Article(id=1322954194440814981, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1322954180431847810, articleNumber=null, orderNo=null, doi=10.16016/j.2097-0927.202507093, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1753632000000, receivedDateStr=2025-07-28, revisedDate=1763308800000, revisedDateStr=2025-11-17, acceptedDate=null, acceptedDateStr=null, onlineDate=1793346459255, onlineDateStr=2026-10-30, pubDate=1765728000000, pubDateStr=2025-12-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793346459255, onlineIssueDateStr=2026-10-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793346459255, creator=13701087609, updateTime=1793346459255, updator=13701087609, issue=Issue{id=1322954180431847810, tenantId=1146029695717560320, journalId=1319373162502258839, year='2025', volume='47', issue='23', pageStart='2881', pageEnd='3008', issueExtLink='null', onlineDate='null', pubDate='1765728000000', pubDateStr='2025-12-15', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1793346455915, creator='13701087609', updateTime=1793346455915, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=2903, endPage=2912, ext={EN=ArticleExt(id=1322954194822496646, articleId=1322954194440814981, tenantId=1146029695717560320, journalId=1319373162502258839, language=EN, title=Armored IL15 or IL15 receptor adaptors enhance proliferation and anti-tumor activity of CD19-targeted CAR-NK cells, columnId=1320434418116837551, journalTitle=Journal of Army Medical University, columnName=Basic Medicine, runingTitle=null, highlight=null, articleAbstract=
Objective

To construct CD19-targeted chimeric antigen receptor natural killer (CAR-NK) cells armored with interleukin15 (IL15) or IL15 receptor-linker (RLI) and preliminarily validate their proliferative capacity and anti-tumor activity in vitro.

Methods

Natural killer cells (NK92 and NK92MI) from patients with human malignant non-Hodgkin lymphoma were cultured, and CD19-targeted CAR-NK cells armored with IL15 or RLI were prepared using retroviral vector particles. IL15 secretion was measured by ELISA, and proliferative capacity was assessed via CFSE dilution assays. Human B-lymphocytic leukemia cells (Nalm6-GFP-Luc) and human colon cancer cells overexpressing CD19 (hCD19-SW620-GFP-Luc) were cultured, with surface CD19 expression confirmed (>99% positivity for both). Anti-tumor activity was evaluated by measuring cytotoxicity at effector-to-target (E:T) ratios using luciferase-based assays (4/12 h), detecting surface CD107a expression via flow cytometry, and quantifying cytokine release using CBA assays.

Results

NK92/NK92MI-CD19 CAR cells armored with IL15 or RLI were successfully generated. IL15 secretion was significantly higher in armored groups versus non-armored controls (P<0.01). Without IL-2 stimulation, IL15/RLI enhanced CAR-NK proliferation (P<0.05). Both armored designs significantly increased tumor-killing efficiency (P<0.05) and CD107a degranulation. IL15/RLI-armored NK92-CD19 CAR cells exhibited elevated release of IL2, IL10, IL6, TNF-α, sFas, IFN-γ, and Granulysin (P<0.05), while armored NK92MI-CD19 CAR cells showed additional increases in Granzyme A, Granzyme B, and Perforin (P<0.05).

Conclusion

IL15/RLI-armored NK92/NK92MI-CD19 CAR cells demonstrate potent anti-tumor activity, supporting their combined clinical therapeutic potential for tumors.

, authors=Mengyuan YU, Xiuying LIU, Xiaotian ZHANG, Jiaying WANG, Zhiming LING, Jianxun WANG, authorsList=Mengyuan YU, Xiuying LIU, Xiaotian ZHANG, Jiaying WANG, Zhiming LING, Jianxun WANG, authorCompany=null, correspAuthors=Jianxun WANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=© 2025 Journal of Army Medical University, copyrightOwner=Editorial Office of Journal of Army Medical University, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1322954199583031700, articleId=1322954194440814981, tenantId=1146029695717560320, journalId=1319373162502258839, language=CN, title=装甲IL15或IL15受体连接体增强靶向CD19的CAR-NK细胞增殖与抗肿瘤活性, columnId=1322954194918965639, journalTitle=陆军军医大学学报, columnName=基础医学·论著, runingTitle=null, highlight=null, articleAbstract=
目的

构建靶向CD19的装甲白细胞介素15(interleukin 15,IL15)或IL15受体连接体(receptor-linker-IL15,RLI)嵌合抗原受体NK细胞(chimeric antigen receptor NK cell,CAR-NK),并进行CAR-NK细胞体外增殖与抗肿瘤活性的初步验证。

