Monoclonal Antibodies That Specifically Bind To And Neutralize Bacillus Anthracis Toxin, Compositions, And Methods Of Use

An expression vector comprising a polynucleotide sequence encoding operably linked components, from 5′ to 3′:

• a promoter;
• a first signal sequence;
• a first polypeptide comprising at least a variable region of a light chain polypeptide of an antibody chosen from 1A5, 4A12, 24B1, 24G4, 32E12, or 33F4;
• a second leader sequence;
• a second polypeptide comprising at least a variable region of a heavy chain polypeptide of an antibody chosen from 1A5, 4A12, 24B1, 24G4, 32E12, or 33F4;
• and
• optionally, a detectable label.

An isolated antibody comprising:

• a heavy chain complementarity determining region (CDR) comprising contiguous amino acid sequences of a CDRH1, a CDRH2, and a CDRH3 of a heavy chain polypeptide of an antibody chosen from 1A5, 4A12, 24B1, 24G4, 32E12, or 33F4;
• and
• a light chain CDR comprising contiguous amino acid sequences of a CDRL1, a CDRL2, and a CDRL3 of a light chain polypeptide of an antibody chosen from 1A5, 4A12, 24B1, 24G4, 32E12, or 33F4;
• wherein the heavy chain CDR and light chain CDR are selected from the same antibody.

A method for producing a library of expression vectors containing immunoglobulin heavy and light chain-encoding sequences, which when expressed provide for production of an antigen-binding polypeptide that specifically binds an antigen of interest, the method comprising:

• subjecting RNA isolated from B lymphocytes to nucleic acid amplification in the presence of primer set, wherein
• said B lymphocytes are obtained from a subject exposed to an amount of antigen of interest effective to elicit an immune response to the antigen in the subject,
• said primer set comprises primers that specifically bind a collection of immunoglobulin (Ig) heavy chain and light chain variable genes, wherein the primers further contain a restriction enzyme recognition sequences of at least 8 bases, and
• said subjecting results in production of DNA amplification products comprising primer-introduced restriction enzyme recognition sequences and a nucleic acid sequence of a Ig heavy chain or light chain variable gene, wherein DNA amplification products comprising a sequence of an Ig heavy chain variable gene and DNA amplification products comprising a sequence of an Ig light chain variable gene contain different primer-introduced restriction enzyme recognition sequences;
• digesting the DNA amplification products with restriction enzymes that recognize said recognition sequences to produced digested DNA;
• and
• ligating said digested DNA into an expression cassette having first and second compatible cloning sites, wherein said ligating provides for directional cloning of DNA amplification products comprising a sequence of an Ig light chain variable gene into said first cloning site and for direction cloning of DNA amplification products comprising a sequence of an Ig heavy chain variable gene into said second cloning site;
• wherein a library of expression vectors containing immunoglobulin heavy and light chain-encoding sequences, which when expressed provide for production of an antigen-binding polypeptide that specifically binds to the antigen of interest.