title: Improvement of anti-Burkholderia mouse monoclonal antibody from various phage-displayed single-chain antibody libraries.
abstract: To improve anti-Burkholderia monoclonal antibody (MAb) binding affinity, six single chain variable fragments (scFvs) constructed previously were used as scaffolds to construct large highly-diversified phage-displayed mouse scFv random and domain libraries. First, we employed random mutagenesis to introduce random point mutations into entire variable regions, generating six random libraries. Additionally, the oligonucleotide-directed mutagenesis was targeted on complementarity-determining region 3 (CDR3) from each variable region of heavy (VH) and light chains (VL) derived from six scFvs, and generated eighteen domain libraries including six VH CDR3, six VL CDR3, and six combined VH/VL CDR3 mutated domains, respectively. We collected high scFvs binders through panning experiment over the large (size ~1 × 10⁹) random and domain libraries. The quality of the libraries was validated by successful selection of high-affinity clones. Random mutagenesis generated many mutant scFv clones having more than one amino acid changes around framework regions, but not many in CDRs. Surprisingly, the resulting eight higher scFv binders were selected from CDR3 mutations, but not from random mutations. Six of them resulted from CDR3 mutations of light chain, except for two scFvs from heavy chain, showing both Burkholderia pseudomallei and Burkholderia mallei had preferentially influenced the VL CDR3. Furthermore, all eight higher scFvs converted to full format human IgG1 antibodies were expressed transiently in 293T cell line. Five chimeric MAbs showed improved higher binding activity, as much as 0.2-0.3 at O.D. 405 nm, than positive control MAbs. These libraries could be valuable sources for selection of anti-Burkholderia antibodies and discovery of the relevant epitope(s) for developing effective vaccines or therapeutics.
authors.affiliation: Department of Environmental and Infectious Disease Sciences, American Registry of Pathology, and Armed Forces Institute of Pathology, Washington, DC 20306, USA.
authors.forename: Ho San
authors.initials: HS
authors.lastname: Kim
authors.forename: Shyh-Ching
authors.initials: SC
authors.lastname: Lo
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Wear
authors.forename: Alexander
authors.initials: A
authors.lastname: Stojadinovic
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Weina
authors.forename: Mina J
authors.initials: MJ
authors.lastname: Izadjoo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D019117:Burkholderia / Q000235:genetics / Q000276:immunology*; D019121:Burkholderia Infections / Q000175:diagnosis / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D004269:DNA, Bacterial; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D057127:Single-Chain Antibodies