Structure-based engineering of a monoclonal antibody for improved solubility

Protein Eng Des Sel. 2010 Aug;23(8):643-51. doi: 10.1093/protein/gzq037. Epub 2010 Jun 11.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / metabolism
  • Binding Sites
  • Calorimetry, Differential Scanning
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Interleukin-13 / antagonists & inhibitors
  • Interleukin-13 / metabolism
  • Isoelectric Focusing
  • Isoelectric Point
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Mapping
  • Protein Conformation*
  • Protein Engineering / methods*
  • Protein Multimerization
  • Protein Stability*
  • Solubility
  • Temperature

Substances

  • Antibodies, Monoclonal
  • Interleukin-13