Site-Specific Antibody鈥揇rug Conjugation through Glycoengineering
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- 作者：Qun Zhou ; James E. Stefano ; Charlene Manning ; Josephine Kyazike ; Bo Chen ; Diego A. Gianolio ; Anna Park ; Michelle Busch ; Julie Bird ; Xiaoyang Zheng ; Helene Simonds-Mannes ; Jennifer Kim ; Rick C. Gregory ; Robert J. Miller ; William H. Brondyk ; Pradeep K. Dhal ; Clark Q. Pan
- 刊名：Bioconjugate Chemistry
- 出版年：2014
- 出版时间：March 19, 2014
- 年：2014
- 卷：25
- 期：3
- 页码：510-520
- 全文大小：470K
- 年卷期：v.25,no.3(March 19, 2014)
- ISSN：1520-4812
文摘
Antibody鈥揹rug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of 1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.