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.

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 - 12 . (canceled)  
     
     
         13 . A method for producing human antibodies in a non-human animal that bind specifically to the RSV F-protein, which comprises the steps of: 
 (i) priming human splenocytes in vitro by culturing said human splenocytes in the presence of RSV F-protein and IL-2;    (ii) transferring said primed human splenocytes into an animal having a severe combined immunodeficiency (SCID) phenotype to produce a reconstituted animal;    (iii) boosting said in vitro primed human splenocytes in vivo by immunizing said reconstituted animal with RSV F-protein; and    (iv) maintaining said reconstituted animal alive for sufficient time to allow said transferred human splenocytes in said animal to produce human antibodies that bind specifically to the RSV F-protein.    
     
     
         14 - 20 . (canceled)  
     
     
         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 - 34 . (canceled)  
     
     
         35 . The method of  claim 13 , wherein the priming in vitro step is effected in the presence of at least one cytokine in addition to IL-2.  
     
     
         36 . The method of  claim 35 , wherein the additional cytokine is IL-4 or IL-6, or a combination thereof.  
     
     
         37 . The method of  claim 13 , wherein the priming in vitro step comprises exposing the human splenocytes for about one to ten days to RSV F-protein at a concentration sufficient to induce an antibody response specific to RSV F-protein.  
     
     
         38 . The method of  claim 13 , wherein the transferring step comprises injecting intraperitoneally the primed human splenocytes into a SCID or SCID/Beige mouse.  
     
     
         39 . The method of  claim 13 , wherein the boosting step occurs from three to fourteen days after the transferring step.  
     
     
         40 . The method of  claim 13 , wherein the boosting step further includes administering RSV F-protein in conjunction with at least one adjuvant.  
     
     
         41 . The method of  claim 13 , further comprising the step of isolating human antibody-producing cells from said reconstituted animal.  
     
     
         42 . The method of  claim 41 , wherein said isolated human antibody-producing cells are obtained from the peritoneum or spleen of the reconstituted animal.  
     
     
         43 . The method of  claim 41 , further comprising the step of immortalizing said isolated human cells in vitro.  
     
     
         44 . The method of  claim 43 , wherein the immortalizing step comprises infecting the isolated human cells with Epstein-Barr Virus (EBV).  
     
     
         45 . The method of  claim 44  wherein said EBV immortalized cells are cloned using a mouse thyoma cell line as a feeder layer.  
     
     
         46 . The method of  claim 41 , wherein said isolated human cells are used to generate a combinatorial human antibody gene library that produces human monoclonal antibodies against RSV F-protein.  
     
     
         47 . The method of  claim 13 , further comprising the step of purifying said human antibodies from a tissue isolated from said reconstituted animal.  
     
     
         48 . The method of  claim 13 , further comprising the steps of detecting tumor formation in said reconstituted animal and isolating human antibody-producing cells from said tumor.  
     
     
         49 . The method of  claim 13 , further comprising the step of detecting increased relative affinity of the RSV F-protein-specific human antibodies produced in the reconstituted animal.  
     
     
         50 . The method of  claim 13 , further comprising the step of introducing fresh non-primed human spleen cells to the reconstituted animal prior to the boosting step.  
     
     
         51 . The method of  claim 13 , wherein the human splenocytes are naive.