Cells expressing both human CD4 and a human fusion accessory factor associated with HIV infection

Abstract

The susceptibility to human immunodeficiency virus (HIV) infection depends on the cell surface expression of the human CD4 molecule and a human fusion accessory factor associated with HIV infection (CXCR4). CXCR4 is a member of the 7-transmembrane segment superfamily of G-protein-coupled cell surface molecules. CXCR4 plays an essential role in the membrane fusion step of HIV infection. The establishment of stable, nonhuman cell lines and transgenic mammals having cells that coexpress human CD4 and CXCR4 provides valuable tools for the continuing research of HIV infection and the development of more effective anti-HIV therapeutics. In addition, antibodies against CXCR4, isolated and purified peptide fragments of CXCR4, and CXCR4-binding biologic agents, capable of blocking membrane fusion between HIV and target cells represent potential anti-HIV therapeutics.

1 . A recombinant cell line that expresses CXCR4 polypeptide.  
     
     
         2 . The cell line of  claim 1 , wherein the cell further expresses CD4 polypeptide.  
     
     
         3 . A recombinant host cell stably transformed with a polynucleotide encoding CXCR4 polypeptide, wherein the cell co-expresses CXCR4 and CD4 polypeptide.  
     
     
         4 . A recombinant host cell stably transformed with a polynucleotide encoding CXCR4 polypeptide and a polynucleotide encoding CD4 polypeptide, wherein the cell co-expresses CXCR4 and CD4 polypeptide.  
     
     
         5 . The cell as in any of claims  1 - 4 , wherein the cell is a human cell.  
     
     
         6 . The cell as in any of claims  1 - 4 , wherein the cell is a non-human cell.  
     
     
         7 . An antibody which specifically binds to CXCR4 polypeptide or fragments thereof.  
     
     
         8 . The antibody of  claim 7 , wherein the antibody is a monoclonal antibody.  
     
     
         9 . A substantially purified peptide fragment of CXCR4, wherein the peptide inhibits cell membrane fusion between HIV and a target cell or between an HIV-infected cell and a CD4 positive uninfected cell.  
     
     
         10 . A substantially purified CXCR4-binding agent, wherein the biologic agent inhibits membrane fusion between HIV and a target cell or between an HIV-infected cell and a CD4 positive uninfected cell.  
     
     
         11 . The agent of  claim 10 , wherein the agent is selected from a biologic agent and a chemical compound.  
     
     
         12 . The agent of  claim 10 , wherein the biologic agent is a chemokine.  
     
     
         13 . The agent of  claim 12 , wherein the agent is stromal cell derived factor (SDF1) derivative, analog or binding fragment thereof.  
     
     
         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 a CXCR4 binding or blocking agent.  
     
     
         15 . The method of  claim 14 , wherein the agent is SDF1 or derivative, analog or binding fragment thereof.  
     
     
         16 . The method of  claim 14 , wherein the agent is a anti-CXCR4 antibody or epitope binding fragment thereof.  
     
     
         17 . The method of  claim 16 , wherein the antibody is a monoclonal antibody or a polyclonal antibody.  
     
     
         18 . The method of  claim 14 , wherein the contacting is by in vivo administration to a subject.  
     
     
         19 . The method of  claim 18 , wherein the anti-CXCR4 antibody is administered by intravenous, intra-muscular or subcutaneous injections.  
     
     
         20 . The method of  claim 19 , wherein the anti-CXCR4 antibody is administered within a dose range of 0.1 ug/kg to 100 mg/kg.  
     
     
         21 . The method of  claim 16 , wherein the antibody is formulated in a pharmaceutically acceptable carrier.  
     
     
         22 . A method for identifying a composition which binds to CXCR4 polypeptide comprising: 
 a) incubating components comprising the composition and CXCR4 polypeptide under conditions sufficient to allow the components to interact; and    b) measuring the binding of the composition to CXCR4 polypeptide.    
     
     
         23 . The method of  claim 22 , wherein the composition is a peptide.  
     
     
         24 . The method of  claim 22 , wherein the composition is a peptidomimetic.  
     
     
         25 . The method of  claim 22 , wherein the CXCR4 polypeptide is expressed in a cell.  
     
     
         26 . The method of  claim 25 , wherein the cell is the cell of  claim 1 .  
     
     
         27 . A method for identifying a composition which blocks membrane fusion between HIV and a target cell or between an HIV-infected cell and a CXCR4 positive uninfected cell comprising: 
 a) incubating components comprising the composition and a CXCR4 positive cell under conditions sufficient to allow the components to interact;    b) contacting the components of step a) with HIV or an HIV-infected cell; and    c) measuring the ability of the composition to block membrane fusion between HIV and the CXCR4 positive cell or between an HIV-infected cell and a CXCR4 positive uninfected cell.    
     
     
         28 . The method of  claim 27 , wherein the CXCR4 postivie cell is a CD4 positive cell.  
     
     
         29 . The method of  claim 27 , wherein measuring the ability of the composition to block membrane fusion is by detection of a reporter means.  
     
