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- Title
Structure-based engineering of a monoclonal antibody for improved solubility.
- Authors
Sheng-Jiun Wu; Jinquan Luo; O'Neil, Karyn T.; Kang, James; Lacy, Eilyn R.; Canziani, Gabriela; Baker, Audrey; Huang, Maggie; Tang, Qing Mike; Raju, T. Shantha; Jacobs, Steven A.; Teplyakov, Alexey; Gilliland, Gary L.; Yiqing Feng
- 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 (99FHW100a). 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.
- Subjects
MONOCLONAL antibodies; SOLUBILITY; GLYCOSYLATION; PROTEIN engineering; ANTIGEN-antibody reactions; PROTEINS
- Publication
PEDS: Protein Engineering, Design & Selection, 2010, Vol 23, Issue 8, p643
- ISSN
1741-0126
- Publication type
Article
- DOI
10.1093/protein/gzq037