Efficient Preparation of Site-Specific Antibody鈥揇rug Conjugates Using Phosphopantetheinyl Transferases

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• 作者：Jan Gr眉newald ; Heath E. Klock ; Susan E. Cellitti ; Badry Bursulaya ; Daniel McMullan ; David H. Jones ; Hsien-Po Chiu ; Xing Wang ; Paula Patterson ; Huanfang Zhou ; Julie Vance ; Edward Nigoghossian ; Hung Tong ; Dylan Daniel ; William Mallet ; Weijia Ou ; Tetsuo Uno ; Ansgar Brock ; Scott A. Lesley ; Bernhard H. Geierstanger
• 刊名：Bioconjugate Chemistry
• 出版年：2015
• 出版时间：December 16, 2015
• 年：2015
• 卷：26
• 期：12
• 页码：2554-2562
• 全文大小：402K
• ISSN：1520-4812

文摘

Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody鈥揹rug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.