title: In vitro improvement of a shark IgNAR antibody by Qbeta replicase mutation and ribosome display mimics in vivo affinity maturation.
abstract: We have employed a novel mutagenesis system, which utilizes an error-prone RNA dependent RNA polymerase from Qbeta bacteriophage, to create a diverse library of single domain antibody fragments based on the shark IgNAR antibody isotype. Coupling of these randomly mutated mRNA templates directly to the translating ribosome allowed in vitro selection of affinity matured variants showing enhanced binding to target, the apical membrane antigen 1 (AMA1) from Plasmodium falciparum. One mutation mapping to the IgNAR CDR1 loop was not readily additive to other changes, a result explained by structural analysis of aromatic interactions linking the CDR1, CDR3, and Ig framework regions. This combination appeared also to be counter-selected in experiments, suggesting that in vitro affinity maturation is additionally capable of discriminating against incorrectly produced protein variants. Interestingly, a further mutation was directed to a position in the IgNAR heavy loop 4 which is also specifically targeted during the in vivo shark response to antigen, providing a correlation between natural processes and laboratory-based affinity maturation systems.
authors.affiliation: EvoGenix Limited, 343 Royal Parade, Parkville, Vic. 3052, Australia.
authors.forename: George
authors.initials: G
authors.lastname: Kopsidas
authors.forename: Anthony S
authors.initials: AS
authors.lastname: Roberts
authors.forename: Gregory
authors.initials: G
authors.lastname: Coia
authors.forename: Victor A
authors.initials: VA
authors.lastname: Streltsov
authors.forename: Stewart D
authors.initials: SD
authors.lastname: Nuttall
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity / Q000235:genetics*; D000953:Antigens, Protozoan / Q000276:immunology; D015374:Biosensing Techniques; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D019151:Peptide Library; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000276:immunology; D011777:Q beta Replicase / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D012754:Sharks / Q000276:immunology*
chemical_list: D000906:Antibodies; D000953:Antigens, Protozoan; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D008565:Membrane Proteins; D019151:Peptide Library; D015800:Protozoan Proteins; D012333:RNA, Messenger; C090077:apical membrane antigen I, Plasmodium; D011777:Q beta Replicase