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Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry
Published on: July 20, 2016
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High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based
Rachana Ohri1, Sunil Bhakta1, Aimee Fourie-O'Donohue1
1Genentech Inc. , 1 DNA Way, South San Francisco, California 94080, United States.
Bioconjugate Chemistry
|February 10, 2018
Summary
This study identified stable antibody conjugation sites for drug delivery using THIOMAB technology. These findings enhance the development of antibody-drug conjugates with improved stability and efficacy.
Area of Science:
- Bioconjugation Chemistry
- Antibody Engineering
- Pharmacology
Background:
- THIOMAB antibody technology enables site-specific conjugation via engineered cysteines.
- Conjugation site location impacts linkage stability and antibody conjugate properties.
- Stable linkages are crucial for maximizing drug delivery to target sites.
Purpose of the Study:
- To systematically evaluate drug conjugation stabilities at all engineered cysteine positions on an anti-HER2 antibody (trastuzumab).
- To identify optimal conjugation sites for enhanced stability of antibody-drug conjugates (ADCs).
Main Methods:
- Engineered cysteine residues into every position of trastuzumab, creating 648 THIOMAB antibody variants.
- Conjugated variants to two monomethyl auristatin E (MMAE) derivatives (MC-vc-PAB-MMAE and PDS-MMAE) using high-throughput methods.
- Assessed in vitro plasma stability using ELISAs and confirmed stable sites with LC/MS.
Main Results:
- Over 50% of THIOMAB variants achieved successful conjugation with desired drug-to-antibody ratios and low aggregation.
- Identified numerous highly stable conjugation sites on both heavy and light chains for both MMAE derivatives.
- Maleimide conjugates showed greater stability than disulfide conjugates, yet stable sites were found for both types.
Conclusions:
- Highly stable conjugation sites for antibody-drug conjugates were identified across the antibody structure.
- The stability of identified sites translated across different payloads, target antibodies, and in vivo conditions.
- This systematic mapping advances the rational design of more stable and effective antibody-drug conjugates.
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