title: Affinity maturation to improve human monoclonal antibody neutralization potency and breadth against hepatitis C virus.
abstract: A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development.
authors: Department of Pathology, Stanford University School of Medicine, Stanford, California 94305.
authors: Yong
authors: Y
authors: Wang
authors: Department of Pathology, Stanford University School of Medicine, Stanford, California 94305.
authors: Zhen-Yong
authors: ZY
authors: Keck
authors: Department of Pathology, Stanford University School of Medicine, Stanford, California 94305.
authors: Anasuya
authors: A
authors: Saha
authors: Department of Pathology, Stanford University School of Medicine, Stanford, California 94305.
authors: Jinming
authors: J
authors: Xia
authors: Department of Anesthesia, University of California, San Francisco, California 94110.
authors: Fraser
authors: F
authors: Conrad
authors: Department of Anesthesia, University of California, San Francisco, California 94110.
authors: Jianlong
authors: J
authors: Lou
authors: Protein and Nucleic Acid Facility, Stanford University School of Medicine, Stanford, California 94305.
authors: Michael
authors: M
authors: Eckart
authors: Department of Anesthesia, University of California, San Francisco, California 94110.
authors: James D
authors: JD
authors: Marks
authors: Department of Pathology, Stanford University School of Medicine, Stanford, California 94305. Electronic address: sfoung@stanford.edu.
authors: Steven K H
authors: SKH
authors: Foung
mesh_terms: D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000915:Antibody Affinity; D002469:Cell Separation; D000939:Epitopes / Q000737:chemistry; D057809:HEK293 Cells; D016174:Hepacivirus / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D007167:Immunotherapy / Q000379:methods; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D009500:Neutralization Tests / Q000379:methods*
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G