方法

培养人恶性非霍奇金淋巴瘤患者的自然杀伤细胞(NK92和NK92MI),并用逆转录病毒载体颗粒制备靶向CD19且装甲IL15或RLI的CAR-NK细胞。采用ELISA法检测CAR-NK细胞IL15分泌水平、CFSE法检测CAR-NK细胞的增殖能力。培养人B淋巴白血病细胞Nalm6-GFP-Luc和过表达人类CD19蛋白的人结肠癌细胞hCD19-SW620-GFP-Luc,并检测其表面CD19靶抗原的表达水平。在评估CAR-NK抗肿瘤活性时,使用荧光素酶发光法检测4或12 h时CAR-NK细胞在不同效应细胞与靶细胞数量比例时的杀伤效率,并利用流式细胞术检测CAR-NK细胞肿瘤杀伤时表面CD107a表达,同时使用CBA法检测其细胞因子释放水平。

结果

成功构建了装甲IL15或RLI的NK92/NK92MI-CD19 CAR细胞,且装甲IL15或RLI的细胞组IL15分泌水平显著高于没有装甲的细胞(P<0.01)。在无IL-2刺激的情况下,IL15或RLI能够增强CAR-NK增殖能力(P<0.05)。Nalm6-GFP-Luc和hCD19-SW620-GFP-Luc细胞表面CD19高表达,阳性率分别为100.0%和99.7%,可以作为CD19 CAR的靶细胞。IL15或RLI能够增强NK92/NK92MI细胞来源的CAR-NK细胞的杀伤能力(P<0.05),以及CD107a的表达水平。装甲IL15或RLI的NK92-CD19 CAR细胞有更高的IL2、IL10、IL6、TNF-α、sFasL、IFN-γ、Granulysin释放水平(P<0.05),而装甲IL15或RLI的NK92MI-CD19 CAR细胞有更高的IL2、IL10、IL6、TNF-α、sFas、IFN-γ、Granzyme A、Granzyme B、Perforin、Granulysin释放水平(P<0.05)。

结论

装甲IL15或RLI的NK92/NK92MI-CD19 CAR细胞具有良好的抗肿瘤活性,IL15细胞因子与CAR-NK联用具有强大的临床抗肿瘤治疗应用潜力。

, authors=于梦圆, 刘秀盈, 章笑甜, 王佳颖, 凌志明, 王建勋, authorsList=于梦圆, 刘秀盈, 章笑甜, 王佳颖, 凌志明, 王建勋, authorCompany=null, correspAuthors=王建勋, authorNote=

同等贡献者

YU Mengyuan and LIU Xiuying contributed equally to the artricle.

, correspAuthorsNote=
王建勋,E-mail:
WANG Jianxun,E-mail:
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A:The transfection efficiency of BaEV-PackRV cells ;B:The transduction efficiency of CAR-NK92 cells;C:The secretion level of IL15 in CAR-NK92 cells;D:The transduction efficiency of CAR-NK92MI cells;E:The secretion level of IL15 in CAR-NK92MI cells

, figureFileSmall=RwCQL4HcfNIEdZDjz2jHEA==, figureFileBig=qfByRmLVdkx/nrNllFr4yQ==, tableContent=null), ArticleFig(id=1322954206834983363, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954194440814981, language=EN, label=Figure 3, caption=The proliferation ability of CD19 CAR-NK cells with armor IL15 or RLI, figureFileSmall=2smyMm+6TtROw9WqArsDdQ==, figureFileBig=oqQoBepcJG9wJUXnIQMQQw==, tableContent=null), ArticleFig(id=1322954206927258052, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954194440814981, language=CN, label=图3, caption=装甲IL15或RLI的CD19 CAR-NK细胞的增殖能力

A:The proliferation and viability curves of CAR-NK92 cells ;B:The proliferation ability of CAR-NK92 cells ;C:The proliferation and viability curves of CAR-NK92MI cells ;D:The proliferation ability of CAR-NK92MI cells

, figureFileSmall=2smyMm+6TtROw9WqArsDdQ==, figureFileBig=oqQoBepcJG9wJUXnIQMQQw==, tableContent=null), ArticleFig(id=1322954207023727045, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954194440814981, language=EN, label=Figure 4, caption=Expression level of CD19 on the surface of tumor cells, figureFileSmall=KebE83tUPo4ZVvX8YHoISg==, figureFileBig=Mj4LeTel2MWMPAmKG8o0LA==, tableContent=null), ArticleFig(id=1322954207095030214, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954194440814981, language=CN, label=图4, caption=肿瘤细胞表面CD19表达水平, figureFileSmall=KebE83tUPo4ZVvX8YHoISg==, figureFileBig=Mj4LeTel2MWMPAmKG8o0LA==, tableContent=null), ArticleFig(id=1322954207174721991, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954194440814981, language=EN, label=Figure 5, caption=The anti-tumor activity of CD19 CAR-NK92 cells armored with IL15 or RLI, figureFileSmall=1e1rKryvyzad4JGSx+UL2w==, figureFileBig=LI1pYZjPc5BZVOdw8yIVNw==, tableContent=null), ArticleFig(id=1322954207250219464, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954194440814981, language=CN, label=图5, caption=装甲IL15或RLI的CD19 CAR-NK92细胞的抗肿瘤活性

