Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays.
Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays.
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DOI：
10.1158/1078-0432.ccr-15-2323
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发表时间：
2016-07-15
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影响因子：
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通讯作者：
Parchment RE
中科院分区：
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作者：
Srivastava AK;Hollingshead MG;Weiner J;Navas T;Evrard YA;Khin SA;Ji JJ;Zhang Y;Borgel S;Pfister TD;Kinders RJ;Bottaro DP;Linehan WM;Tomaszewski JE;Doroshow JH;Parchment RE
合理开发用于癌症治疗的靶向MET抑制剂需要定量了解靶向药效学，包括分子靶点接合、作用机制和作用持续时间。开发并验证了夹心免疫测定法和酶处理程序，用于定量核心肿瘤活检中的全长MET及其关键磷酸化物质（pMET）。使用针对细胞外结构域的抗体捕获MET，然后使用针对其C末端（全长）和含有pY 1234/1235、pY 1235和pY 1356的表位的抗体进行探测。使用pMET：MET比率作为测定终点，在MET扩增和补偿（VEGFR阻断）模型中表征MET抑制剂药效学。通过将冷缺血时间限制在2分钟以内，MET抑制剂PHA 665752和PF 02341066（克唑替尼）的药效学作用可使用人胃癌异种移植物（GTL-16和SNU 5）的芯针活检进行定量。一次给药在4小时内使pY 1234/1235 MET：MET、pY 1235-MET：MET和pY 1356-MET：MET比率降低60%-80%，但尽管持续每日给药，该作用并未完全持续。帕唑帕尼的VEGFR阻断增加了pY 1235-MET：MET和pY 1356-MET：MET比率，这被tivantinib逆转。在从切除的遗传性乳头状肾癌中获得的5个芯针样本中，全长MET可定量，但pMET物质的水平接近测定的定量下限。这些经验证的MET信号传导药效学生物标志物的免疫测定适用于研究扩增癌症中的MET应答以及对VEGFR阻断的代偿应答。将药效学生物标志物研究纳入MET抑制剂的临床试验可以为该领域提供关键的机制验证和概念验证。
Rational development of targeted MET inhibitors for cancer treatment requires a quantitative understanding of target pharmacodynamics, including molecular target engagement, mechanism of action, and duration of effect. Sandwich immunoassays and specimen-handling procedures were developed and validated for quantifying full-length MET and its key phosphospecies (pMET) in core tumor biopsies. MET was captured using an antibody to the extracellular domain and then probed using antibodies to its C-terminus (full-length) and epitopes containing pY1234/1235, pY1235, and pY1356. Using pMET:MET ratios as assay endpoints, MET inhibitor pharmacodynamics were characterized in MET-amplified and -compensated (VEGFR blockade) models. By limiting cold ischemia time to less than two min, the pharmacodynamic effects of the MET inhibitors PHA665752 and PF02341066 (crizotinib) were quantifiable using core needle biopsies of human gastric carcinoma xenografts (GTL-16 and SNU5). One dose decreased pY1234/1235 MET:MET, pY1235-MET:MET, and pY1356-MET:MET ratios by 60%-80% within 4 h, but this effect was not fully sustained despite continued daily dosing. VEGFR blockade by pazopanib increased pY1235-MET:MET and pY1356-MET:MET ratios, which was reversed by tivantinib. Full-length MET was quantifiable in 5 of 5 core needle samples obtained from a resected hereditary papillary renal carcinoma, but the levels of pMET species were near the assay lower limit of quantitation. These validated immunoassays for pharmacodynamic biomarkers of MET signaling are suitable for studying MET responses in amplified cancers as well as compensatory responses to VEGFR blockade. Incorporating pharmacodynamic biomarker studies into clinical trials of MET inhibitors could provide critical proof-of-mechanism and proof-of-concept for the field.