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

優(yōu)化生產(chǎn)用于過繼免疫治療的臨床級 NK 細(xì)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.

自然殺傷細(xì)胞已顯示出治療不同惡性腫瘤的希望。已經(jīng)描述了幾種方法來獲得完全活化的 NK 細(xì)胞以供臨床使用。在這里，我們使用不同的細(xì)胞培養(yǎng)基和不同的人工抗原呈遞細(xì)胞來優(yōu)化符合GMP的制造方法，以獲得適合臨床使用的活化和擴增的NK細(xì)胞。

細(xì)胞工廠

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.

自然殺傷 (NK) 細(xì)胞代表了癌癥免疫治療的有希望的工具。我們報告了 NK 細(xì)胞活化-擴增過程的優(yōu)化及其在臨床規(guī)模上的驗證。方法：使用RPMI-1640、干細(xì)胞生長培養(yǎng)基（SCGM）、NK MACS和TexMACS作為培養(yǎng)基。通過將總外周血單核細(xì)胞 (PBMC) 或 CD45RA +細(xì)胞與照射過的 K562mbIL15-41BBL 或 K562mbIL21-41BBL 共培養(yǎng)，獲得活化和擴增的 NK 細(xì)胞 (NKAE)。分析了倍數(shù)增加、NK 細(xì)胞純度、活化狀態(tài)、細(xì)胞毒性和轉(zhuǎn)錄組譜。臨床級 NKAE 細(xì)胞在 CliniMACS Prodigy 中制造。結(jié)果：NK MACS 和 TexMACs 實現(xiàn)了最高的 NK 細(xì)胞純度和最低的 T 細(xì)胞污染。從 CD45RA +獲得 NKAE 細(xì)胞細(xì)胞是可行的，盡管 PBMC 產(chǎn)生的總細(xì)胞數(shù)和 NK 細(xì)胞純度高于 CD45RA +細(xì)胞。使用 PBMC 和 K562mbIL21-41BBL 細(xì)胞實現(xiàn)了最高倍數(shù)擴增和 NK 純度。然而，當(dāng)使用 NK 細(xì)胞源或激活細(xì)胞系時，沒有發(fā)現(xiàn)激活和細(xì)胞毒性方面的差異。基礎(chǔ) NK 細(xì)胞和用 K562mbIL21-41BBL 或 K562mbIL15-41BBL 擴增的 NKAE 細(xì)胞之間的轉(zhuǎn)錄組譜顯示不同。臨床級制造的 NKAE 細(xì)胞符合西班牙監(jiān)管機構(gòu)的規(guī)范。結(jié)論：使用不同的起始細(xì)胞和aAPC可以獲得GMP級NK細(xì)胞供臨床使用。

高效搖瓶

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.

三角細(xì)胞搖瓶

在本報告中，我們優(yōu)化了一種方案，通過使用四種不同的培養(yǎng)生長培養(yǎng)基（RPMI、SCGM、TexMACs 和 NKMACs）、兩種不同的 NK 細(xì)胞來源：PBMC 或 CD45RA +細(xì)胞和兩種不同的輻照 aAPC（K562mbIL15 或 K562mbIL21）來獲得 NKAE 細(xì)胞. 我們確定 TexMACs 是最適合擴增 NK 細(xì)胞的細(xì)胞培養(yǎng)基。NK 細(xì)胞可以從那些 CD45RA +激活和擴增盡管使用 PBMC 作為 NK 細(xì)胞來源獲得了最高數(shù)量的純化 NKAE 細(xì)胞，但從非動員單采獲得的細(xì)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. 從用 K562mbIL15 或 K562mbIL21 擴增的 PBMC 或 CD45RA+ 獲得的所有 NKAE 細(xì)胞對肉瘤、T-ALL、CML、神經(jīng)母細(xì)胞瘤和橫紋肌肉瘤細(xì)胞顯示出相當(dāng)?shù)目鼓[瘤能力。最后，我們使用 CD56 +在自動化封閉系統(tǒng) CliniMACS Prodigy 中完成了 NKAE 細(xì)胞的臨床制造。細(xì)胞和輻照 K562mbIL15 或 K562mbIL21。在這兩個過程中，在培養(yǎng) 14 天后，制造了足夠數(shù)量的高純度和低 T 細(xì)胞污染的 NKAE 細(xì)胞。進(jìn)行的不同釋放測試表明，制造的 NKAE 細(xì)胞符合監(jiān)管機構(gòu)的要求和規(guī)范，因此適合臨床使用。

關(guān)鍵詞： NK細(xì)胞免疫治療,NK細(xì)胞活化和擴增,NKAE細(xì)胞,臨床級制造,CliniMACS 神童,NK cell immunotherapy,NK cell activation and expansion,NKAE cells,clinical-grade manufacturing,CliniMACS Prodigy

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