Antibodies against anthrax protective antigen

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 - 20 . (canceled) 
     
     
         21 . An isolated antibody or antigen-binding fragment thereof which specifically binds to the same protective antigen (PA) epitope as an anti-anthrax toxin monoclonal antibody, wherein said anti-anthrax toxin monoclonal antibody is 21D9. 
     
     
         22 . (canceled) 
     
     
         23 . The isolated antibody or fragment thereof of  claim 21 , wherein the heavy chain variable region (VH) of said antibody or fragment thereof comprises at least one heavy chain complementarity determining region (VH-CDR) which is identical, except for variations or modifications that do not alter specific binding activity of said antibody or fragment thereof to anthrax toxin, to a VH-CDR selected from the group consisting of VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NO: 2. 
     
     
         24 - 27 . (canceled) 
     
     
         28 . The isolated antibody or fragment thereof of  claim 21 , wherein the light chain variable region (VL) of said antibody or fragment thereof comprises at least one light chain complementarity determining region (VL-CDR) which is identical, except for variations or modifications that do not alter specific binding activity of said antibody or fragment thereof to anthrax toxin, to a VL-CDR selected from the VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NO: 4. 
     
     
         29 - 32 . (canceled) 
     
     
         33 . The isolated antibody or fragment thereof of  claim 23 , wherein the VH of said antibody or fragment thereof comprises a VH-CDR3 amino acid sequence which is identical, except for variations or modifications that do not alter specific binding activity of said antibody or fragment thereof to anthrax toxin, to amino acids 99-110 of SEQ ID NO: 2. 
     
     
         34 . The isolated antibody or fragment thereof of  claim 33 , wherein the VH-CDR3 amino acid sequence comprises amino acids 99-110 of SEQ ID NO: 2. 
     
     
         35 . The isolated antibody or fragment thereof of  claim 28 , wherein the VL of said antibody or fragment thereof comprises a VL-CDR3 amino acid sequence which is identical, except for variations or modifications that do not alter specific binding activity of said antibody or fragment thereof to anthrax toxin, to amino acids 89-96 of SEQ ID NO: 4. 
     
     
         36 . The isolated antibody or fragment thereof of  claim 35 , wherein the VL-CDR3 amino acid sequence comprises amino acids 89-96 of SEQ ID NO: 4. 
     
     
         37 . The isolated antibody or fragment thereof of  claim 21 , wherein the VH of said antibody or fragment thereof comprises VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences which are identical, except for variations or modifications that do not alter specific binding activity of said antibody or fragment thereof to anthrax toxin, to amino acids 31-35, 50-66, and 99-110 of SEQ ID NO: 2, respectively. 
     
     
         38 . The isolated antibody or fragment thereof of  claim 37 , wherein the VH-CDR1 amino acid sequence comprises amino acids 31-35 of SEQ ID NO: 2. 
     
     
         39 . The isolated antibody or fragment thereof of  claim 37 , wherein the VH-CDR2 amino acid sequence comprises amino acids 50-66 of SEQ ID NO: 2. 
     
     
         40 . The isolated antibody or fragment thereof of  claim 37 , wherein the VH-CDR3 amino acid sequence comprises amino acids 99-110 of SEQ ID NO: 2. 
     
     
         41 . The isolated antibody or fragment thereof of  claim 37 , wherein the VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences comprise amino acids 31-35, 50-66, and 99-110 of SEQ ID NO: 2, respectively. 
     
     
         42 . The isolated antibody or fragment thereof of  claim 21 , wherein the VL of said antibody or fragment thereof comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences which are identical, except for variations or modifications that do not alter specific binding activity of said antibody or fragment thereof to anthrax toxin, to amino acids 24-34, 50-56, and 89-96 of SEQ ID NO: 4, respectively. 
     
     
         43 . The isolated antibody or fragment thereof of  claim 42 , wherein the VL-CDR1 amino acid sequence comprises amino acids 24-34 of SEQ ID NO: 4. 
     
     
         44 . The isolated antibody or fragment thereof of  claim 42 , wherein the VL-CDR2 amino acid sequence comprises amino acids 50-56 of SEQ ID NO: 4. 
     
     
         45 . The isolated antibody or fragment thereof of  claim 42 , wherein the VL-CDR3 amino acid sequence comprises amino acids 89-96 of SEQ ID NO: 4. 
     
     
         46 . The isolated antibody or fragment thereof of  claim 42 , wherein the VL-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences comprise amino acids 24-34, 50-56, and 89-96 of SEQ ID NO: 4, respectively. 
     
     
         47 - 63 . (canceled) 
     
     
         64 . An isolated polynucleotide comprising a nucleic acid sequence which encodes an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises at least one VH-CDR of a VH polypeptide which specifically binds to the same protective antigen (PA) epitope as an anti-anthrax toxin monoclonal antibody, wherein said anti-anthrax toxin monoclonal antibody is 21D9;
 wherein said VH-CDR is encoded by a polynucleotide sequence that is identical, except for variations or modifications due to degeneracy in the genetic code,   to a polynucleotide sequence selected from the group consisting of: VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NO: 1.   
     
     
         65 . An isolated polynucleotide comprising a nucleic acid sequence which encodes an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises at least one VL-CDR of a VL polypeptide which specifically binds to the same protective antigen (PA) epitope as an anti-anthrax toxin monoclonal antibody, wherein said anti-anthrax toxin monoclonal antibody is 21D9;
 wherein said VL-CDR is encoded by a polynucleotide sequence that is identical, except for variations or modifications due to degeneracy in the genetic code,   to a polynucleotide sequence selected from the group consisting of: VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NO: 3.   
     
     
         66 . (canceled) 
     
     
         67 . (canceled) 
     
     
         68 . A polypeptide encoded by the polynucleotide of  claim 64 . 
     
     
         69 . A vector comprising the polynucleotide of  claim 64 . 
     
     
         70 . A host cell comprising the vector of  claim 69 . 
     
     
         71 . A method of producing an antibody or fragment thereof which specifically binds PA, comprising culturing the host cell of  claim 70 , and recovering said antibody or fragment thereof. 
     
     
         72 . An antibody or fragment thereof recovered by the method of  claim 71 . 
     
     
         73 . A composition comprising a first antibody or fragment thereof of  claim 21  and one or more additional therapeutic agents, wherein said additional therapeutic agent is selected from the group consisting of a vaccine, an antibiotic, and a second antibody or fragment thereof. 
     
     
         74 - 79 . (canceled) 
     
     
         80 . An isolated antibody or antigen-binding fragment thereof which specifically binds to a protective antigen (PA) and inhibits PA63 assembly, wherein said isolated antibody or antigen-binding fragment thereof binds the distal linear epitope within PA47 polypeptide of PA83. 
     
     
         81 . The isolated antibody or antigen-binding fragment thereof of  claim 80 , wherein said isolated antibody or antigen-binding fragment has a binding affinity of about 1×10 −11  to about 1×10 −10  M. 
     
     
         82 . A method of preventing PA63 assembly in a mammal comprising administration of the isolated antibody or antigen-binding fragment thereof of  claim 80  to said mammal thereby preventing PA63 assembly. 
     
     
         83 . A polypeptide encoded by the polynucleotide of  claims 65 . 
     
     
         84 . A vector comprising the polynucleotide of  65 . 
     
     
         85 . A host cell comprising the vector of  claim 84 . 
     
     
         86 . A method of producing an antibody or fragment thereof which specifically binds PA, comprising culturing the host cell of  claim 85 , and recovering said antibody or fragment thereof. 
     
     
         87 . An antibody or fragment thereof recovered by the method of  claim 86 .