Clinical grade purification and expansion of NK cell products for an optimized manufacturing protocol
Clinical grade purification and expansion of NK cell products for an optimized manufacturing protocol
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DOI：
10.3389/fonc.2013.00118
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发表时间：
2013-01-01
影响因子：
4.7
通讯作者：
Esser, Ruth
中科院分区：
文献类型：
--
作者：
Koehl, Ulrike;Brehm, Claudia;Esser, Ruth
同种异体自然杀伤（NK）细胞用于干细胞移植后的过继免疫治疗。为了克服 NK 细胞纯化和激活的技术限制，以下研究调查了良好生产规范 (GMP) 级 NK 细胞选择过程中不同变量对 NK 细胞回收、细胞毒性和 T 细胞耗竭的影响。使用免疫磁性 CD3 T 细胞耗竭，然后进行 CD56 细胞富集步骤，从 54 个未刺激的供体白细胞中提取出 40 种 NK 细胞产品。对于 T 细胞去除，在 CliniMACS 仪器上使用单程序或双程序中的去除 2.1 程序（D2.1(1depl)，n = 18；D2.1(2depl)，n = 13）或更快的去除 3.1（D3.1，n = 9）。 17 种纯化的 NK 细胞产物在体外被 IL-2 激活 12 天。整个过程产生中位数为 7.59 x 10(8) CD56(+)CD3(-) 细胞，纯度和活力分别为 94%。与 D3.1 相比，使用 D2.1(1depl/2depl) 的 T 细胞消耗明显更好（log 4.6/log 4.9 与 log 3.7；p < 0.01），并且两个阶段的双重程序总是导致残留 T 细胞低于 0.1%。相反，在CD56(+)CD3(-)NK细胞的回收方面，D3.1优于D2.1(1depl/2depl)(68％对41％/38％)。与未刺激的 NK 细胞相比，伴随的单核细胞特别是 IL-2 激活导致 NK 细胞针对恶性靶细胞的活性增加，这与天然细胞毒性受体和细胞内信号传导的上调相关。总体而言，NK 细胞扩增率和 NK 细胞亚群分布存在很大差异。总之，我们的结果表明 NK 细胞的 GMP 级纯化可以通过 T 细胞消除程序 D2.1 和 D3.1 的连续处理得到改善。此外，NK 细胞扩增方案需要进一步优化。
Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.1(1depl), n = 18; D2.1(2depl), n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 x 10(8) CD56(+)CD3(-) cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.1(1depl/2depl) compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.1(1depl/2depl) with regard to recovery of CD56(+)CD3(-) NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.