NK-92及NK92/MI細(xì)胞系在研究及臨床應(yīng)用中一直備受關(guān)注，其完全培養(yǎng)基也一直是賽庫生物暢銷產(chǎn)品。我們將出系列文章，分析NK-92的來歷和應(yīng)用。
2022年是NK-92細(xì)胞的建系30周年，其建系科學(xué)家 Hans klingemann在CYTOTHERAPY雜志發(fā)表文章“The NK-92 cell line-30 years later:its impact on natural killer cell research and treatment of cancer”,文中不僅總結(jié)了過往30年中NK-92細(xì)胞系的廣泛使用給細(xì)胞研究及腫瘤治療領(lǐng)域帶來的影響，也回顧了NK-92細(xì)胞的發(fā)現(xiàn)歷史。為了方便科研工作者更好了解NK-92細(xì)胞系的特性，我們截取了文中關(guān)于NK-92細(xì)胞系發(fā)現(xiàn)簡史的章節(jié)并翻譯成中文，供大家在使用這個細(xì)胞系的過程中參考。
2022年是NK-92細(xì)胞的建系30周年，其建系科學(xué)家 Hans klingemann在CYTOTHERAPY雜志發(fā)表文章“The NK-92 cell line-30 years later:its impact on natural killer cell research and treatment of cancer”,文中不僅總結(jié)了過往30年中NK-92細(xì)胞系的廣泛使用給細(xì)胞研究及腫瘤治療領(lǐng)域帶來的影響，也回顧了NK-92細(xì)胞的發(fā)現(xiàn)歷史。為了方便科研工作者更好了解NK-92細(xì)胞系的特性，我們截取了文中關(guān)于NK-92細(xì)胞系發(fā)現(xiàn)簡史的章節(jié)并翻譯成中文，供大家在使用這個細(xì)胞系的過程中參考。
The NK-92 cell line, named after the year of discovery (1992), was established in the author’s laboratory at the British Columbia Cancer Agency in Vancouver, Canada, from mononuclear blood cells from a patient who had been diagnosed with an aggressive NK-cell lymphoma [11]. The patient had a relatively high number of circulating NK cells that displayed characteristics of early NK cells. To establish the cell line, various culture media were tested that were available at that time at the Terry Fox Laboratory in Vancouver, Canada, whose major scientific focus is on stem cell biology. The initial culture medium consisted of Minimum Essential Medium Alpha with 12.5% horse serum and 12.5% fetal calf serum in addition to other additives. The medium is commercially available under the name Myelocult (Stemcell Technologies, Vancouver, Canada). NK-92 cells are grown in suspension, forming clumps with individual cells being 10-12 micron in diameter.
NK-92細(xì)胞系的名字來源于其發(fā)現(xiàn)年份（1992年），它是由本文作者在加拿大溫哥華的不列顛哥倫比亞癌癥署的實驗室中，從一位被診斷為侵襲性NK細(xì)胞淋巴瘤患者的單核血細(xì)胞中建立起來的。該患者血液中存在一定數(shù)量的循環(huán)NK細(xì)胞，這些細(xì)胞具有早期NK細(xì)胞的特征。為了建立這個細(xì)胞系，當(dāng)時在溫哥華的特里·福克斯實驗室（該實驗室的主要科學(xué)焦點是干細(xì)胞生物學(xué)）可獲得的各種培養(yǎng)基都經(jīng)過了測試。最初的培養(yǎng)基MEMα，其中含有12.5%的馬血清和12.5%的小牛血清，以及其他添加劑。這種培養(yǎng)基以Myelocult（Stemcell Technologies, Vancouver, Canada）的名稱進(jìn)行商業(yè)銷售。NK-92細(xì)胞以懸浮方式生長，形成細(xì)胞團(tuán)，單個細(xì)胞直徑為10~12微米。
Since animal-based media are unsuitable for any clinical development, efforts were made in the ensuing years to identify an animal-free medium that would maintain both optimal growth/
expansion and functionality of NK-92. The X-Vivo 10 medium (Lonza Bioscience, Basel, Switzerland) supplemented with 5% human serum was found to meet those requirements. Of note, the medium in which the cells are maintained can affect the expression of surface molecules and the functionality of NK-92 cells. Since the American Type Culture Collection recommends—on their website—the use of the original culture medium containing horse and fetal calf serum, many researchers continue to use that particular medium. An example for the effect of the culture medium used is reflected in the surface molecule expression of NKp44, which is only expressed when NK-92 cells are maintained in culture medium that contains animal serum (Boissel, L. (2016)). Figure 1 summarizes the surface antigen profile of clinical-grade NK-92 when maintained in X-Vivo 10 with 5% human serum.
