PDF(Pubmed)
Abstract：
For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 -) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.
摘要：
对于先进的治疗药物，需要开发和验证效力测定，根据国际准则，表征产品并获得可靠和一致的数据。我们的目的是验证用于评估自体抗CD19嵌合抗原受体(CAR)T效力的杀伤测定。我们使用CD4+和CD8+淋巴细胞或抗CD19CAR-T细胞作为效应细胞,使用REH(CD19+)或MOLM-13(CD19-)细胞系作为靶细胞。在共培养靶细胞和效应细胞（1：1比例）24小时后，样品用7-AAD标记，通过流式细胞术评估抗CD3和抗CD19抗体以及CD19+死细胞的频率。为了验证CAR-T对CD19+靶标的特异性，安排了CAR-T和REH或MOLM-13以不同的效应器-靶标比进行共培养.此外，测试未转导的CD4+和CD8+淋巴细胞与来自同一供体的CAR-T进行比较,以证明测定特异性.线性和准确性进行了评估，并为这两个参数编制了既定的验收标准(线性为r2≥0.97，准确性为平均相对误差≤10%)。此外，在共培养23到25小时之间进行时，该方法被认为是稳健的，和内部测定，获得了测定间和日间精确度。最后,为了验证分析师之间的精确度，该检验由3个不同的操作者执行,两种情况下类内相关系数均>0.4.总之,我们认为该CAR-T效价测定在产品开发和质量控制的所有步骤中都是有效的.
