Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy

优化生产用于过继免疫治疗的临床级 NK 细胞的程序

Natural Killer cells have shown promise to treat different malignancies. Several methods have been described to obtain fully activated NK cells for clinical use. Here, we use different cell culture media and different artificial antigen presenting cells to optimize a GMP compliant manufacturing method to obtain activated and expanded NK cells suitable for clinical use.

自然杀伤细胞已显示出治疗不同恶性肿瘤的希望。已经描述了几种方法来获得完全活化的 NK 细胞以供临床使用。在这里，我们使用不同的细胞培养基和不同的人工抗原呈递细胞来优化符合GMP的制造方法，以获得适合临床使用的活化和扩增的NK细胞。

细胞工厂

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation–expansion process and its validation on clinical-scale. Methods: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Conclusions: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

自然杀伤 (NK) 细胞代表了癌症免疫治疗的有希望的工具。我们报告了 NK 细胞活化-扩增过程的优化及其在临床规模上的验证。方法：使用RPMI-1640、干细胞生长培养基（SCGM）、NK MACS和TexMACS作为培养基。通过将总外周血单核细胞 (PBMC) 或 CD45RA +细胞与照射过的 K562mbIL15-41BBL 或 K562mbIL21-41BBL 共培养，获得活化和扩增的 NK 细胞 (NKAE)。分析了倍数增加、NK 细胞纯度、活化状态、细胞毒性和转录组谱。临床级 NKAE 细胞在 CliniMACS Prodigy 中制造。结果：NK MACS 和 TexMACs 实现了最高的 NK 细胞纯度和最低的 T 细胞污染。从 CD45RA +获得 NKAE 细胞细胞是可行的，尽管 PBMC 产生的总细胞数和 NK 细胞纯度高于 CD45RA +细胞。使用 PBMC 和 K562mbIL21-41BBL 细胞实现了最高倍数扩增和 NK 纯度。然而，当使用 NK 细胞源或激活细胞系时，没有发现激活和细胞毒性方面的差异。基础 NK 细胞和用 K562mbIL21-41BBL 或 K562mbIL15-41BBL 扩增的 NKAE 细胞之间的转录组谱显示不同。临床级制造的 NKAE 细胞符合西班牙监管机构的规范。结论：使用不同的起始细胞和aAPC可以获得GMP级NK细胞供临床使用。

高效摇瓶

In this report, we optimized a protocol to obtain NKAE cells by using four different culture growth media (RPMI, SCGM, TexMACs and NKMACs), two different NK cell sources: PBMC or CD45RA+ cells and two distinct irradiated aAPC (K562mbIL15 or K562mbIL21). We determined that TexMACs was the most suitable cell culture medium to expand NK cells. NK cells could be activated and expanded from those CD45RA+ cells obtained from non-mobilized apheresis, although the use of PBMC as the NK cell source yielded the highest numbers of purified NKAE cells. When K562mbIL21 was chosen as aAPC, the highest numbers of NKAE cells with less contamination of T cells were achieved regardless of the NK cell source used. All NKAE cells obtained from either PBMC or CD45RA+ expanded with K562mbIL15 or K562mbIL21 showed comparable antitumor ability against sarcoma, T-ALL, CML, neuroblastoma and rhabdomyosarcoma cells. Finally, we fulfilled clinical manufacturing of NKAE cells in an automated closed system CliniMACS Prodigy by using CD56+ cells and either irradiated K562mbIL15 or K562mbIL21. In both processes, sufficient numbers of NKAE cells with high purity and low T cell contamination were manufactured after 14 days of culture. The different release tests performed showed that manufactured NKAE cells met the requirements and specifications from the regulatory agency, and thus were suitable for clinical use.

三角细胞摇瓶

在本报告中，我们优化了一种方案，通过使用四种不同的培养生长培养基（RPMI、SCGM、TexMACs 和 NKMACs）、两种不同的 NK 细胞来源：PBMC 或 CD45RA +细胞和两种不同的辐照 aAPC（K562mbIL15 或 K562mbIL21）来获得 NKAE 细胞. 我们确定 TexMACs 是最适合扩增 NK 细胞的细胞培养基。NK 细胞可以从那些 CD45RA +激活和扩增尽管使用 PBMC 作为 NK 细胞来源获得了最高数量的纯化 NKAE 细胞，但从非动员单采获得的细胞。When K562mbIL21 was chosen as aAPC, the highest numbers of NKAE cells with less contamination of T cells were achieved regardless of the NK cell source used. 从用 K562mbIL15 或 K562mbIL21 扩增的 PBMC 或 CD45RA+ 获得的所有 NKAE 细胞对肉瘤、T-ALL、CML、神经母细胞瘤和横纹肌肉瘤细胞显示出相当的抗肿瘤能力。最后，我们使用 CD56 +在自动化封闭系统 CliniMACS Prodigy 中完成了 NKAE 细胞的临床制造。细胞和辐照 K562mbIL15 或 K562mbIL21。在这两个过程中，在培养 14 天后，制造了足够数量的高纯度和低 T 细胞污染的 NKAE 细胞。进行的不同释放测试表明，制造的 NKAE 细胞符合监管机构的要求和规范，因此适合临床使用。

关键词： NK细胞免疫治疗,NK细胞活化和扩增,NKAE细胞,临床级制造,CliniMACS 神童,NK cell immunotherapy,NK cell activation and expansion,NKAE cells,clinical-grade manufacturing,CliniMACS Prodigy

来源：MDPI https://www.mdpi.com/2072-6694/13/3/577/htm