title: Directed evolution of an anti-human red blood cell antibody.
abstract: We have earlier described a haemagglutination-based assay for on-site detection of antibodies to HIV using whole blood. The reagent in this assay comprises of monovalent Fab fragment of an anti-human RBC antibody fused to immunodominant antigens of HIV-1 and HIV-2. In the present work, we describe a rational and systematic method for directed evolution of scFv and Fab antihuman RBC antibody fragments. Based on homology modeling and germline sequence alignments of antibodies, target residues in the anti-RBC MAb B6 sequence were identified for mutagenesis.A combinatorial library of 107 clones was constructed and subjected to selection on whole RBC under competitive binding conditions to identify several phage-displayed B6 scFv clones with improved binding as determined in an agglutination assay.Selected VL and VH sequences were shuffled to generate a second generation phage-displayed Fab library which on panning yielded Fab clones with several fold better binding than wild type. The mutants with better binding also displayed more Fab molecules per phage particle indicating improved in vivo folding which was also confirmed by their increased periplasmic secretion compared to the wild type. The mutant Fab molecules also showed superior characteristics in large scale production by in vitro folding of LC and Fd. The biophysical measurements involving thermal and chemical denaturation and renaturation kinetics clearly showed that two of the mutant Fab molecules possessed significantly improved characteristics as compared to the wild type B6 Fab.Structural modelling revealed that B6 Fab mutants had increased hydrogen bonding resulting in increased stability. Our approach provides a novel and useful strategy to obtain recombinant antibodies with improved characteristics.
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi, India.
authors.forename: Amita
authors.initials: A
authors.lastname: Gupta
authors.forename: Vijay K
authors.initials: VK
authors.lastname: Chaudhary
authors.forename: Rajiv
authors.initials: R
authors.lastname: Bhat
mesh_terms: D000372:Agglutination Tests; D000915:Antibody Affinity; D020650:Combinatorial Chemistry Techniques; D019020:Directed Molecular Evolution; D004912:Erythrocytes / Q000276:immunology*; D015723:Gene Library; D015658:HIV Infections / Q000175:diagnosis* / Q000276:immunology; D015497:HIV-1 / Q000276:immunology*; D015498:HIV-2 / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics*; D040681:Structural Homology, Protein
chemical_list: D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins