Neutralizing high affinity human monoclonal antibodies specific to RSV F-protein and methods for their manufacture and therapeutic use thereof

Abstract

A highly efficient method for generating human antibodies in particular which are specific to be RSV fusion protein which combines in vitro primary of human spleen cells and antigen boosting in SCID mice is taught. This method provides for very high human antibody titers which are predominantly of the IgG isotype which contain antibodies of high specificity and affinity to desired antigens. This method is well suited for generating human monoclonal antibodies for therapeutic and diagnostic applications as well as for rescue of human cells for generation of combinational human antibody gene libraries. Two human monoclonal antibodies, RF-1 and RF-2 which each possess an affinity for RSV F-protein ≦2×10 −9 Molar are taught as well as their corresponding amino acid and DNA sequences. These antibodies are to be used therapeutically and prophylactically for treating or preventing RSV infection, as well as for diagnosis of RSV in analytes.

In the claims:  
     
         1 . A human monoclonal antibody which specifically binds the RSV fusion protein and which possesses an affinity for the RSV F-protein of ≦2×10 −9  molar.  
     
     
         2 . The human monoclonal antibody of  claim 1  which neutralizes RSV in vitro.  
     
     
         3 . A human monoclonal antibody which specifically binds to the RSV fusion protein which is selected from the group consisting of RF-1, RF-2 and recombinant human monoclonal antibodies which contain the variable heavy and light domains of either RF-1 or RF-2.  
     
     
         4 . The human monoclonal antibody of  claim 3  wherein said antibody is RF-1.  
     
     
         5 . The human monoclonal antibody of  claim 3  wherein said antibody is RF-2.  
     
     
         6 . The human antibody of  claim 3 , wherein said antibody is a recombinant antibody which contains either the human gamma 1, human gamma 4, or human gamma 4 PE constant region.  
     
     
         7 . Eukaryotic cells which have been transfected with DNA sequences which encode for the heavy and light variable domains of either RF-1 or RF-2.  
     
     
         8 . The cells of  claim 7  wherein said eukaryotic cells are CHO cells.  
     
     
         9 . The eukaryotic cells of  claim 7  wherein said DNA sequences are selected from the DNA sequences set forth in any one of FIGS. 7 a,    7   b,    8   a,    8   b,    9   a,    9   b,    10   a  or  10   b.    
     
     
         10 . An Epstein-Barr immortalized B cell line which secretes a human monoclonal antibody which possesses an affinity for the RSV fusion protein of ≦2×10 −9  molar.  
     
     
         11 . The cell line of  claim 10  wherein said antibody neutralizes RSV in vitro.  
     
     
         12 . The cell line of  claim 10  wherein said cell line is selected from the group consisting of RF-1 and RF-2.  
     
     
         13 . A method for producing human antibodies specific to the RSV fusion (F) protein which comprises: 
 (i) priming human splenocytes in vitro in the presence of IL-2;    (ii) transferring said primed human splenocytes into a SCID mouse;    (iii) boosting said SCID mouse with RSV F-protein; and    (iv) isolating human B cells from said SCID mouse which secrete human monoclonal antibodies specific for the RSV F-protein.    
     
     
         14 . The method of  claim 13  wherein said isolated human B cells are immortalized.  
     
     
         15 . The method of  claim 14  wherein immortalization is effected using Epstein-Barr Virus (EBV).  
     
     
         16 . The method of  claim 15  wherein said EBV immortalized cells are cloned using the mouse thyoma cell line EL-4 B5 as a feeder layer.  
     
     
         17 . The method of  claim 13  wherein the priming step is effected in the presence of IL-4 or IL-6.  
     
     
         18 . A DNA sequence which encodes for the variable heavy and/or variable light domain of RP-1 or RF-2.  
     
     
         19 . An expression vector which provides for the expression of a DNA sequence according to  claim 18 .  
     
     
         20 . The DNA sequence of  claim 18  which is selected from the group consisting of the DNA sequences set forth in FIGS. 7 a,    7   b,    8   a,    8   b,    9   a,    9   b,    10   a  and  10   b.    
     
     
         21 . A method for preventing or treating RSV infection in susceptible or RSV infected persons which comprises administering a prophylactically or therapeutically effective amount of one or more human monoclonal antibodies which possess an affinity to the RSV F-protein of ≦2×10 −9  molar and which also neutralize RSV in vitro.  
     
     
         22 . The method of  claim 21  wherein said antibodies are selected from the group consisting of RF-1, RF-2 and recombinant human monoclonal antibodies which contain the variable heavy and light domains of RF-1 or RF-2.  
     
     
         23 . The method of  claim 21  wherein said antibodies are administered by injection or by aerosol.  
     
     
         24 . The method of  claim 21  wherein said antibodies are administered in combination with an adjuvant.  
     
     
         25 . The method of  claim 24  wherein said adjuvant is Complete Freund's Adjuvant (CFA), Alum or a combination thereof.  
     
     
         26 . A pharmaceutical composition suitable for preventing or treating RSV infection in susceptible or RSV infected persons which comprises a prophylactically or therapeutically effective amount of human monoclonal antibodies which neutralize RSV in vitro and which possess an affinity for the RSV F-protein of ≦2×10 −9  molar and a pharmaceutically acceptable carrier.  
     
     
         27 . The pharmaceutical composition of  claim 26  wherein said human monoclonal antibodies are selected from the group consisting of RF-1, RF-2 and recombinant human monoclonal antibodies which contain the variable heavy and light domains of either RF-1 or RF-2.  
     
     
         28 . A method of detecting the presence of RSV in an analyte which comprises incubating, said analyte with a human monoclonal antibody which possesses an affinity for the RSV F-protein of ≦2×10 −9  molar under conditions which provide for the formation of RSV F-protein antibody immune complexes; and 
 detecting the presence of said RSV F-protein antibody immune complexes to determine whether RSV is present in the analyte.  
 
     
     
         29 . The method of  claim 28  wherein said antibody is RF-1 or RF-2.  
     
     
         30 . The method of  claim 29  wherein said antibody is directly or indirectly attached to a reporter molecule.  
     
     
         31 . The method of  claim 30  wherein said reporter molecule is a detectable enzyme or radionuclide.  
     
     
         32 . The method of  claim 28  wherein the analyte comprises fluid obtained from respiratory tissue.  
     
     
         33 . A test kit for assaying the presence of RSV in an analyte which comprises: 
 (i) a human monoclonal antibody having an affinity for the RSV F-protein of ≦2×10 −9  molar; and    (ii) a reporter molecule which is directly or indirectly attached to said human monoclonal antibody.    
     
     
         34 . The test kit of  claim 33  wherein said human monoclonal antibody is RF-1 or RF-2.