A:The killing efficiency of CAR-NK92 on target cells at different effector-to-target ratios and different time periods;B:The expression level of CD107a on the surface of CAR-NK92 cells;C:The secretion level of CAR-NK92 cytokin-related cytokines

, figureFileSmall=1e1rKryvyzad4JGSx+UL2w==, figureFileBig=LI1pYZjPc5BZVOdw8yIVNw==, tableContent=null), ArticleFig(id=1322954207317328329, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954194440814981, language=EN, label=Figure 6, caption=The anti-tumor activity of CD19 CAR-NK92MI cells with armor IL15 or RLI, figureFileSmall=my6b1G+TehJsyhP1omwi4g==, figureFileBig=kHEr2Zr6SvIgUPiIDTsWQQ==, tableContent=null), ArticleFig(id=1322954207388631498, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1322954194440814981, language=CN, label=图6, caption=装甲IL15或RLI的CD19 CAR-NK92MI细胞的抗肿瘤活性

A:The killing efficiency of CAR-NK92MI on target cells at different effector-to-target ratios;B:The expression level of CD107a on the surface of CAR-NK92MI cells;C:The secretion level of CAR-NK92MI cytokin-related cytokines

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装甲IL15或IL15受体连接体增强靶向CD19的CAR-NK细胞增殖与抗肿瘤活性
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于梦圆 , 刘秀盈 , 章笑甜 , 王佳颖 , 凌志明 , 王建勋
陆军军医大学学报 | 基础医学·论著 2025,47(23): 2903-2912
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陆军军医大学学报 |基础医学·论著 2025 , 47 (23) : 2903 -2912
装甲IL15或IL15受体连接体增强靶向CD19的CAR-NK细胞增殖与抗肿瘤活性
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于梦圆, 刘秀盈, 章笑甜, 王佳颖, 凌志明, 王建勋
作者信息
  • 1北京中医药大学生命科学学院,北京
通讯作者:
王建勋,E-mail:
WANG Jianxun,E-mail:
作者简介:

同等贡献者

YU Mengyuan and LIU Xiuying contributed equally to the artricle.

Armored IL15 or IL15 receptor adaptors enhance proliferation and anti-tumor activity of CD19-targeted CAR-NK cells
Mengyuan YU, Xiuying LIU, Xiaotian ZHANG, Jiaying WANG, Zhiming LING, Jianxun WANG
Affiliations
  • 1College of Life Sciences,Beijing University of Chinese Medicine,Beijing,China
出版时间: 2025-12-15 doi: 10.16016/j.2097-0927.202507093
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目的

构建靶向CD19的装甲白细胞介素15(interleukin 15,IL15)或IL15受体连接体(receptor-linker-IL15,RLI)嵌合抗原受体NK细胞(chimeric antigen receptor NK cell,CAR-NK),并进行CAR-NK细胞体外增殖与抗肿瘤活性的初步验证。

方法

培养人恶性非霍奇金淋巴瘤患者的自然杀伤细胞(NK92和NK92MI),并用逆转录病毒载体颗粒制备靶向CD19且装甲IL15或RLI的CAR-NK细胞。采用ELISA法检测CAR-NK细胞IL15分泌水平、CFSE法检测CAR-NK细胞的增殖能力。培养人B淋巴白血病细胞Nalm6-GFP-Luc和过表达人类CD19蛋白的人结肠癌细胞hCD19-SW620-GFP-Luc,并检测其表面CD19靶抗原的表达水平。在评估CAR-NK抗肿瘤活性时,使用荧光素酶发光法检测4或12 h时CAR-NK细胞在不同效应细胞与靶细胞数量比例时的杀伤效率,并利用流式细胞术检测CAR-NK细胞肿瘤杀伤时表面CD107a表达,同时使用CBA法检测其细胞因子释放水平。

结果

成功构建了装甲IL15或RLI的NK92/NK92MI-CD19 CAR细胞,且装甲IL15或RLI的细胞组IL15分泌水平显著高于没有装甲的细胞(P<0.01)。在无IL-2刺激的情况下,IL15或RLI能够增强CAR-NK增殖能力(P<0.05)。Nalm6-GFP-Luc和hCD19-SW620-GFP-Luc细胞表面CD19高表达,阳性率分别为100.0%和99.7%,可以作为CD19 CAR的靶细胞。IL15或RLI能够增强NK92/NK92MI细胞来源的CAR-NK细胞的杀伤能力(P<0.05),以及CD107a的表达水平。装甲IL15或RLI的NK92-CD19 CAR细胞有更高的IL2、IL10、IL6、TNF-α、sFasL、IFN-γ、Granulysin释放水平(P<0.05),而装甲IL15或RLI的NK92MI-CD19 CAR细胞有更高的IL2、IL10、IL6、TNF-α、sFas、IFN-γ、Granzyme A、Granzyme B、Perforin、Granulysin释放水平(P<0.05)。