由于含有動物成分的培養(yǎng)基不適合任何臨床開發(fā)，因此在隨后的幾年里，人們努力尋找一種無動物成分的培養(yǎng)基，以維持NK-92細(xì)胞的最佳生長/擴(kuò)增和功能。研究發(fā)現(xiàn)，用5%人血清補(bǔ)充的X-Vivo 10培養(yǎng)基（Lonza Bioscience, Basel, Switzerland）能夠滿足這些要求。值得注意的是，細(xì)胞所處的培養(yǎng)基會影響NK-92細(xì)胞表面分子的表達(dá)及其功能。由于美國ATCC在其網(wǎng)站上推薦使用含有馬血清和小牛血清的原始培養(yǎng)基，許多研究人員仍在使用這種特定的培養(yǎng)基。培養(yǎng)基的影響體現(xiàn)在NK-92細(xì)胞表面分子表達(dá)的一個例子是NKp44，只有當(dāng)NK-92細(xì)胞在含有動物血清的培養(yǎng)基中培養(yǎng)時才會表達(dá)該分子（Boissel, L. (2016)）。圖1總結(jié)了在用5%人血清的X-Vivo 10培養(yǎng)時臨床級NK-92的表面抗原譜。
Since animal-based media are unsuitable for any clinical development, efforts were made in the ensuing years to identify an animal-free medium that would maintain both optimal growth/
expansion and functionality of NK-92. The X-Vivo 10 medium (Lonza Bioscience, Basel, Switzerland) supplemented with 5% human serum was found to meet those requirements. Of note, the medium in which the cells are maintained can affect the expression of surface molecules and the functionality of NK-92 cells. Since the American Type Culture Collection recommends—on their website—the use of the original culture medium containing horse and fetal calf serum, many researchers continue to use that particular medium. An example for the effect of the culture medium used is reflected in the surface molecule expression of NKp44, which is only expressed when NK-92 cells are maintained in culture medium that contains animal serum (Boissel, L. (2016)). Figure 1 summarizes the surface antigen profile of clinical-grade NK-92 when maintained in X-Vivo 10 with 5% human serum.
由于含有動物成分的培養(yǎng)基不適合任何臨床開發(fā)，因此在隨后的幾年里，人們努力尋找一種無動物成分的培養(yǎng)基，以維持NK-92細(xì)胞的最佳生長/擴(kuò)增和功能。研究發(fā)現(xiàn)，用5%人血清補(bǔ)充的X-Vivo 10培養(yǎng)基（Lonza Bioscience, Basel, Switzerland）能夠滿足這些要求。值得注意的是，細(xì)胞所處的培養(yǎng)基會影響NK-92細(xì)胞表面分子的表達(dá)及其功能。由于美國ATCC在其網(wǎng)站上推薦使用含有馬血清和小牛血清的原始培養(yǎng)基，許多研究人員仍在使用這種特定的培養(yǎng)基。培養(yǎng)基的影響體現(xiàn)在NK-92細(xì)胞表面分子表達(dá)的一個例子是NKp44，只有當(dāng)NK-92細(xì)胞在含有動物血清的培養(yǎng)基中培養(yǎng)時才會表達(dá)該分子（Boissel, L. (2016)）。圖1總結(jié)了在用5%人血清的X-Vivo 10培養(yǎng)時臨床級NK-92的表面抗原譜。
NK-92 cells express the full spectrum of activating receptors, with CD94/NKG2A and LIR1 being the only known inhibitory receptor expressed [14]. Most importantly, the cells are largely negative for killer cell immunoglobulin-like receptors (KIR), except for KIR2DL4 [14]. Since KIR receptors are known to inhibit cytotoxic activity when engaged with human leukocyte antigen (HLA), this may explain, to some extent, why NK-92 cells show only moderate immunogenicity in patients even after repeated infusions [15].
