Neutralizing human antibodies to anthrax toxin

Abstract

A highly efficient method for generating human antibodies using recall technology is provided. In one aspect, human antibodies which are specific to the anthrax toxin are provided. In one aspect, human peripheral blood cells that have been pre-exposed to anthrax toxin are used in the SCID mouse model. This method results in high human antibody titers which are primarily of the IgG isotype and which contain antibodies of high specificity and affinity to desired antigens. The antibodies generated by this method can be used therapeutically and prophylactically for preventing or treating mammals exposed to anthrax. Thus, in one embodiment, a prophylactic or therapeutic agent used to counter the effects of anthrax toxin, released as a mechanism of bioterrorism, is provided. In one embodiment, a formulation and method for preventing and/or treating anthrax infection comprising a binding agent that prevents the assembly of the PA63 heptamer is also provided. Methods for diagnosis and methods to determine anthrax contamination are also described.

1 . A fully human monoclonal antibody or fragment thereof that recognizes at least a portion of an anthrax exotoxin, wherein said antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of one or more of the following: SEQ ID 2, SEQ ID 4, SEQ ID 6, SEQ ID 8, SEQ ID 10, SEQ ID 12, SEQ ID 14, and SEQ ID 16.  
     
     
         2 . The fully human antibody of  claim 1 , wherein said antibody or fragment thereof comprises the amino acid sequences of SEQ ID 2 and SEQ ID 4.  
     
     
         3 . The fully human antibody of  claim 1 , wherein said antibody or fragment thereof comprises the amino acid sequences of SEQ ID 6 and SEQ ID8.  
     
     
         4 . The fully human antibody of  claim 1 , wherein said antibody or fragment thereof comprises the amino acid sequences of SEQ ID 10 and SEQ ID 12.  
     
     
         5 . The fully human antibody of  claim 1 , wherein said antibody or fragment thereof comprises the amino acid sequences of SEQ ID 14 and SEQ ID 16.  
     
     
         6 . The filly human antibody of  claim 1 , comprising a first antibody and a second antibody, wherein said first antibody comprises the amino acid sequences of SEQ ID 2 and SEQ ID 4, and wherein said second antibody comprises the amino acid sequences of SEQ ID 6 and SEQ ID 8.  
     
     
         7 . The fully human antibody of  claim 1 , wherein the PA portion of said anthrax exotoxin is recognized by said antibody or fragment thereof.  
     
     
         8 . A pharmaceutical composition for passively immunizing a mammal against anthrax, wherein said pharmaceutical composition comprises said fully human antibody or fragment thereof of  claim 1 .  
     
     
         9 . The pharmaceutical composition of  claim 8 , wherein said mammal has not been previously exposed to anthrax.  
     
     
         10 . The pharmaceutical composition of  claim 8 , wherein said pharmaceutical composition comprises at least two different fully human antibodies or fragments thereof.  
     
     
         11 . The pharmaceutical composition of  claim 8 , wherein said pharmaceutical composition further comprises an additional agent, wherein said additional agent is an antibody that comprises less than 100% human protein sequences.  
     
     
         12 . The pharmaceutical composition of  claim 8 , wherein said pharmaceutical composition further comprises AVA or rPA.  
     
     
         13 . A pharmaceutical composition for treating a mammal exposed to anthrax, wherein said pharmaceutical composition comprises said fully human monoclonal antibody or fragment thereof of  claim 1 .  
     
     
         14 . The pharmaceutical composition of  claim 13 , further comprising an antibiotic.  
     
     
         15 . The pharmaceutical composition of  claim 13 , wherein said pharmaceutical composition comprises at least two different fully human monoclonal antibodies or fragments thereof.  
     
     
         16 . The pharmaceutical composition of  claim 13 , wherein said pharmaceutical composition further comprises an antibody comprising less than 100% human protein sequences.  
     
     
         17 . A fully human monoclonal antibody that binds to anthrax, where said antibody comprises at least one complementarity determining region (CDR) selected from the group consisting of one or more of the following: amino acids 31-35 of SEQ ID 2, amino acids 50-66 of SEQ ID 2, and amino acids 99-110 of SEQ ID 2.  
     
     
         18 . The fully human monoclonal antibody of  claim 17 , wherein said fully human monoclonal antibody binds to the PA portion of an anthrax exotoxin.  
     
     
         19 . A fully human monoclonal antibody, wherein said antibody or fragment thereof is encoded at least in part by a polynucleotide comprising a nucleotide sequence selected from the group consisting of one or more of the following: SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQ ID 7, SEQ ID 9, SEQ ID 11, SEQ ID 13, and SEQ ID 15.  
     
     
         20 . A hybridoma comprising the polynucleotide of  claim 19 .  
     
     
         21 . A fully human monoclonal antibody that recognizes anthrax exotoxin, wherein said fully human monoclonal antibody is made by a method comprising: 
 administering peripheral blood cells from one or more human donors exposed to said anthrax exotoxin to a SCID mouse;    administering one or more doses of an anthrax antigen to said mouse;    isolating at least one lymphocytic cell from said mouse;    fusing said at least one lymphocytic cell with a fusion partner, thereby generating a hybridoma, wherein said hybridoma produces a fully human monoclonal antibody which recognizes at least a portion of said anthrax exotoxin.    
     
     
         22 . A fully human monoclonal antibody or fragment thereof that recognizes at least a portion of an anthrax exotoxin, wherein said antibody or fragment thereof comprises the amino acid sequence of SEQ ID 2.  
     
     
         23 . A hybridoma cell that produces a fully human monoclonal antibody or fragment thereof, wherein said antibody or fragment thereof comprises the amino acid sequences of SEQ ID 2 and SEQ ID 4.  
     
     
         24 . A fully human monoclonal antibody that recognizes at least a portion of an anthrax exotoxin, wherein said anthrax exotoxin comprises a protective antigen (PA), and wherein said fully human monoclonal antibody binds to said protective antigen (PA) and interferes with the assembly of a protective antigen (PA) heptamer.  
     
     
         25 . A fully human monoclonal antibody that recognizes at least a portion of an anthrax exotoxin, wherein said antibody comprises a heavy chain or a light chain encoded by a polynucleotide obtainable from a human peripheral blood cell engrafted in a SCID mouse.