结论

装甲IL15或RLI的NK92/NK92MI-CD19 CAR细胞具有良好的抗肿瘤活性,IL15细胞因子与CAR-NK联用具有强大的临床抗肿瘤治疗应用潜力。

嵌合抗原受体NK细胞  /  白细胞介素15  /  抗肿瘤活性  /  细胞增殖
Objective

To construct CD19-targeted chimeric antigen receptor natural killer (CAR-NK) cells armored with interleukin15 (IL15) or IL15 receptor-linker (RLI) and preliminarily validate their proliferative capacity and anti-tumor activity in vitro.

Methods

Natural killer cells (NK92 and NK92MI) from patients with human malignant non-Hodgkin lymphoma were cultured, and CD19-targeted CAR-NK cells armored with IL15 or RLI were prepared using retroviral vector particles. IL15 secretion was measured by ELISA, and proliferative capacity was assessed via CFSE dilution assays. Human B-lymphocytic leukemia cells (Nalm6-GFP-Luc) and human colon cancer cells overexpressing CD19 (hCD19-SW620-GFP-Luc) were cultured, with surface CD19 expression confirmed (>99% positivity for both). Anti-tumor activity was evaluated by measuring cytotoxicity at effector-to-target (E:T) ratios using luciferase-based assays (4/12 h), detecting surface CD107a expression via flow cytometry, and quantifying cytokine release using CBA assays.

Results

NK92/NK92MI-CD19 CAR cells armored with IL15 or RLI were successfully generated. IL15 secretion was significantly higher in armored groups versus non-armored controls (P<0.01). Without IL-2 stimulation, IL15/RLI enhanced CAR-NK proliferation (P<0.05). Both armored designs significantly increased tumor-killing efficiency (P<0.05) and CD107a degranulation. IL15/RLI-armored NK92-CD19 CAR cells exhibited elevated release of IL2, IL10, IL6, TNF-α, sFas, IFN-γ, and Granulysin (P<0.05), while armored NK92MI-CD19 CAR cells showed additional increases in Granzyme A, Granzyme B, and Perforin (P<0.05).

Conclusion

IL15/RLI-armored NK92/NK92MI-CD19 CAR cells demonstrate potent anti-tumor activity, supporting their combined clinical therapeutic potential for tumors.

chimeric antigen receptor NK cells  /  interleukin 15  /  anti-tumor activity  /  proliferation
于梦圆, 刘秀盈, 章笑甜, 王佳颖, 凌志明, 王建勋. 装甲IL15或IL15受体连接体增强靶向CD19的CAR-NK细胞增殖与抗肿瘤活性. 陆军军医大学学报, 2025 , 47 (23) : 2903 -2912 . DOI: 10.16016/j.2097-0927.202507093
Mengyuan YU, Xiuying LIU, Xiaotian ZHANG, Jiaying WANG, Zhiming LING, Jianxun WANG. Armored IL15 or IL15 receptor adaptors enhance proliferation and anti-tumor activity of CD19-targeted CAR-NK cells[J]. Journal of Army Medical University, 2025 , 47 (23) : 2903 -2912 . DOI: 10.16016/j.2097-0927.202507093
近年来,过继性细胞疗法(adoptive cell therapy,ACT)已在肿瘤免疫治疗中发挥了重要作用,而其中的嵌合抗原受体T细胞(chimeric antigen receptor T cell,CAR-T)细胞疗法,在血液瘤治疗领域已表现出显著的疗效。NK细胞是除T细胞和B细胞之外的第三大类淋巴细胞[1],对免疫应答有重要贡献,其对异常细胞、感染细胞和肿瘤细胞有很强的杀伤能力[2]。与T细胞不同,NK细胞的激活无需抗原提呈或主要组织相溶性复合体限制性,其功能依赖于表面激活性受体与抑制性受体的动态平衡[3]。CAR-NK疗法在临床治疗恶性肿瘤,特别是实体瘤时,增殖和持久性较差,已有许多策略来改善CAR-NK功能,其中一种是利用刺激性细胞因子来增加NK细胞的存活和扩增。
白细胞介素是一组主要由白细胞产生的细胞因子,在各种免疫细胞(包括NK细胞)的发育、分化和功能中起关键作用。白细胞介素15(interleukin 15,IL15)于1994年作为与IL2高度相关的分子而被发现的,在2008年被美国国家癌症研究所列为最具潜力的癌症免疫治疗的第一位[4]。IL15是细胞因子4-α螺旋束家族的一员,是一种分子量约为14~15 kDa的小蛋白[5]。IL15能刺激NK细胞、CD8+ T细胞等多种免疫细胞增殖,尤其对NK细胞的激活、增殖、存活和成熟具有显著的促进作用。自1994年IL15被发现以来,已有多种天然存在的IL15和IL15Rα异构体被发现。不同异构体在细胞内发挥不同功能,从调节内部信号转导到增加可溶性细胞因子分泌等。IL15受体连接体(receptor-linker-IL15,RLI)是一种由IL15和IL15Rα sushi组成的融合蛋白,sushi结构域是成熟IL15Rα的n端区域(1-66aa),包含所有负责IL15高亲和力结合的结构氨基酸。在小鼠模型中,RLI表现了有效的抗转移作用,可恢复非小细胞肺癌(non-small cell lung cancer,NSCLC)中功能受损的NK细胞,恢复肺微环境中NK细胞和中性粒细胞的平衡,增加活化受体NKp 30、NKp 44、CD107a和细胞内TNF-α的表达,使肿瘤难以逃避NK细胞的细胞毒性,在乳腺癌、结肠癌等癌症治疗中具有潜在应用价值[6]。细胞扩增与持久性是目前CAR-NK疗法发展的局限,共表达IL15来提升CAR-NK细胞活性是有效的解决方案,但目前该方案仍处早期探索阶段,疗效与安全性待进一步临床验证。
本研究以应用广泛的CD19为靶点,NK92细胞与NK92MI细胞为来源,分别构建装甲IL15或RLI的CD19 CAR-NK细胞,增强CAR-NK细胞的增殖能力与杀伤能力,以期改善CAR-NK细胞缺乏持久性的问题并提高其疗效,为CAR-NK细胞疗法的临床转化提供理论依据和实验支持。
逆转录病毒包装BaEV-PackRV细胞系由深圳细胞谷公司馈赠[7];人恶性非霍奇金淋巴瘤患者的自然杀伤细胞NK92和NK92MI细胞系购自美国典型微生物保藏中心;人B淋巴白血病细胞(Nalm6)和人结肠癌细胞(SW620)购自中国医学科学院细胞库,并均经过北京中医药大学生命科学学院整合医学中心改造携带GFP-Luc标签,SW620细胞过表达人类CD19抗原[8]。
RPMI 1640 培养基、DMEM培养基、胰蛋白酶、胎牛血清(fetal bovine serum,FBS)、PBS缓冲液购自美国Gibco公司;X-VIVO 15培养基购自美国Lonza公司;FuGene HD转染试剂购自美国Promega公司;白细胞介素2(interleukin 2,IL2)购自北京义翘神州科技股份有限公司;内切酶和CFSE 荧光染料购自北京兰博利德科技有限公司;胶回收试剂盒、T4连接酶、DH-5α感受态、质粒提取试剂盒购自南京诺唯赞生物科技股份有限公司;荧光素酶报告基因检测试剂盒购自中国碧云天生物公司;人IL15双抗夹心ELISA检测试剂盒、CBA检测试剂盒、MYC-PE、MYC-APC、CD19-APC、CD107a-APC抗体购自美国Biolegend公司。
