accelerating angiogenesis or wound healing in a subject, comprising administering to the subject an effective amount of an inhibitor of Dipeptidyl peptidase IV (DPPIV) enzyme activity or gene expression or a DPPIV-insensitive chemokine, such that chemokine processing is inhibited, thereby accelerating angiogenesis or wound healing.  
     
     
         38 . The method of  claim 37 , wherein the chemokine is IP-10.  
     
     
         39 . The method of  claim 37 , wherein the DPPIV-insensitive chemokine is a wild-type chemokine with the proviso that alanine or proline at position 2 is replaced with any amino acid other than alanine or proline.  
     
     
         40 . A method for inhibiting HIV-1 replication in a host cell susceptible to HIV-1 infection, comprising contacting the cell or the host with an effective amount of Dipeptidyl peptidase IV (DPPIV) enzyme such that RANTES is cleaved to produce truncated RANTES, thereby providing antiviral activity and inhibiting HIV-1 replication.  
     
     
         41 . The method as in any of claims  7 ,  28 ,  32 ,  33 ,  34 ,  37 , or  39 , wherein the DPPIV enzyme is CD26.  
     
     
         42 . A method of diagnosis of a subject having a chemokine-associated disorder comprising: 
 identifying the presence of a chemokine of interest from a specimen isolated from the subject;    determining the amino-terminal sequence of the chemokine,    wherein a full-length amino acid sequence is indicative of the presence of a wild-type chemokine polypeptide and a truncated amino-terminal sequence is indicative of the presence of a variant chemokine; and    determining the concentration of wild-type chemokine as compared to variant chemokine, thereby providing a diagnosis of the subject.    
     
     
         43 . The method of  claim 42 , wherein the determining of the amino-terminal sequence of the chemokine is by contacting the chemokine with an antibody which distinguishes wild-type from variant chemokine polypeptide.  
     
     
         44 . The method of  claim 42 , wherein the specimen is selected from the group consisting of blood, sputum, urine, saliva, cerebrospinal fluid, and serum.  
     
     
         45 . Antibodies which bind to wild-type chemokine but not to DPPIV-truncated chemokine.  
     
     
         46 . Antibodies which bind to DPPIV-truncated chemokine but not to wild-type chemokine.  
     
     
         47 . The antibodies as in claims  45  or  46 , wherein the chemokine is RANTES.