Bridging Disulfides for Stable and Defined Antibody Drug Conjugates
Bridging Disulfides for Stable and Defined Antibody Drug Conjugates
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DOI：
10.1021/bc500148x
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发表时间：
2014-06-01
影响因子：
4.7
通讯作者：
Godwin, Antony
中科院分区：
文献类型：
--
作者：
Badescu, George;Bryant, Penny;Godwin, Antony
为了提高ADC的均一性和稳定性，我们开发了位点特异性药物缀合试剂，其共价地重新桥接还原的二硫键。新试剂包含药物、接头和双反应性缀合部分，所述双反应性缀合部分能够与来源于抗体和抗体片段中还原的二硫键的硫原子进行反应。制备包含单甲基澳瑞他汀E（MMAE）的二硫键再桥接试剂并将其缀合至曲妥珠单抗（TRA）。实现了抗体向ADC的78%转化，药物与抗体比率（DAR）为4，没有剩余未缀合的抗体。还将MMAE再桥接试剂缀合至源自TRA的蛋白水解消化的Fab的链间二硫键，以得到均匀的单一药物缀合产物。所得偶联物保留抗原结合，在血清中稳定，并在体外和体内癌症模型中表现出强效且抗原选择性的细胞杀伤作用。二硫键再桥接缀合是制备稳定ADC的一般方法，其不需要将抗体重组再工程化用于位点特异性缀合。
To improve both the homogeneity and the stability of ADCs, we have developed site-specific drug-conjugating reagents that covalently rebridge reduced disulfide bonds. The new reagents comprise a drug, a linker, and a bis-reactive conjugating moiety that is capable of undergoing reaction with both sulfur atoms derived from a reduced disulfide bond in antibodies and antibody fragments. A disulfide rebridging reagent comprising monomethyl auristatin E (MMAE) was prepared and conjugated to trastuzumab (TRA). A 78% conversion of antibody to ADC with a drug to antibody ratio (DAR) of 4 was achieved with no unconjugated antibody remaining. The MMAE rebridging reagent was also conjugated to the interchain disulfide of a Fab derived from proteolytic digestion of TRA, to give a homogeneous single drug conjugated product. The resulting conjugates retained antigen-binding, were stable in serum, and demonstrated potent and antigen-selective cell killing in in vitro and in vivo cancer models. Disulfide rebridging conjugation is a general approach to prepare stable ADCs, which does not require the antibody to be recombinantly re-engineered for site-specific conjugation.