以CD19 scFv(抗体号:FMC63)序列为基础,构建CD19 CAR质粒。scFv序列前是短信号肽SP段和MYC标签,其中MYC标签用于检测CAR的表达水平,后续是CD8铰链区和跨膜区、CD28后续和CD3ζ胞内区依次串联,构成经典的二代CAR结构。通过文献与专利查找合适的IL15/IL15Rα结构,在GeneBank与专利中查找IL15、IL15Rα sushi结构域,由生物公司合成P2A-IL15、P2A-RLI片段,通过分子克隆技术将片段与CD19 CAR连接,质粒结构见图1。将构建成功且测序正确的3组CD19 CAR转染至BaEV-PackRV包装细胞中,收获逆转录病毒颗粒,并检测MYC的表达以检测转染效率。
从液氮中取出并复苏NK92和NK92MI细胞,在37 ℃、5%CO2培养箱中无菌培养。NK92的培养基为含有200 IU/mL的IL2的X-VIVO 15培养基,NK92MI的培养基则不含IL2。定期观察细胞状态,当细胞处于对数增殖期且细胞存活率达到90%以上时表示细胞状态良好,可以进行后续实验。分别用3组CD19 CAR逆转录病毒载体颗粒转导NK92和NK92MI细胞制备CAR-NK细胞。对照组细胞不做处理,正常传代。转导72 h后用MYC-APC抗体染色,通过流式细胞术检测MYC表达效率,即为CAR阳性率。此后定期用培养基传代。
收集不添加IL2的情况下培养NK/CAR-NK细胞的上清,用样品稀释液稀释5倍,采用ELISA法检测上清IL15的含量。通过标准曲线推算出拟合浓度,再乘以样本的稀释倍数,即可得到上清中IL15的实测浓度。
在不添加IL2的情况下培养NK细胞,观察装甲的细胞因子是否可刺激NK细胞继续扩增。CAR-NK细胞制备成功后,取部分细胞更换为无IL2的培养基,每48小时计数,并调整细胞密度至5×105个/mL,根据细胞总数与细胞存活率绘制曲线。
取NK/CAR-NK细胞,采用CFSE染剂(用X-VIVO 15培养基稀释成 10 µmol/mL)37 ℃避光染色 30 min 后,调整细胞密度为5×105个/mL培养24 h,剩余细胞用(1×)PBS缓冲液清洗后上机检测FITC通道信号强度,并记为0 h。培养结束,1×PBS缓冲液清洗后上机检测FITC通道信号强度,记为24 h。按照以下公式计算增殖指数:增殖指数=(1-24 h FITC通道信号强度/0 h FITC通道信号强度)×100%。
从液氮中取出并复苏Nalm6-GFP-Luc和hCD19-SW620-GFP-Luc细胞,在37 ℃、5%CO2细胞培养箱中无菌培养。Nalm6-GFP-Luc细胞用含有10%FBS的RPMI 1640完全培养基培养,hCD19-SW620-GFP-Luc细胞用含有10%FBS的DMEM完全培养基培养。每隔48小时计数,并传代1次,观察细胞状态,当细胞处于对数增殖期且细胞存活率达到90%以上时表示细胞状态良好,可以进行后续实验。CD19-APC抗体染色,并通过流式细胞术检测其细胞表面CD19的表达水平。
在不透明全白的96孔板接种肿瘤细胞2×104个/孔,第2天将NK细胞和3组CAR-NK细胞按照不同效应细胞与靶细胞数量比例(简称“效靶比”)稀释,接种至96孔板和带有GFP-Luc标签的肿瘤细胞共培养。以仅有肿瘤细胞的孔作为最大释放孔,以只有培养基的孔作为空白孔。4或12 h后,每孔加入100 µL配置好的荧光素酶底物,室温避光培养5 min。设置化学发光模式(luminescence,LUM)、全白酶标板、震板5 s,光电倍增管增益设置为1 000,检测每个孔的荧光数(D)值。计算靶细胞死亡率,即CAR-NK细胞杀伤效率:杀伤效率=1-(D实验孔-D空白孔)/(D最大释放孔-D空白孔)×100%。
将4组NK/CAR-NK细胞按照1∶1的效靶比和肿瘤细胞Nalm6-GFP-Luc共培养,4 h后收集共培养的细胞,用APC-CD107a抗体染色,通过流式细胞术检测GFP-的细胞群(即NK/CAR-NK细胞)中APC阳性的细胞比例。
铺板方式参考“1.2.7”,共孵育4 h后将上清收集并保存于-20 ℃冰箱,收集3次上清后按照CBA试剂盒说明书操作,通过流式细胞仪检测磁珠信号,使用Biolegend官网软件分析数据。
使用GraphPad Prism 8.0软件对IL15的浓度、增殖指数、杀伤效率、细胞因子分泌水平数据进行统计学分析处理,计量资料符合正态分布以x¯±s表示,两组间比较采用独立样本t检验。P<0.05为差异具有统计学意义。
通过分子克隆技术构建CAR分子包装质粒,流式细胞术检测病毒包装细胞和NK92/NK92MI细胞的CAR阳性率,ELISA法检测CAR-NK细胞的IL15分泌水平。成功构建了装甲IL15或RLI的CD19 CAR分子(图1),并转染病毒包装细胞系,转染效率均超过45%(图2A)。流式细胞术检测显示,反复转导后基于NK92细胞构建的CAR-NK细胞CAR阳性率均超过95%(图2B),并且装甲IL15或RLI的NK92-CAR较原始的NK92-CD19 CAR能够分泌高浓度的IL15(P<0.001,图2C)。流式细胞术检测显示,反复转导后基于NK92MI细胞构建的CAR-NK细胞CAR阳性率均超过95%(图2D),并且装甲IL15或RLI的NK92MI-CAR较原始的NK92MI-CD19 CAR能够分泌高浓度的IL15(P<0.001,图2E)。这些结果提示CD19 CAR在NK92/NK92MI细胞表面成功表达,且IL15分泌功能正常,CAR-NK细胞制备成功。
