Reduction−Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides
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- 1 September 2005
- journal article
- research article
- Published by American Chemical Society (ACS) in Bioconjugate Chemistry
- Vol. 16 (5), 1282-1290
- https://doi.org/10.1021/bc050201y
Abstract
Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogeneous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60−90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated at efficacious doses.Keywords
This publication has 32 references indexed in Scilit:
- cAC10-vcMMAE, an anti-CD30–monomethyl auristatin E conjugate with potent and selective antitumor activityBlood, 2003
- Contrasting IgG Structures Reveal Extreme Asymmetry and FlexibilityJournal of Molecular Biology, 2002
- Construction, Expression, and Activities of L49-sFv-β-Lactamase, a Single-Chain Antibody Fusion Protein for Anticancer Prodrug ActivationBioconjugate Chemistry, 1997
- A single expression system for the display, purification and conjugation of single-chain antibodiesGene, 1995
- Photoaffinity Labeling of Antibodies for Applications in Homogeneous fluoroimmunoassaysAnalytical Chemistry, 1995
- Cure of Xenografted Human Carcinomas by BR96-Doxorubicin ImmunoconjugatesScience, 1993
- Carbodiimide crosslinking of human Clq and rabbit IgGMolecular Immunology, 1990
- Acquistion of the covalent quaternary structure of an immunoglobulin G molecule. Theoretical reoxidation modelsBiochemistry, 1977
- Enzyme‐Linked ImmunoassayEuropean Journal of Biochemistry, 1976
- Sensitivity to reduction of human immunoglobulin G of different heavy chain sub-classesImmunochemistry, 1973