p53 Laboratory (p53Lab), Agency for Science, Technology, and Research (A(∗)STAR), Singapore 138648, Singapore.
Division of Cellular & Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore, Singapore 169610, Singapore.
Cell Rep. 2018 Jan 2;22(1):299-312. doi: 10.1016/j.celrep.2017.11.112.
The large number of mutations identified across all cancers represents an untapped reservoir of targets that can be useful for therapeutic targeting if highly selective, mutation-specific reagents are available. We report here our attempt to generate such reagents: monoclonal antibodies against the most common R175H, R248Q, and R273H hotspot mutants of the tumor suppressor p53. These antibodies recognize their intended specific alterations without any cross-reactivity against wild-type (WT) p53 or other p53 mutants, including at the same position (as exemplified by anti-R248Q antibody, which does not recognize the R248W mutation), evaluated by direct immunoblotting, immunoprecipitation, and immunofluorescence methods on transfected and endogenous proteins. Moreover, their clinical utility to diagnose the presence of specific p53 mutants in human tumor microarrays by immunohistochemistry is also shown. Together, the data demonstrate that antibodies against specific single-amino-acid alterations can be generated reproducibly and highlight their utility, which could potentially be extended to therapeutic settings.
在所有癌症中发现的大量突变代表了一个未开发的靶点库，如果有高度选择性、针对突变的试剂可用，这些靶点可以用于治疗性靶向。我们在此报告我们生成此类试剂的尝试：针对肿瘤抑制因子 p53 的最常见 R175H、R248Q 和 R273H 热点突变体的单克隆抗体。这些抗体识别其预期的特定改变，而与野生型 (WT) p53 或其他 p53 突变体（包括相同位置的突变，如抗 R248Q 抗体不识别 R248W 突变）没有任何交叉反应，通过直接免疫印迹、免疫沉淀和转染和内源性蛋白质的免疫荧光方法进行评估。此外，还通过免疫组织化学在人类肿瘤微阵列中显示了它们用于诊断特定 p53 突变体存在的临床实用性。总之，这些数据表明可以可重复地生成针对特定单个氨基酸改变的抗体，并强调了它们的实用性，这可能扩展到治疗环境中。