通过定期细胞计数、存活率检测和CFSE法检测CAR-NK细胞的增殖情况。在无IL2刺激的情况下,未转导的NK92及NK92-CD19 CAR无法长时间增殖,细胞存活率持续下降,第4天存活率下降至50%以下,而装甲细胞因子的细胞均能稳定增长,且RLI对NK92-CD19 CAR的增殖促进作用更具优势,细胞总数与细胞存活率均高于NK92-CD19 CAR-IL15组(图3A)。CFSE染色24 h后,所有CAR-NK92细胞荧光信号强度明显降低,分泌细胞因子的NK92-CD19 CAR细胞增殖能力有明显提升(P<0.05),且对比IL15,RLI对NK92-CD19 CAR细胞的增殖促进作用更具优势(P=0.001,图3B)。
不同于在无IL2刺激的情况下,NK92无法长时间增殖,NK92MI细胞可自分泌IL2,但装甲RLI对NK92MI-CD19 CAR细胞的增殖有促进作用,细胞总数与细胞存活率均高于NK92MI-CD19 CAR-IL15组(图3C)。CFSE染色24 h后,所有CAR-NK92MI细胞荧光信号强度明显降低,分泌细胞因子NK92MI-CD19 CAR增殖能力有明显提升(P<0.05),且对比IL15,RLI对NK92MI-CD19 CAR的增殖促进作用更具优势(P=0.020 5,图3D)。以上结果提示IL15和RLI的分泌能够显著增强CAR-NK细胞的增殖能力。
流式细胞术检测肿瘤细胞表面的CD19靶抗原表达水平。Nalm6-GFP-Luc和hCD19-SW620-GFP-Luc细胞表面CD19的阳性率分别为100.0%和99.7%(图4),可以作为后续杀伤实验的靶细胞使用。
通过荧光素酶化学发光法检测靶细胞的裂解率,即CAR-NK细胞的杀伤效率,采用流式细胞术检测CAR-NK细胞CD107a的表达水平,CBA法检测CAR-NK细胞的细胞因子分泌水平。对实体瘤与血液瘤两种CD19阳性的靶细胞,与NK92细胞比较,转导后的NK92-CD19 CAR杀伤能力有明显提升(P=0.010 8),且装甲IL15或RLI可提升NK92-CD19 CAR细胞的杀伤能力(P=0.031 9)。将效靶比调整至1∶10、1∶20、1∶40,将杀伤时间延长至12 h,以Nalm6-GFP-Luc细胞为靶细胞进行杀伤能力检测,与NK92细胞相比,转导后的NK92-CD19 CAR杀伤能力有明显提升,且装甲RLI的NK92-CD19 CAR细胞的杀伤能力高于装甲IL15的NK92-CD19 CAR细胞(P=0.004,图5A)。NK92细胞表面CD107a的表达为15.5%,NK92-CD19 CAR细胞表面CD107a的表达为10.4%,NK92-CD19 CAR-IL15细胞表面CD107a的表达为22.4%,NK92-CD19 CAR-RLI细胞表面CD107a的表达为33.4%,装甲IL15或RLI的NK92-CD19 CAR细胞杀伤活性更强(图5B)。装甲IL15或RLI的NK92-CD19 CAR细胞有更高的IL2、IL10、IL6、TNF-α、sFas、IFN-γ、Granulysin释放水平(P<0.05,图5C)。上述结果提示,装甲IL15或RLI可增强NK92-CD19 CAR细胞的抗肿瘤活性。
对实体瘤与血液瘤两种CD19阳性的靶细胞,与NK92MI细胞比较,转导后的NK92MI-CD19 CAR杀伤能力提升,且装甲IL15或RLI可提升NK92MI-CD19 CAR细胞的杀伤能力(P<0.05,图6A)。NK92MI细胞表面CD107a表达10.3%,NK92MI-CD19 CAR细胞表面CD107a的表达为10.2%,NK92MI-CD19 CAR-IL15细胞表面CD107a的表达为15.7%,NK92MI-CD19 CAR-RLI细胞表面CD107a的表达为19.6%,装甲IL15或RLI的NK92MI-CD19 CAR细胞杀伤活性更强(图6B)。装甲IL15或RLI的NK92MI-CD19 CAR细胞有更高的IL2、IL10、IL6、TNF-α、sFasL、IFN-γ、Granzyme A、Granzyme B、Perforin、Granulysin释放水平(P<0.05,图6C)。上述结果提示装甲IL15或RLI可增强NK92MI-CD19 CAR细胞的抗肿瘤活性。
NK细胞是大型颗粒淋巴细胞,是先天性(非特异性)免疫防御系统的组成部分。它们在骨髓、淋巴结、脾脏、胸腺和扁桃体中成熟,约占外周血循环淋巴细胞的5%~10%[9]。在临床治疗中,相比目前新兴的CAR-T细胞疗法,CAR-NK细胞疗法引发细胞因子释放综合征(cytokine release syndrome,CRS)和神经毒性等副反应的可能性更低,且NK细胞被认为是更安全的癌症治疗效应细胞,NK细胞不需要严格的人类白细胞抗原(human leukocyte antigen,HLA)配型,不会引发移植物抗宿主病(graft versus-host disease,GVHD),临床使用更加便捷安全,能够极大程度地降低临床治疗中的风险[10]。
