NK-92及NK92/MI细胞系在研究及临床应用中一直备受关注，其完全培养基也一直是赛库生物畅销产品。我们将出系列文章，分析NK-92的来历和应用。
2022年是NK-92细胞的建系30周年，其建系科学家 Hans klingemann在CYTOTHERAPY杂志发表文章“The NK-92 cell line-30 years later:its impact on natural killer cell research and treatment of cancer”,文中不仅总结了过往30年中NK-92细胞系的广泛使用给细胞研究及肿瘤治疗领域带来的影响，也回顾了NK-92细胞的发现历史。为了方便科研工作者更好了解NK-92细胞系的特性，我们截取了文中关于NK-92细胞系发现简史的章节并翻译成中文，供大家在使用这个细胞系的过程中参考。
2022年是NK-92细胞的建系30周年，其建系科学家 Hans klingemann在CYTOTHERAPY杂志发表文章“The NK-92 cell line-30 years later:its impact on natural killer cell research and treatment of cancer”,文中不仅总结了过往30年中NK-92细胞系的广泛使用给细胞研究及肿瘤治疗领域带来的影响，也回顾了NK-92细胞的发现历史。为了方便科研工作者更好了解NK-92细胞系的特性，我们截取了文中关于NK-92细胞系发现简史的章节并翻译成中文，供大家在使用这个细胞系的过程中参考。
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细胞系的名字来源于其发现年份（1992年），它是由本文作者在加拿大温哥华的不列颠哥伦比亚癌症署的实验室中，从一位被诊断为侵袭性NK细胞淋巴瘤患者的单核血细胞中建立起来的。该患者血液中存在一定数量的循环NK细胞，这些细胞具有早期NK细胞的特征。为了建立这个细胞系，当时在温哥华的特里·福克斯实验室（该实验室的主要科学焦点是干细胞生物学）可获得的各种培养基都经过了测试。最初的培养基MEMα，其中含有12.5%的马血清和12.5%的小牛血清，以及其他添加剂。这种培养基以Myelocult（Stemcell Technologies, Vancouver, Canada）的名称进行商业销售。NK-92细胞以悬浮方式生长，形成细胞团，单个细胞直径为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.
由于含有动物成分的培养基不适合任何临床开发，因此在随后的几年里，人们努力寻找一种无动物成分的培养基，以维持NK-92细胞的最佳生长/扩增和功能。研究发现，用5%人血清补充的X-Vivo 10培养基（Lonza Bioscience, Basel, Switzerland）能够满足这些要求。值得注意的是，细胞所处的培养基会影响NK-92细胞表面分子的表达及其功能。由于美国ATCC在其网站上推荐使用含有马血清和小牛血清的原始培养基，许多研究人员仍在使用这种特定的培养基。培养基的影响体现在NK-92细胞表面分子表达的一个例子是NKp44，只有当NK-92细胞在含有动物血清的培养基中培养时才会表达该分子（Boissel, L. (2016)）。图1总结了在用5%人血清的X-Vivo 10培养时临床级NK-92的表面抗原谱。
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.
由于含有动物成分的培养基不适合任何临床开发，因此在随后的几年里，人们努力寻找一种无动物成分的培养基，以维持NK-92细胞的最佳生长/扩增和功能。研究发现，用5%人血清补充的X-Vivo 10培养基（Lonza Bioscience, Basel, Switzerland）能够满足这些要求。值得注意的是，细胞所处的培养基会影响NK-92细胞表面分子的表达及其功能。由于美国ATCC在其网站上推荐使用含有马血清和小牛血清的原始培养基，许多研究人员仍在使用这种特定的培养基。培养基的影响体现在NK-92细胞表面分子表达的一个例子是NKp44，只有当NK-92细胞在含有动物血清的培养基中培养时才会表达该分子（Boissel, L. (2016)）。图1总结了在用5%人血清的X-Vivo 10培养时临床级NK-92的表面抗原谱。
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细胞表达完整的激活受体谱，CD94/NKG2A和LIR1是唯一已知的抑制受体[14]。最重要的是，这些细胞几乎不表达杀伤细胞免疫球蛋白样受体（KIR），除了KIR2DL4[14]。由于KIR受体已知会在与人类白细胞抗原（HLA）结合时抑制细胞毒性活性，这在一定程度上可以解释为什么NK-92细胞在患者体内即使经过多次输注也只表现出适度的免疫原性[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细胞系需要在培养基中存在白介素（IL）-2（或IL-15）来支持其持续生长和扩增，因此通过将IL-2基因转染到亲本细胞中，产生了两种不依赖IL-2的NK-92变体[16]。NK-92ci细胞系是以用于生成该变体的pCEP4-LTR质粒载体命名的，而NK-92mi细胞系是以MFG载体构建物命名的。这两种构建物都是通过颗粒介导基因转移（Bio-Rads“基因枪”）的方法转染到亲本细胞中的，这种方法通常具有较低的转染效率。然而，由于转染后的细胞是在缺乏IL-2的培养基中生长的，因此只有那些成功转染并表达IL-2基因的少数细胞能够生长和扩增。所选的NK-92ci和NK-92mi变体能够产生足够的IL-2来维持自身的生长、扩增和细胞毒性活性[16]（表1）。值得注意的是，与NK-92ci细胞相比，NK-92mi细胞产生和分泌的IL-2浓度显著更高。
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.