NK-92細(xì)胞表達(dá)完整的激活受體譜，CD94/NKG2A和LIR1是唯一已知的抑制受體[14]。最重要的是，這些細(xì)胞幾乎不表達(dá)殺傷細(xì)胞免疫球蛋白樣受體（KIR），除了KIR2DL4[14]。由于KIR受體已知會在與人類白細(xì)胞抗原（HLA）結(jié)合時抑制細(xì)胞毒性活性，這在一定程度上可以解釋為什么NK-92細(xì)胞在患者體內(nèi)即使經(jīng)過多次輸注也只表現(xiàn)出適度的免疫原性[15]。
Because the original NK-92 cell line requires the presence of interleukin (IL)-2 (or IL-15) in the culture medium to support continuous growth and expansion, two IL-2 independent variants of NK-92 were generated by transfecting the gene for IL-2 into the parental cells [16]. The NK-92ci cell line was named after the pCEP4-LTR plasmid vector used to generate the variant, and the NK-92mi cell line was named after the MFG vector construct. Both constructs were transfected into the parental cells using particle mediated gene transfer (Bio-Rad’s “gene gun”), a method that generally has a low transfection efficiency. However, since the transfected cells were left to grow in the absence of IL-2 in the culture medium, only those few successfully transfected cells expressing the IL-2 gene grew out and expanded. The selected NK-92ci and NK-92mi variants were able to produce IL-2 in sufficient quantities to maintain their own growth, expansion and cytotoxic activity [16] (Table 1). Of note, the NK-92mi cells produce and secrete significantly greater concentrations of IL-2 than the NK-92ci cells.
由于原始的NK-92細(xì)胞系需要在培養(yǎng)基中存在白介素（IL）-2（或IL-15）來支持其持續(xù)生長和擴(kuò)增，因此通過將IL-2基因轉(zhuǎn)染到親本細(xì)胞中，產(chǎn)生了兩種不依賴IL-2的NK-92變體[16]。NK-92ci細(xì)胞系是以用于生成該變體的pCEP4-LTR質(zhì)粒載體命名的，而NK-92mi細(xì)胞系是以MFG載體構(gòu)建物命名的。這兩種構(gòu)建物都是通過顆粒介導(dǎo)基因轉(zhuǎn)移（Bio-Rads“基因槍”）的方法轉(zhuǎn)染到親本細(xì)胞中的，這種方法通常具有較低的轉(zhuǎn)染效率。然而，由于轉(zhuǎn)染后的細(xì)胞是在缺乏IL-2的培養(yǎng)基中生長的，因此只有那些成功轉(zhuǎn)染并表達(dá)IL-2基因的少數(shù)細(xì)胞能夠生長和擴(kuò)增。所選的NK-92ci和NK-92mi變體能夠產(chǎn)生足夠的IL-2來維持自身的生長、擴(kuò)增和細(xì)胞毒性活性[16]（表1）。值得注意的是，與NK-92ci細(xì)胞相比，NK-92mi細(xì)胞產(chǎn)生和分泌的IL-2濃度顯著更高。
All three NK-92 cell lines were deposited into the American Type Culture Collection cell line repository (Manassas, VA, USA) and the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Researchers have had access to these cell lines since the mid-1990s. The wide availability of those cells and their use as a research tool have resulted in a plethora of scientific publications that not only have contributed to a better understanding of NK-92 cell biology but also to NK cell biology in general.
所有三種NK-92細(xì)胞系都已存入美國ATCC和德國DSMZ。自20世紀(jì)90年代中期以來，研究人員一直可以使用這些細(xì)胞系。這些細(xì)胞作為研究工具的廣泛可用性，導(dǎo)致了大量科學(xué)出版物的出現(xiàn)，這些出版物不僅有助于更好地理解NK-92細(xì)胞特性，而且有助于更好地理解NK細(xì)胞特性。
For example, the Johnston Space Center (Houston, TX, USA) used NK-92 cells to determine whether long space flights can impair NKcell function [17]. Blood samples collected from astronauts returning from long space flights were co-incubated with NK-92 to measure whether the body would generate any blood/serum changes that could negatively affect the function of NK cells. The study confirmed that this was not the case.
例如，美國約翰遜航天中心（美國德克薩斯州休斯頓）使用NK-92細(xì)胞來確定長期太空飛行是否會損害NK細(xì)胞的功能[17]。從長期太空飛行返回的宇航員的血液樣本與NK-92細(xì)胞共孵育，以測量身體是否會因太空飛行產(chǎn)生任何血液/血清變化，從而對NK細(xì)胞的功能產(chǎn)生負(fù)面影響。研究證實并非如此。
NK-92 cells show consistently high cytotoxic activity against malignant cells even in comparison with enriched and optimally activated blood-derived NK cells. There are several reasons for this broad and consistently high cytolytic activity: (i) NK-92 cells have a greater content of granzymes than blood-derived IL-2 dependent NK cells [15,16], hence the cytotoxic effect with NK-92 occurs at a much lower effector-to-target ratio compared with blood NK cells; (ii) NK-92 cells express only one of the inhibitory KIR family of receptors that silence NK cell function when they encounter unrelated (allogeneic) cells [13]; (iii) NK-92 express a number of activating receptors, like NKG2D as well as adhesion receptors (leukocyte function-associated antigen-1, intercellular adhesion molecule) that are relevant for engaging with tumor targets [14,16]; (iv) NK-92 cells do not display a loss of activity in a hypoxic tumor tissue microenvironment [18], and (v) NK-92 can perform serial killing of target cells to a much greater extent that blood NK cells [19].
