Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings. Wild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section. Wild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622–2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127–2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0–18% CV across consecutive tissue sections and 5–20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625). This work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.

中文翻译：

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质谱法量化 KRASG12C 抑制剂在 FFPE 肿瘤组织中的靶标参与情况

药物与靶点结合的量化对于确认药物达到预期的蛋白质靶点、了解作用机制和解释剂量反应关系至关重要。对于共价抑制剂，可以通过治疗前后的游离靶标水平来推断靶标接合。靶向质谱分析可对复杂生物样品中的蛋白质进行精确定量，并已常规应用于临床前研究中，以量化冷冻肿瘤组织中的靶点参与情况，以用于肿瘤药物开发。然而，临床试验中通常无法获得冷冻组织，因此至关重要的是，检测适用于福尔马林固定石蜡包埋 (FFPE) 组织，以便将基于质谱的靶标参与研究扩展到临床环境。通过高度优化的靶向质谱分析对 FFPE 组织中的野生型 RAS 和 RASG12C 进行定量，该分析将高场不对称波形离子迁移谱 (FAIMS) 和平行反应监测 (PRM) 与内标相结合。在样本子集中，通过分析来自同一肿瘤块的连续组织切片来评估技术再现性，并获取同一组织切片中相邻肿瘤区域之间的生物学变异。通过 FAIMS-PRM 质谱法测量 32 个临床非小细胞肺癌肿瘤 (622–2525 amol/μg) 中的野生型 RAS 蛋白。具有已知 KRASG12C 突变的肿瘤 (n = 17) 表达多种 RASG12C 突变蛋白 (127–2012 amol/μg)。野生型 RAS 和 RASG12C 测量值的变化在连续组织切片中的 CV 范围为 0-18%，在相邻组织区域中的 CV 范围为 5-20%。然后在来自用 RASG12C 抑制剂 (AZD4625) 处理的 2 个异种移植模型（MIA PaCa-2 和 NCI-H2122）的 FFPE 组织中证明了定量靶点参与。这项工作说明了通过分析 FFPE 肿瘤活检来扩展基于质谱的蛋白质组学在临床前和临床肿瘤药物开发中的潜力。