Structure-based engineering of a monoclonal antibody for improved solubility.
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.
Wu
Sheng-Jiun
SJ
Biologics Research, Centocor R&D, 145 King of Prussia Radnor, PA 19087-4557, USA. swu4@its.jnj.com
Luo
Jinquan
J
O'Neil
Karyn T
KT
Kang
James
J
Lacy
Eilyn R
ER
Canziani
Gabriela
G
Baker
Audrey
A
Huang
Maggie
M
Tang
Qing Mike
QM
Raju
T Shantha
TS
Jacobs
Steven A
SA
Teplyakov
Alexey
A
Gilliland
Gary L
GL
Feng
Yiqing
Y
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
D000911:Antibodies, Monoclonal; D018793:Interleukin-13