所有三种NK-92细胞系都已存入美国ATCC和德国DSMZ。自20世纪90年代中期以来，研究人员一直可以使用这些细胞系。这些细胞作为研究工具的广泛可用性，导致了大量科学出版物的出现，这些出版物不仅有助于更好地理解NK-92细胞特性，而且有助于更好地理解NK细胞特性。
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，美国约翰逊航天中心（美国德克萨斯州休斯顿）使用NK-92细胞来确定长期太空飞行是否会损害NK细胞的功能[17]。从长期太空飞行返回的宇航员的血液样本与NK-92细胞共孵育，以测量身体是否会因太空飞行产生任何血液/血清变化，从而对NK细胞的功能产生负面影响。研究证实并非如此。
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].
与经过富集和最佳激活的血液来源NK细胞相比，NK-92细胞对恶性细胞表现出持续的高细胞毒性活性。NK-92细胞具有广泛的、持续的高细胞毒性活性，原因有以下几点：（i）与血液来源的IL-2依赖性NK细胞相比，NK-92细胞含有更多的颗粒酶[15,16]，因此NK-92细胞的细胞毒性效应在远低于血液NK细胞的效应细胞与靶细胞比例下即可发生；（ii）NK-92细胞仅表达一种抑制性KIR家族受体，这种受体在遇到无关（异体）细胞时会抑制NK细胞的功能[13]；（iii）NK-92细胞表达多种激活受体，如NKG2D以及与肿瘤靶标结合相关的黏附受体（白细胞功能相关抗原-1、细胞间黏附分子）[14,16]；（iv）NK-92细胞在缺氧肿瘤组织微环境中不会失去活性[18]；（v）与血液NK细胞相比，NK-92细胞能够更大程度地连续杀伤靶细胞[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].
人们很早就认识到NK-92细胞有可能被开发用于治疗疾病，特别是癌症。大量的体外和体内研究证实，未经改造的亲本NK-92能够有效杀伤人类癌细胞系以及原发癌细胞[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.
为了成功地将这些细胞开发用于患者治疗，需要解决两个主要问题：（i）由于NK-92细胞源自一位淋巴瘤患者的细胞，因此存在接受者发生淋巴瘤的风险；（ii）由于NK-92细胞的异体性质，可能会被患者排斥。为了应对第一个问题，体外研究表明，1000 cGy的辐射足以完全阻止NK-92细胞的增殖[14,25]。当免疫缺陷的NSG小鼠皮下注射经过至少500 cGy辐射的NK-92细胞时，没有观察到肿瘤生长，这使得人们得出结论，1000 cGy的辐射足以安全地防止患者体内细胞的增殖。重要的是，经过辐射的细胞在输注前至少16~24小时内保持了完整的细胞毒性，包括细胞因子的产生。因此，在迄今为止进行的所有使用亲本NK-92细胞或其改造变体的临床研究中，细胞在输注前都经过了1000~1500 cGy的辐射处理。
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.
为了应对免疫原性问题，UCLA免疫遗传学中心进行了实验，将异体供体淋巴细胞（分为CD4和CD8细胞）暴露于NK-92细胞。没有观察到T细胞的显著增殖，并且T细胞产生的IFN-γ作为异体刺激的第二个指标也没有显著增加[未发表数据]。在多伦多[15]和法兰克福[26]进行的I期研究中也观察到了相对温和的异体反应。尽管进行了重复输注，但在多伦多研究的12名患者中，只有6名患者产生了针对NK-92的HLA抗体，而且以患者淋巴细胞作为反应细胞的混合淋巴细胞培养在所有患者样本中均为阴性。法兰克福研究也报告了类似的结果。
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细胞具有可预测的快速生长特性（24~36小时的倍增时间）和易于扩增的特点，NK-92细胞已成为CAR基因工程的“热门”效应NK细胞，尤其是用于引入CAR。德国法兰克福的Winfried Wels博士和Torsten Tonn博士团队是第一个描述使用HER-2作为CAR的CAR改造临床级NK-92细胞的研究团队[27]。一些关于CAR改造NK-92细胞的研究已在最近的综述论文中进行了总结[13,28~30]。研究人员通常使用第一代和第二代CAR构建体，这些构建体通常通过慢病毒或逆转录病毒载体进行传递。尽管这些研究表明CAR改造的NK-92细胞具有疗效，但由于安全性和监管原因，病毒载体并不适合临床应用。因此，目前ImmunityBio公司进行的CAR改造NK-92细胞的临床试验（ImmunityBio公司拥有NK-92及其变体的全球权利）使用非病毒的、基于质粒的CAR基因构建物，通过简单的电穿孔进行转染。
原文链接：
https://www.isct-cytotherapy.org/article/S1465-3249(22)01075-1/fulltext
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