Ponte Jose F, Sun Xiuxia, Yoder Nicholas C, Fishkin Nathan, Laleau Rassol, Coccia Jennifer, Lanieri Leanne, Bogalhas Megan, Wang Lintao, Wilhelm Sharon, Widdison Wayne, Pinkas Jan, Keating Thomas A, Chari Ravi, Erickson Hans K, Lambert John M
ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451-1477, United States.
Bioconjug Chem. 2016 Jul 20;27(7):1588-98. doi: 10.1021/acs.bioconjchem.6b00117. Epub 2016 Jun 20.
Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.
抗体药物偶联物（ADCs）已成为癌症治疗中广泛研究的一种治疗方式，部分原因是ado曲妥珠单抗（赫赛汀）的临床研究结果。ado曲妥珠单抗采用Ab-SMCC-DM1形式，其中硫醇功能化的美登素类细胞毒性药物DM1通过异双功能SMCC连接子的马来酰亚胺部分与抗体（Ab）相连。ado曲妥珠单抗的药代动力学（PK）数据表明，与总抗体相比，ADC的清除速度更快。据报道，非美登素类、通过硫醇-马来酰亚胺交换连接的半胱氨酸连接的ADCs（如brentuximab vedotin）在血浆中会释放细胞毒性药物。然而，对于Ab-SMCC-DM1 ADCs，报道的主要代谢产物是赖氨酸-SMCC-DM1，这是细胞内抗体蛋白水解的预期产物。为了更好地理解这些观察结果，我们进行了一系列研究，以研究硫醇-马来酰亚胺连接的稳定性，使用靶向表皮生长因子受体（EGFR）的偶联物J2898A-SMCC-DM1，并将其与用缺乏硫醇-马来酰亚胺加合物的不可裂解连接子制成的对照ADC（J2898A-(CH2)3-DM）进行比较。我们使用放射性标记的ADCs直接测量血浆中的抗体和ADC成分。两种偶联物J2898A-SMCC-DM1和J2898A-(CH2)3-DM（每种抗体平均含有3.0至3.4个美登素类分子）的偶联抗体部分的PK特性似乎与未偶联抗体的PK特性相似。完整偶联物的清除值略快于Ab成分的清除值。此外，J2898A-SMCC-DM1的清除速度略快于J2898A-(CH2)3-DM，这表明SMCC-DM1 ADC中存在一部分美登素类药物损失，可能是通过硫醇-马来酰亚胺依赖性机制。体外稳定性实验证实Ab-SMCC-DM1偶联物中的一些美登素类药物损失可通过硫醇消除发生，但速度比报道的由抗体中内源性半胱氨酸残基还原产生的硫醇形成的硫醇-马来酰亚胺连接的相应损失速度慢，这与硫醇-马来酰亚胺稳定性与硫醇pKa相关的预期差异一致。这些发现为未来ADCs的设计策略提供了参考。