與經(jīng)過富集和最佳激活的血液來源NK細(xì)胞相比，NK-92細(xì)胞對惡性細(xì)胞表現(xiàn)出持續(xù)的高細(xì)胞毒性活性。NK-92細(xì)胞具有廣泛的、持續(xù)的高細(xì)胞毒性活性，原因有以下幾點：（i）與血液來源的IL-2依賴性NK細(xì)胞相比，NK-92細(xì)胞含有更多的顆粒酶[15,16]，因此NK-92細(xì)胞的細(xì)胞毒性效應(yīng)在遠(yuǎn)低于血液NK細(xì)胞的效應(yīng)細(xì)胞與靶細(xì)胞比例下即可發(fā)生；（ii）NK-92細(xì)胞僅表達(dá)一種抑制性KIR家族受體，這種受體在遇到無關(guān)（異體）細(xì)胞時會抑制NK細(xì)胞的功能[13]；（iii）NK-92細(xì)胞表達(dá)多種激活受體，如NKG2D以及與腫瘤靶標(biāo)結(jié)合相關(guān)的黏附受體（白細(xì)胞功能相關(guān)抗原-1、細(xì)胞間黏附分子）[14,16]；（iv）NK-92細(xì)胞在缺氧腫瘤組織微環(huán)境中不會失去活性[18]；（v）與血液NK細(xì)胞相比，NK-92細(xì)胞能夠更大程度地連續(xù)殺傷靶細(xì)胞[19]。
It was recognized early on that NK-92 cells could potentially be developed for the treatment of diseases, in particular cancer. Numerous studies in vitro and in vivo had confirmed that parental, nonengineered NK-92 effectively kill human cancer cell lines as well as primary cancer cells [20 24].
人們很早就認(rèn)識到NK-92細(xì)胞有可能被開發(fā)用于治療疾病，特別是癌癥。大量的體外和體內(nèi)研究證實，未經(jīng)改造的親本NK-92能夠有效殺傷人類癌細(xì)胞系以及原發(fā)癌細(xì)胞[20~24]。
For successful development of the cells for patient treatment, two major issues had to be addressed: (i) the risk for lymphoma developing in recipients due to NK-92 cells originating from a patient with lymphoma and (ii) the issue of its potential rejection due to the allogeneic nature of NK-92. To address the first concern, in vitro studies were performed that confirmed that 1000 cGy of radiation was sufficient to completely arrest the proliferation of NK-92 cells [14,25]. When immuno-compromised NSG mice were injected subcutaneously with increasing numbers of NK-92 cells that had been irradiated with at least 500 cGy, no tumor outgrowth was seen, leading to the conclusion that 1000 cGy of radiation is sufficient and safe to prevent proliferation in patients. Importantly, the irradiated cells maintained full cytotoxicity for at least 16 24 h, including cytokine production. Hence, in all clinical studies conducted so far with the parental NK-92 cells or its engineered variants, the cells have been subjected to 1000-1500 cGy of irradiation before infusion.
