Lai Katharine C, Deckert Jutta, Setiady Yulius Y, Shah Prerak, Wang Lintao, Chari Ravi, Lambert John M
ImmunoGen, Inc., Waltham, Massachusetts, USA.
Pharm Res. 2015 Nov;32(11):3593-603. doi: 10.1007/s11095-015-1633-2. Epub 2015 Jan 29.
Many antibody-drug conjugates (ADCs) become active only after antigen-mediated internalization and release of the cytotoxic agent via antibody degradation. Quantifying these processes can provide critical information on the suitability of a particular receptor target or antibody for ADC therapy by providing insight into the amount of cytotoxic agent released. We describe a simple and inexpensive radiolabel assay to monitor this process in cultured cancer cells.
Monoclonal antibodies were trace-labeled at their lysine residues by treatment with the N-hydroxysuccinimide ester of [(3)H]propionic acid. Human cancer cell cultures were treated with the labeled antibody at concentrations sufficient to saturate the targeted antigen. After washing to remove unbound antibody, cells were incubated and analyzed for antigen expression, conjugate degradation and catabolite formation. Results were compared with data obtained from similar assays run with radiolabeled antibody-[(3)H]maytansinoid conjugates ([(3)H]AMCs). To exemplify the method, studies were conducted with a panel of [(3)H]propionamide-antibodies to evaluate processing efficiency in EGFR-expressing SCCHN cell lines, and in NHL cell lines expressing the B-cell targets CD19, CD20, CD22 and CD37.
Use of the [(3)H]propionamide-antibody assay yielded cell-mediated processing results similar to those obtained with corresponding maytansinoid ADCs. Further exploration allowed comparison of expression levels, antigen-dependent degradation, and catabolite formation across a panel of EGFR-expressing SCCHN cell lines, and for multiple targets in various B-cell cancer indications.
The [(3)H]propionamide-antibody assay described here is a sensitive, facile method which enables rapid and robust assessment of relative antibody processing amounts for target, antibody, and cell line evaluation.
许多抗体药物偶联物（ADC）只有在抗原介导的内化以及通过抗体降解释放细胞毒性药物后才会变得具有活性。通过深入了解细胞毒性药物的释放量，对这些过程进行定量分析可为特定受体靶点或抗体用于ADC治疗的适用性提供关键信息。我们描述了一种简单且成本低廉的放射性标记检测方法，用于监测培养的癌细胞中的这一过程。
通过用[（3）H]丙酸的N-羟基琥珀酰亚胺酯处理，对单克隆抗体的赖氨酸残基进行微量标记。用人癌细胞培养物以足以饱和靶向抗原的浓度处理标记的抗体。洗涤以去除未结合的抗体后，将细胞孵育并分析抗原表达、偶联物降解和分解代谢物形成。将结果与用放射性标记的抗体-[（3）H]美登素类偶联物（[（3）H]AMC）进行的类似检测获得的数据进行比较。为了举例说明该方法，用一组[（3）H]丙酰胺-抗体进行了研究，以评估表达表皮生长因子受体（EGFR）的头颈部鳞状细胞癌（SCCHN）细胞系以及表达B细胞靶点CD19、CD20、CD22和CD37的非霍奇金淋巴瘤（NHL）细胞系中的处理效率。
使用[（3）H]丙酰胺-抗体检测获得的细胞介导的处理结果与相应的美登素类ADC获得的结果相似。进一步的探索使得能够比较一组表达EGFR的SCCHN细胞系中的表达水平、抗原依赖性降解和分解代谢物形成，以及各种B细胞癌适应症中的多个靶点。
本文所述的[（3）H]丙酰胺-抗体检测是一种灵敏、简便的方法，能够快速且可靠地评估用于靶点、抗体和细胞系评估的相对抗体处理量。
