title: Structure-based engineering of a monoclonal antibody for improved solubility.
abstract: Protein aggregation is of great concern to pharmaceutical formulations and has been implicated in several diseases. We engineered an anti-IL-13 monoclonal antibody CNTO607 for improved solubility. Three structure-based engineering approaches were employed in this study: (i) modifying the isoelectric point (pI), (ii) decreasing the overall surface hydrophobicity and (iii) re-introducing an N-linked carbohydrate moiety within a complementarity-determining region (CDR) sequence. A mutant was identified with a modified pI that had a 2-fold improvement in solubility while retaining the binding affinity to IL-13. Several mutants with decreased overall surface hydrophobicity also showed moderately improved solubility while maintaining a similar antigen affinity. Structural studies combined with mutagenesis data identified an aggregation 'hot spot' in heavy-chain CDR3 (H-CDR3) that contains three residues ((99)FHW(100a)). The same residues, however, were found to be essential for high affinity binding to IL-13. On the basis of the spatial proximity and germline sequence, we reintroduced the consensus N-glycosylation site in H-CDR2 which was found in the original antibody, anticipating that the carbohydrate moiety would shield the aggregation 'hot spot' in H-CDR3 while not interfering with antigen binding. Peptide mapping and mass spectrometric analysis revealed that the N-glycosylation site was generally occupied. This variant showed greatly improved solubility and bound to IL-13 with affinity similar to CNTO607 without the N-linked carbohydrate. All three engineering approaches led to improved solubility and adding an N-linked carbohydrate to the CDR was the most effective route for enhancing the solubility of CNTO607.
authors.affiliation: Biologics Research, Centocor R&D, 145 King of Prussia Radnor, PA 19087-4557, USA. swu4@its.jnj.com
authors.forename: Sheng-Jiun
authors.initials: SJ
authors.lastname: Wu
authors.forename: Jinquan
authors.initials: J
authors.lastname: Luo
authors.forename: Karyn T
authors.initials: KT
authors.lastname: O'Neil
authors.forename: James
authors.initials: J
authors.lastname: Kang
authors.forename: Eilyn R
authors.initials: ER
authors.lastname: Lacy
authors.forename: Gabriela
authors.initials: G
authors.lastname: Canziani
authors.forename: Audrey
authors.initials: A
authors.lastname: Baker
authors.forename: Maggie
authors.initials: M
authors.lastname: Huang
authors.forename: Qing Mike
authors.initials: QM
authors.lastname: Tang
authors.forename: T Shantha
authors.initials: TS
authors.lastname: Raju
authors.forename: Steven A
authors.initials: SA
authors.lastname: Jacobs
authors.forename: Alexey
authors.initials: A
authors.lastname: Teplyakov
authors.forename: Gary L
authors.initials: GL
authors.lastname: Gilliland
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018793:Interleukin-13 / Q000037:antagonists & inhibitors / Q000378:metabolism; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D055550:Protein Stability; D012995:Solubility; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D018793:Interleukin-13