為了成功地將這些細(xì)胞開發(fā)用于患者治療，需要解決兩個主要問題：（i）由于NK-92細(xì)胞源自一位淋巴瘤患者的細(xì)胞，因此存在接受者發(fā)生淋巴瘤的風(fēng)險；（ii）由于NK-92細(xì)胞的異體性質(zhì)，可能會被患者排斥。為了應(yīng)對第一個問題，體外研究表明，1000 cGy的輻射足以完全阻止NK-92細(xì)胞的增殖[14,25]。當(dāng)免疫缺陷的NSG小鼠皮下注射經(jīng)過至少500 cGy輻射的NK-92細(xì)胞時，沒有觀察到腫瘤生長，這使得人們得出結(jié)論，1000 cGy的輻射足以安全地防止患者體內(nèi)細(xì)胞的增殖。重要的是，經(jīng)過輻射的細(xì)胞在輸注前至少16~24小時內(nèi)保持了完整的細(xì)胞毒性，包括細(xì)胞因子的產(chǎn)生。因此，在迄今為止進(jìn)行的所有使用親本NK-92細(xì)胞或其改造變體的臨床研究中，細(xì)胞在輸注前都經(jīng)過了1000~1500 cGy的輻射處理。
To address the immunogenicity question, the UCLA Immunogenetics Center conducted experiments exposing allogeneic donor lymphocytes (separated into CD4 and CD8 cells) to NK-92. No signifi- cantly increased proliferation of T cells was noted and INF-g production by T cells as a second readout for allogeneic stimulation was not significantly increased [unpublished data]. A relatively moderate allogeneic response was also seen in phase I studies in Toronto [15] and Frankfurt [26]. Despite repeated infusion, only six of 12 patients in the Toronto study developed HLA antibodies against NK-92, and the mixed lymphocyte culture with patient lymphocytes as
responder cells was negative in all patient samples. Similar results were reported in the Frankfurt study.
為了應(yīng)對免疫原性問題，UCLA免疫遺傳學(xué)中心進(jìn)行了實驗，將異體供體淋巴細(xì)胞（分為CD4和CD8細(xì)胞）暴露于NK-92細(xì)胞。沒有觀察到T細(xì)胞的顯著增殖，并且T細(xì)胞產(chǎn)生的IFN-γ作為異體刺激的第二個指標(biāo)也沒有顯著增加[未發(fā)表數(shù)據(jù)]。在多倫多[15]和法蘭克福[26]進(jìn)行的I期研究中也觀察到了相對溫和的異體反應(yīng)。盡管進(jìn)行了重復(fù)輸注，但在多倫多研究的12名患者中，只有6名患者產(chǎn)生了針對NK-92的HLA抗體，而且以患者淋巴細(xì)胞作為反應(yīng)細(xì)胞的混合淋巴細(xì)胞培養(yǎng)在所有患者樣本中均為陰性。法蘭克福研究也報告了類似的結(jié)果。
Largely because of its predictable fast growth characteristics (doubling time of 24 36 h) and ease of expansion, NK-92 cells have become “popular” effector NK cells for genetic engineering, particularly for introducing CARs. The groups of Dr. Winfried Wels and Dr. Torsten Tonn in Frankfurt, Germany, were the first to describe CAR engineered clinical grade NK-92 cells using HER-2 as the CAR [27]. Some of the research with CAR-engineered NK-92 cells has been summarized in recent review papers [13,28-30]. Investigators used first- and second-generation CAR constructs generally in lentiviral or retroviral constructs. Although those studies confirmed the efficacy of CAR-modified NK-92, viral vectors are not ideally suited for clinical applications for safety and regulatory reasons. Consequently, current clinical trials with CAR engineered NK-92 cells by ImmunityBio Inc., which holds the worldwide rights to NK-92 and its variants—use non-viral, plasmid-based CAR gene constructs, transfected by simple electroporation.
由于NK-92細(xì)胞具有可預(yù)測的快速生長特性（24~36小時的倍增時間）和易于擴(kuò)增的特點，NK-92細(xì)胞已成為CAR基因工程的“熱門”效應(yīng)NK細(xì)胞，尤其是用于引入CAR。德國法蘭克福的Winfried Wels博士和Torsten Tonn博士團(tuán)隊是第一個描述使用HER-2作為CAR的CAR改造臨床級NK-92細(xì)胞的研究團(tuán)隊[27]。一些關(guān)于CAR改造NK-92細(xì)胞的研究已在最近的綜述論文中進(jìn)行了總結(jié)[13,28~30]。研究人員通常使用第一代和第二代CAR構(gòu)建體，這些構(gòu)建體通常通過慢病毒或逆轉(zhuǎn)錄病毒載體進(jìn)行傳遞。盡管這些研究表明CAR改造的NK-92細(xì)胞具有療效，但由于安全性和監(jiān)管原因，病毒載體并不適合臨床應(yīng)用。因此，目前ImmunityBio公司進(jìn)行的CAR改造NK-92細(xì)胞的臨床試驗（ImmunityBio公司擁有NK-92及其變體的全球權(quán)利）使用非病毒的、基于質(zhì)粒的CAR基因構(gòu)建物，通過簡單的電穿孔進(jìn)行轉(zhuǎn)染。
原文鏈接：
https://www.isct-cytotherapy.org/article/S1465-3249(22)01075-1/fulltext
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