Light Chain Q166C Mutation Permits One-step Site Specific Conjugation on Monoclonal Antibodies.
Chembiochem
; 24(13): e202200780, 2023 07 03.
Article
in En
| MEDLINE
| ID: mdl-37079449
ABSTRACT
Engineered cysteines are frequently used for site-specific conjugation in antibody-drug conjugate (ADC) development. When cysteine-engineered mAbs are produced in the cell culture process, the sulfhydryl groups on the engineered cysteines are mostly in an oxidized form. The oxidized cysteines require multiple steps (such as reduction, reoxidation, and buffer exchanges) to reactivate for bioconjugation, which complicates the ADC production process and reduces yields. In this study, we identified a Q166C mutation in the light chain that allows the presence of free sulfhydryl groups during cell culture and purification process. This mutation is in the constant region and away from sites involved in antigen binding or Fc-mediated functions. The free sulfhydryl reacts readily with maleimide in a mild solution at a high conjugation rate. This is only the second such site reported (the first one is Q124C in the light chain). Using the Q166C mutation, we conjugated an anti-angiopoietin-2 (Ang-2) peptide on bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, to construct a peptide antibody conjugate, Ava-Plus, which could block two pro-angiogenic factors simultaneously. Ava-Plus showed high affinity for both VEGF and Ang-2 and demonstrated higher activity than bevacizumab in inâ
vitro cell migration and inâ
vivo mouse xenograft models.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Immunoconjugates
/
Antibodies, Monoclonal
Type of study:
Prognostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
Chembiochem
Journal subject:
BIOQUIMICA
Year:
2023
Document type:
Article
Affiliation country:
China