     
         30 . The method of  claim 29 , wherein the reporter means is selected from the group consisting of a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.  
     
     
         31 . The method of  claim 30 , wherein the reporter means is a lacZ gene.  
     
     
         32 . A transgenic non-human animal having a phenotype characterized by expression of CXCR4 polypeptide and CD4 polypeptide otherwise not naturally occurring in the animal, the phenotype being conferred by a transgene contained in the somatic and germ cells of the animal, the transgene comprising a nucleic acid sequence which encodes CXCR4 polypeptide and a nucleic acid sequence which encodes CD4 polypeptide.  
     
     
         33 . The transgenic non-human animal of  claim 32 , wherein the animal is a mouse.  
     
     
         34 . The transgenic non-human animal of  claim 32 , wherein the animal is a rabbit.  
     
     
         35 . A transgenic non-human animal having a phenotype characterized by expression of CXCR4 polypeptide otherwise not naturally occurring in the animal, the phenotype being conferred by a transgene contained in the somatic and germ cells of the animal, the transgene comprising a nucleic acid sequence which encodes CXCR4 polypeptide.  
     
     
         36 . A method for producing a transgenic non-human animal having a phenotype characterized by expression of CXCR4 polypeptide and CD4 polypeptide otherwise not naturally occurring in the animal, the method comprising: 
 (a) introducing at least one transgene into a zygote of an animal, the transgene(s) comprising a DNA construct encoding CXCR4,    (b) transplanting the zygote into a pseudopregnant animal,    (c) allowing the zygote to develop to term, and    (d) identifying at least one transgenic offspring containing the transgene.    
     
     
         37 . The method of  claim 36 , further comprising a DNA construct encoding CD4.  
     
     
         38 . The method of  claim 36 , wherein the introducing of the transgene into the embryo is by introducing an embryonic stem cell containing the transgene into the embryo.  
     
     
         39 . The method of  claim 36 , wherein the introducing of the transgene into the embryo is by infecting the embryo with a retrovirus containing the transgene.  
     
     
         40 . The method of  claim 36 , wherein the animal is selected from the group consisting of a mouse and a rabbit.  
     
     
         41 . A transgenic non-human animal having a transgene disrupting or interfering with expression of CXCR4 chromosomally integrated into the germ cells of the animal.  
     
     
         42 . The transgenic animal of  claim 41 , wherein the animal is selected from the group consisting of a mouse and a rabbit.  
     
     
         43 . The transgenic non-human animal of  claim 41 , wherein the transgene comprises CXCR4 antisense polynucleotide.  
     
     
         44 . 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 an anti-CXCR4 antibody, wherein the antibody inhibits cell-cell fusion in cells infected with HIV.  
     
     
         45 . The method of  claim 44 , wherein the antibody is a monoclonal antibody.  
     
     
         46 . The method of  claim 45 , wherein the monoclonal antibody is a humanized monoclonal antibody.  
     
     
         47 . The method of  claim 44 , wherein the monoclonal antibody is administered to a patient suffering from AIDS or ARC.  
     
     
         48 . The method of  claim 44 , wherein the monoclonal antibody is administered within a dose range between about 0.1/kg to about 100 mg/kg.  
     
     
         49 . The method of  claim 44 , wherein the monoclonal antibody is formulated in a pharmaceutically acceptable carrier.  
     
     
         50 . A method of treating a subject having an HIV-related disorder associated with expression of CXCR4 comprising administering to an HIV infected or susceptible cell of the subject, a agent that suppresses CXCR4.  
     
     
         51 . The method of  claim 50 , wherein the agent is an anti-CXCR4 antibody.  
     
     
         52 . The method of  claim 50 , wherein the agent is an antisense nucleic acid that hybridizes to a CXCR4 nucleic acid.  
     
     
         53 . The method of  claim 50 , wherein the agent is introduced into the cell using a carrier.  
     
     
         54 . The method of  claim 50 , wherein the carrier is a vector.  
     
     
         55 . The method of  claim 50 , wherein the administering is ex vivo.  
     
     
         56 . The method of  claim 50 , wherein the administering is in vivo.  
     
     
         57 . A method for detecting susceptibility of a first cell to HIV infection comprising: 
 incubating the first cell with a second cell which expresses HIV-env, under conditions to allow fusion of the two cells; and detecting fusion of the cells, wherein fusion is indicative of susceptibility to HIV infection.    
     
     
         58 . The method of  claim 57 , wherein the first or second cell further comprises a reporter means for detection of cell fusion.  
     
     
         59 . The method of  claim 57 , wherein the first cell is a T cell.  
     
     
         60 . The method of  claim 58 , wherein the T-cell is a CXCR4- and CD4+cell.  
     
     
         61 . The method of  claim 58 , wherein the T-cell is a CXCR4+ and CD4-cell.  
     
     
         62 . The method of  claim 57 , wherein the T-cell is a CXCR4+ and CD4+cell.  
     
     
         63 . The method of  claim 58 , wherein the reporter means is selected from the group consisting of a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.  
     
     
         64 . The method of  claim 63 , wherein the reporter means is a lacZ gene.