Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab′ Synthesis

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• 作者：Zhan Zhou ; Jing Zhang ; Yan Zhang ; Guanghui Ma ; Zhiguo Su
• 刊名：Bioconjugate Chemistry
• 出版年：2016
• 出版时间：January 20, 2016
• 年：2016
• 卷：27
• 期：1
• 页码：238-246
• 全文大小：479K
• ISSN：1520-4812

文摘

Conventional preparation strategies for antibody–drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab′ fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment–drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab′ fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab′ fragment to form a Fab′-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab′-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.