Digital PCR Assays for Precise Quantification of CD19-CAR-T Cells after Treatment with Axicabtagene Ciloleucel
Digital PCR Assays for Precise Quantification of CD19-CAR-T Cells after Treatment with Axicabtagene Ciloleucel
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DOI：
10.1016/j.omtm.2019.12.018
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发表时间：
2020-03-13
影响因子：
4.7
通讯作者：
Ayuk, Francis A.
中科院分区：
文献类型：
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作者：
Fehse, Boris;Badbaran, Anita;Ayuk, Francis A.
已批准使用Axicabtagene ciloleucel（Axi-cel）CD 19-CAR-T（嵌合抗原受体T）细胞治疗难治性/复发性弥漫性大B细胞淋巴瘤（DLBCL）和原发性纵隔大B细胞淋巴瘤（PMBCL）。由于治疗的成功以及副作用可能取决于体内CAR-T细胞的扩增，我们的目标是开发用于检测和定量CAR-T细胞的数字PCR（dPCR）测定。为此，我们克隆并测序了CAR构建体的完整cDNA。我们设计了位于不同CAR区域的引物和双标记水解探针的不同组合。在与参考基因（REF）的双重反应中，在CAR阳性和阴性细胞上成功测试了三种组合，以同时评估细胞数量。所有试验均表现出良好的专属性和重现性，试验间变异可忽略不计。对于所有三种测定，观察到两种dPCR（Axi-cel与REF）之间几乎完美的相关性，并且检测限是一个单个CAR转导的细胞，对应于100 ng基因组DNA的0.01%的灵敏度。交叉验证后，我们使用一种检测方法来监测患者中的Axi-cel CAR-T数量。CAR-T扩增和收缩遵循预期动力学，在11.3天（中位数）时中位峰值为11.2 Axi-cel CAR-T细胞/μ L。在临床上，我们在CAR-T细胞峰数低于中位数的5名患者中仅观察到2例部分应答（PR），而在第30天，5名扩增相对良好的患者中有4名显示出临床应答（2例完全应答[CR]和2例PR）。总之，我们建立了一种用于转基因CAR-T细胞敏感检测的新型dPCR测定法，这在Axi-cel治疗的背景下应该非常有用。
Treatment with axicabtagene ciloleucel (Axi-cel) CD19-CAR-T (chimeric antigen receptor T) cells has been approved for refractory/relapsed diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). Because treatment success as well as side effects might depend on CAR-T cell expansion in vivo, we aimed at developing digital PCR (dPCR) assays for detection and quantification of CAR-T cells. To this end, we cloned and sequenced the complete cDNA of the CAR construct. We designed different combinations of primers and dual-labeled hydrolysis probes located in various CAR regions. Three combinations were successfully tested on CAR-positive and -negative cells in duplex reactions with a reference gene (REF) to concomitantly assess cell numbers. All assays demonstrated excellent specificity and reproducibility with neglectable inter-assay variations. For all three assays, almost perfect correlation between the two dPCRs (Axi-cel versus REF) was observed, and the limit of detection was one single CAR-transduced cell corresponding to a sensitivity of 0.01% for 100 ng genomic DNA. After cross-validation, we used one assay to monitor Axi-cel CAR-T numbers in patients. CAR-T expansion and contraction followed the expected kinetics with median peak value of 11.2 Axi-cel CAR-T cells/mu L at 11.3 days (median). Clinically, we observed only two partial responses (PRs) in the five patients with CAR-T cell peak numbers below median, whereas four of the five patients with comparatively good expansion showed clinical responses (two complete responses [CRs] and two PRs) on day 30. In conclusion, we established a novel dPCR assay for the sensitive detection of transgenic CAR-T cells, which should be very useful in the context of Axi-cel treatment.