NK细胞来源与杀伤机制多样,以其为细胞来源构建CAR-NK细胞疗法,不仅能在一定程度上解决细胞来源等成本问题,还能通过NK细胞非依赖性CAR杀伤机制对肿瘤细胞进行识别杀伤,更好地应对抗原逃逸等问题,安全性更高[11]。NK细胞来源中,NK92细胞由于其可预测的快速生长特性(倍增时间为24~36 h)和易于扩增的特点,成为了基因工程中“受欢迎”的效应NK细胞,尤其是在引入CAR的应用方面。NK92细胞以发现年份(1992年)命名,来自被诊断患有侵袭性NK细胞淋巴瘤的患者的单核血细胞[12]。NK92细胞除易于扩增、细胞毒性高外,也易于导入基因,转导CAR的阳性率高于其他来源的NK细胞,且无论较同种异体NK细胞,还是T细胞,NK92的制备和使用成本显著更低,使用前不需经过白细胞分离、T细胞清除和复杂耗时长的体外扩增(如选择适宜饲养细胞),其可冷冻保存便于储存及运输的性质也大大降低了成本。NK92MI细胞是通过逆转录病毒转染NK92细胞得到的自分泌IL2的细胞,NK92MI细胞与NK92细胞同样可体外大量扩增、易于转导、细胞毒性较强,且增殖速度更快,扩增无需依赖IL2,可用于研究NK细胞功能及肿瘤免疫实验[13]。在临床治疗中,KLOESS等[14]的一项急性髓系白血病(acute myeloid leukemia,AML)免疫治疗临床前研究证实,工程化的CD123-CAR-NK92细胞显示出比原代人供体来源的CD123-CAR-NK细胞更高水平的颗粒酶和白细胞介素分泌以及更好的细胞毒性活性。
NK92和NK92MI细胞在临床应用前需要提前接受辐照,但这一举措会导致其持久性大幅下降,疗效也会受到影响[15-16],而本研究通过装甲细胞因子的方法,希望能够增强其增殖能力与活性。NK细胞是IL2依赖细胞,但IL2会促进诱导T细胞发生活化诱导的T细胞死亡(activation induced cell death,AICD)并激活调节性T细胞(regulatory T cell,Treg),限制其在肿瘤免疫治疗的应用[17]。而与其结构相似的另一个细胞因子IL15能够作为IL2更好的代替品,IL15可以抑制IL2介导的T细胞应答和AICD过程,对Treg活性没有影响[18]。另一方面,IL15被认为在NK细胞的发育和稳态中起着不可或缺的作用。在小鼠和人类中,缺乏IL15或其受体亚基会导致成熟NK细胞数量显著减少。而且,IL15还能增强NK细胞抗体依赖性的细胞介导的细胞毒作用,上调与NK细胞活性相关的关键蛋白(如INF-γ、NKG2D、Granzyme B),促进其对肿瘤细胞和感染病原体的杀伤作用,进一步增强免疫反应[19-20]。然而,半衰期短(在人体中不到1 h)和严格的转录后调控限制了IL15的应用[21],有研究证明可通过改变IL15的表达形式或结构来应对这一问题,RLI就是一个更好的选择。RLI是由IL15Rα Sushi结构域通过一个包含20个氨基酸的柔性连接体融合到IL15中组成,是IL15激动剂,IL15/IL15Rα半衰期可以由原来的30 min延长至20 h,生物学活性也可提高至少50倍[7,22-24]。且研究表明,RLI通过刺激NK细胞而不刺激T细胞,可以降低原位人结肠癌原位裸鼠模型中的原发肿瘤生长和转移负担,在乳腺癌模型中也观察到抗转移作用[8]。
综上所述,本研究以CD19 CAR为基础,通过采用第四代CAR结构装甲IL15或RLI,从细胞因子分泌、增殖能力与杀伤能力3个方面对其进行初步的体外功能验证,成功制备了装甲IL15与RLI的CD19 CAR-NK92/NK92MI细胞,证明了IL15细胞因子与CAR-NK联用具有巨大潜力,为细胞因子与CAR-NK联用提供了体外实验证据,也为CAR-NK细胞克服临床应用中的低持久性提供了一定思路。本研究所选择的hCD19-SW620-GFP-Luc细胞的原始SW620细胞并不表达CD19,其表面的CD19高表达为课题组前期工作所构建,在实验验证层面上需要客观看待本次实验结果,且本研究所制备的CAR-NK细胞需进一步开展体内验证以确定其有效性。未来将通过进一步动物实验乃至临床试验对装甲IL15或RLI的CD19 CAR-NK细胞相关转化进行完善,进一步推动相关疗法的临床落地。

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2025年第47卷第23期
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doi: 10.16016/j.2097-0927.202507093
  • 接收时间:2025-07-28
  • 首发时间:2026-10-30
  • 出版时间:2025-12-15
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  • 收稿日期:2025-07-28
  • 修回日期:2025-11-17
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    1北京中医药大学生命科学学院,北京

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2种不同金属材料的力学参数
科
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属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)
属
Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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