title: An integrated approach to extreme thermostabilization and affinity maturation of an antibody.
abstract: Antibodies are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. However, in order to be practically useful, antibodies must be highly stable and bind their target antigens with high affinity. We present a combinatorial approach to generate high-affinity, highly stable antibodies through the design of stable frameworks, specificity grafting and maturation via somatic hypermutation in vitro. By collectively employing these methods, we have engineered a highly stable, high-affinity, full-length antibody with a T(m) over 90°C that retains significant activity after heating to 90°C for 1 h, and has ~95-fold improved antigen-binding affinity. The stabilized IgG framework is compatible with affinity maturation, and should provide a broadly useful scaffold for grafting a variety of complementarity-determining region loops for the development of stable antibodies with desired specificities.
authors.affiliation: AnaptysBio, Inc., 10421 Pacific Center Court, Suite 200, San Diego, CA 92121, USA. amcconnell@anaptysbio.com
authors.forename: Audrey D
authors.initials: AD
authors.lastname: McConnell
authors.forename: Vladimir
authors.initials: V
authors.lastname: Spasojevich
authors.forename: John L
authors.initials: JL
authors.lastname: Macomber
authors.forename: Irina P
authors.initials: IP
authors.lastname: Krapf
authors.forename: Andy
authors.initials: A
authors.lastname: Chen
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Sheffer
authors.forename: Ashley
authors.initials: A
authors.lastname: Berkebile
authors.forename: Robert A
authors.initials: RA
authors.lastname: Horlick
authors.forename: Steven
authors.initials: S
authors.lastname: Neben
authors.forename: David J
authors.initials: DJ
authors.lastname: King
authors.forename: Peter M
authors.initials: PM
authors.lastname: Bowers
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D036022:Capsid Proteins / Q000276:immunology; D061505:Cell Surface Display Techniques; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics; D003198:Computer Simulation; D003553:Cystine / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution; D057809:HEK293 Cells; D006358:Hot Temperature; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D017909:Levivirus / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D009010:Monte Carlo Method; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D058767:Protein Unfolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics; D044366:Transition Temperature
chemical_list: D036022:Capsid Proteins; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D003553:Cystine