title: Affinity maturation of a humanized rat antibody for anti-RAGE therapy: comprehensive mutagenesis reveals a high level of mutational plasticity both inside and outside the complementarity-determining regions.
abstract: Antibodies that neutralize RAGE (receptor for advanced glycation end products)-ligand interactions have potential therapeutic applications in both acute and chronic diseases. We generated XT-M4, a rat anti-RAGE monoclonal antibody that has in vivo efficacy in an acute sepsis model. This antibody was subsequently humanized. To improve the affinity of this antibody for the treatment of chronic indications, we used random and targeted mutagenesis strategies in combination with ribosome and phage-display technologies, respectively, to generate libraries of XT-M4 variants. We identified a panel of single-chain Fv antibody fragments (scFv's) that was improved up to 110-fold in a homogeneous time-resolved fluorescence competition assay against parental XT-M4 immunoglobulin G (IgG). After reformatting to bivalent scFv-Fc fusions and IgGs, we observed similar gains in potency in the same assay. Further analysis of binding kinetics as IgG revealed multiple variants with subnanomolar apparent affinity that was dictated primarily by improvements in the off-rate. All variants also had improved binding to cell surface-expressed human RAGE, and all retained, or had improved, apparent affinity for mouse RAGE. F100bL in V(H) (variable region of the heavy chain) complementarity-determining region 3 (CDR3) was one of a number of key mutations that correlated with affinity improvements and was independently identified by both mutagenesis strategies. Random mutagenesis coupled with ribosome display and high-throughput screening revealed an unexpectedly high level of mutational plasticity across the whole length of the humanized scFv, suggesting greater scope for structural optimization outside of the primary antigen-combining site defined by V(H) CDR3 and V(kappa) CDR3. In summary, our comprehensive mutagenesis approach not only achieved the desired affinity maturation of XT-M4 but also defined multiple mutational hotspots across the antibody sequence, provided an insight into the specificity-determining residues of the antibody paratope, and identified additional sites within the CDR loops where human germ-line amino acids may be introduced without affecting function.
authors.affiliation: Wyeth Research Ireland, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: William J
authors.initials: WJ
authors.lastname: Finlay
authors.forename: Orla
authors.initials: O
authors.lastname: Cunningham
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Lambert
authors.forename: Alfredo
authors.initials: A
authors.lastname: Darmanin-Sheehan
authors.forename: Xuemei
authors.initials: X
authors.lastname: Liu
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Fennell
authors.forename: Ciara M
authors.initials: CM
authors.lastname: Mahon
authors.forename: Emma
authors.initials: E
authors.lastname: Cummins
authors.forename: Jason M
authors.initials: JM
authors.lastname: Wade
authors.forename: Cliona M
authors.initials: CM
authors.lastname: O'Sullivan
authors.forename: Xiang Yang
authors.initials: XY
authors.lastname: Tan
authors.forename: Nicole
authors.initials: N
authors.lastname: Piche
authors.forename: Debra D
authors.initials: DD
authors.lastname: Pittman
authors.forename: Janet
authors.initials: J
authors.lastname: Paulsen
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Tchistiakova
authors.forename: Sreekumar
authors.initials: S
authors.lastname: Kodangattil
authors.forename: Davinder
authors.initials: D
authors.lastname: Gill
authors.forename: Simon E
authors.initials: SE
authors.lastname: Hufton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology*; D000915:Antibody Affinity; D022801:Complementarity Determining Regions / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D005470:Fluorometry; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D051381:Rats; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors* / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic