title: A general approach to antibody thermostabilization.
abstract: Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.
authors.affiliation: a AnaptysBio, Inc. ; San Diego , CA USA.
authors.forename: Audrey D
authors.initials: AD
authors.lastname: McConnell
authors.forename: Xue
authors.initials: X
authors.lastname: Zhang
authors.forename: John L
authors.initials: JL
authors.lastname: Macomber
authors.forename: Betty
authors.initials: B
authors.lastname: Chau
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Sheffer
authors.forename: Sorena
authors.initials: S
authors.lastname: Rahmanian
authors.forename: Eric
authors.initials: E
authors.lastname: Hare
authors.forename: Vladimir
authors.initials: V
authors.lastname: Spasojevic
authors.forename: Robert A
authors.initials: RA
authors.lastname: Horlick
authors.forename: David J
authors.initials: DJ
authors.lastname: King
authors.forename: Peter M
authors.initials: PM
authors.lastname: Bowers
mesh_terms: D000068879:Adalimumab; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D002152:Calorimetry, Differential Scanning; D061505:Cell Surface Display Techniques; D000068818:Cetuximab; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology*; D000069448:Denosumab; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D027041:Somatic Hypermutation, Immunoglobulin; D013696:Temperature; D000068878:Trastuzumab
chemical_list: D000906:Antibodies; D061067:Antibodies, Monoclonal, Humanized; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D000069448:Denosumab; D000068879:Adalimumab; D000068878:Trastuzumab; D000068818:Cetuximab
keywords: CDR, complementarity-determining region; CH2, heavy chain constant domain 2; CH3, heavy chain constant domain 3; DSC, differential scanning calorimetry; HC, heavy chain; LC, light chain; NGF, β-nerve growth factor; SHM, somatic hypermutation; SPR, surface plasmon resonance; TNF, tumor necrosis factor; Tm, melting temperature; VH, heavy chain variable region; VL, light chain variable region; affinity maturation; monoclonal antibodies; protein engineering; solubility; somatic hypermutation; thermostability