Rinat-Pfizer Inc. , 230 East Grand Avenue, South San Francisco, California 94080, United States.
Bioconjug Chem. 2014 Feb 19;25(2):240-50. doi: 10.1021/bc4003794. Epub 2014 Jan 8.
Antibody drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical conjugation methods that yield heterogeneous mixtures of varying number of drugs attached at different positions. The potential benefits of site-specific drug conjugation in terms of stability, manufacturing, and improved therapeutic index has recently led to the development of several new site-specific conjugation technologies. However, detailed characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme-based site-specific antibody-drug conjugation technology that we recently developed. We found that, in addition to conjugation of the engineered site, a small amount of aglycosylated antibody present in starting material led to conjugation at position Q295, resulting in approximately 1.3% of off-target conjugation. Based on our detection limits, we show that Q295N mutant eliminates the off-target conjugation yielding highly homogeneous conjugates that are better than 99.8% site-specific. Our study demonstrates the importance of detailed characterization of ADCs and describes methods that can be utilized to characterize not only our enzyme based conjugates, but also ADCs generated by other conjugation technologies.
抗体药物偶联物（ADCs）正在成为治疗癌症的一种重要的新型治疗剂。ADC 是通过将细胞毒性小分子（药物）与靶向肿瘤细胞的单克隆抗体连接而产生的。传统上，大多数 ADC 依赖于化学偶联方法，这些方法产生不同数量的药物连接在不同位置的不均匀混合物。在稳定性、制造和提高治疗指数方面，定点药物偶联的潜在好处最近导致了几种新的定点偶联技术的发展。然而，目前缺乏对定点偶联程度的详细表征。在这项研究中，我们利用质谱法来表征我们最近开发的基于酶的定点抗体药物偶联技术的定点特异性程度。我们发现，除了工程化位点的偶联外，起始材料中存在的少量去糖基化抗体导致 Q295 位点的偶联，导致约 1.3%的非靶标偶联。基于我们的检测限，我们表明 Q295N 突变消除了非靶标偶联，得到了高度均一的偶联物，其特异性优于 99.8%。我们的研究表明了详细表征 ADC 的重要性，并描述了可用于表征不仅基于酶的偶联物，还可用于表征其他偶联技术产生的 ADC 的方法。
