Reduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides

详细信息    查看全文

• 作者：Michael M. C. Sun ; Kevin S. Beam ; Charles G. Cerveny ; Kevin J. Hamblett ; Richard S. Blackmore ; Michael Y. Torgov ; Felicia G. M. Handley ; Nathan C. Ihle ; Peter D. Senter ; and Stephen C. Alley
• 刊名：Bioconjugate Chemistry
• 出版年：2005
• 出版时间：September 2005
• 年：2005
• 卷：16
• 期：5
• 页码：1282 - 1290
• 全文大小：282K
• 年卷期：v.16,no.5(September 2005)
• ISSN：1520-4812

文摘

Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utilityin the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise controlover the location of conjugation would yield highly homogeneous materials that could have improvedbiological properties. We describe for the first time chemical reduction and oxidation methods thatlead 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 thepotent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugationlocation by purification with hydrophobic interaction chromatography followed by analysis usingreversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated thattreating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chaindisulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferentialreformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugateshad isomeric homogeneity as high as 60-90%, allowing for control of the distribution of molecularspecies. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated atefficacious doses.