免疫细胞治疗制剂体外杀伤效力评价方法的分析研究

In vitro cytotoxicity assays for potency evaluation of immune cells prepared for immunotherapy

目的 通过对4种体外杀伤检测方法的方法学优化及分析比较,以期寻找到一种可以替代经典51Cr释放法、相对快速、并可用于多种具有杀伤活性免疫细胞的体外杀伤效力评价方法.方法 以自然杀伤性NK细胞系NK-92为效应细胞,以K562细胞为靶细胞,分别对4种体外杀伤检测方法BATDA法、CAM法、CytoTox-Glo法和PKH法的标记条件、杀伤时间、效靶比等参数进行优化,分析每种方法的实验内及实验间可重复性,以及与51Cr释放法的相关性,并初步用于不同靶细胞、效应细胞的广谱性分析.结果 经方法学优化后,4种体外杀伤方法在一定范围内均可显示出杀伤效力与效靶比的相关性,但与其他3种方法相比,CytoTox-Glo法在高效靶比时（40∶1）表现出明显的倒钩效应;4种方法中,BATDA法的检测时间最短,NK-92细胞与K562细胞作用1 h即可检测出明显的杀伤效力,CAM法及PHK法基本上都需要作用4 h才能测出明显的杀伤效力,而CytoTox-Glo法可能需要6～8 h才能看出明显的杀伤结果;4种方法的实验内与实验间可重复性均相对较好,其中BATDA法及CAM法表现出更好的可重复性;4种方法与51Cr释放法均具有一定的相关性,但BATDA法与51Cr释放法曲线的拟合度更好;与其他3种方法相比,BATDA法可用于贴壁性的靶细胞Caov3的杀伤效力检测,且在自体NK细胞和CD19-CAR T细胞的杀伤检测中亦显示出较好的结果.结论与其他3种方法相比,BATDA法的检测结果与51Cr释放法的线性相关性最好,作用时间短、方法的可重复性好,并且对贴壁性靶细胞的检测也比其他方法有优势,BATDA法具有替代51Cr释放法用于NK及其他免疫细胞的体外杀伤效力评价的应用前景.

Objective To optimize and compare four in vitro cytotoxicity assays in order to find a relatively rapid assay that can replace the traditional 51Cr release assay to evaluate the cytotoxicity of immune cells prepared for immunotherapy.Methods Four assays including BATDA, CAM （calcein acetoxymethyl ester）, CytoTox-Glo and PKH were optimized and used to measure the in vitro cytotoxicity of NK-92 cells to K562 cell line.Intra-and inter-assay reproducibility of these assays and their correlation with 51Cr release assay were analyzed.Results After optimization, all of the four cytotoxicity assays showed good correlation with effector to target （E/T） ratio in a certain range.Compared with the other three assays, CytoTox-Glo assay showed obvious hook effect at a high E/T ratio of 40∶1.BATDA assay could detect the significant cytotoxicity of NK-92 cells to K562 cells after incubating them for one hour and that was the shortest time taken by the four assays to detect in vitro cytotoxicity.Both CAM and PKH assays took about four hours and CytoTox-Glo assay took six to eight hours to detect the significant cytotoxic potency.All of the four assays, especially BATDA and CAM assays, showed good intra-and inter-assay reproducibility.Among these assays, BATDA assay showed the best correlation with the traditional 51Cr release assay.BATDA assay, as compared with the other three assays, could be used to detect the cytotoxicity of Caov3 cells, an adherent cell line, and showed good results in evaluating the cytotoxic potency of autologous primary NK cells and CD19-CAR T cells.Conclusion Compared with the other three assays, BATDA assay shows the best linear correlation with 51Cr release assay and has the advantages of time-saving and good reproducibility.Besides, it is a better assay for detecting the cytotoxicity of adherent cells.BATDA assay is a promising substitute for 51Cr release assay in evaluating the in vitro cytotoxic potency of NK cells and other immune cells.