CN117683723A - 靶向cd5的膜表面工程化nk细胞的制备方法及药物组合物及应用 - Google Patents
靶向cd5的膜表面工程化nk细胞的制备方法及药物组合物及应用 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及细胞免疫治疗领域,提供一种靶向CD5的膜表面工程化NK细胞的制备方法及药物组合物及应用。包括以下步骤:S1、所述NK细胞为从血液中提取单个核细胞,体外激活;S2、采用步骤S1获得的NK细胞与Ac4ManNAz溶液共培养24‑72小时,获得NK‑Ac4ManNAz;S3、制备DBCO‑PEG4‑NH2修饰的Anti‑CD5抗体,通过两步法酶促DBCO‑PEG4‑NH2修饰抗体Fc段;S4、制备DBCO‑PEG4‑NHS修饰的IL‑15细胞因子;S5、通过点击化学将步骤S3得到的Anti‑CD5抗体和步骤S4得到的IL‑15细胞因子共价偶联于步骤S2得到的NK‑Ac4ManNAz,获得NK‑CD5‑IL‑15细胞。优点:NK‑CD5‑IL‑15细胞在赋予NK细胞靶向杀伤T‑ALL肿瘤的同时,靶向递送IL‑15细胞因子,使靶向CD5的NK细胞具有体内扩增能力,二者协同增强NK细胞杀伤T‑ALL以及其他CD5+肿瘤的疗效,具有极大的临床应用前景。
Description
技术领域
本发明涉及细胞免疫治疗领域,更具体地,涉及一种靶向CD5的膜表面工程化NK细胞的制备方法及药物组合物及应用。
背景技术
急性淋巴细胞白血病是儿童肿瘤中最为常见的恶性肿瘤之一,急性T淋巴细胞白血病(T cell acute lymphocyte leukemia,T-ALL)在其中约占10-15%。相较于B型急性淋巴细胞白血病(B cell acute lymphocyte leukemia,B-ALL)儿童T-ALL的治疗效果较差,复发难治T-ALL患者无事件生存率和总生存率低于25%。
抗原嵌合受体T细胞(Chimeric antigen receptor T cell,CAR-T)在B型血液恶性肿瘤中取得了令人瞩目的疗效,但CAR-T在治疗复发难治T-ALL上存在T细胞功能异常、肿瘤污染、靶点重叠等问题,因此使用受到明显的限制。
自然杀伤细胞(Natural killer cell,NK)可以非特异性识别和杀伤肿瘤细胞,且在抗肿瘤的同时没有明显GVHD反应,最重要的是NK细胞与T细胞在许多膜表面蛋白存在差异,这使得NK相关的细胞免疫治疗或能成为治疗复发难治急性T淋巴细胞白血病的突破点。目前,已有研究者尝试使用慢病毒制备靶向CD5、CD7的CAR-NK细胞。然而,CAR-NK在治疗急性T淋巴细胞白血病的实际的应用中并未获得期待的疗效。首先,NK细胞慢病毒转导效率较低,病毒感染后扩增能力下降,导致制备CAR-NK困难;其次,缺乏IL-15等细胞因子的CAR-NK细胞在体内扩增不佳,无法达到长期的抗肿瘤疗效,这些因素都限制了CAR-NK的应用。因此,寻找更安全、高效、简易的方式构建具有靶向杀伤T淋巴细胞肿瘤并能够在体内增殖的NK细胞对于进一步提升NK抗肿瘤的疗效和促进NK细胞药物的“商品化”显得尤为重要。
另外,目前应用点击化学和生物正交反应制备细胞免疫药物正成为新的研究热点。生物正交反应是可以在活细胞、活体动物等环境下与某一分子快速发生化学反应的过程,该过程不对周围的生物体系产生影响,具有反应迅速、反应环境简单等特点。当前,生物正交反应在活细胞成像、药物可控靶向释放、抗体药物偶联上运用广泛。叠氮和炔烃(DBCO、TCO、BCN、四嗪等)是可以发生生物正交反应的常见物质,叠氮在生物体系中非常少见,这保证了反应的特异性。
发明内容
本发明旨在克服上述现有技术的至少一种缺陷(不足),提供一种靶向CD5的膜表面工程化NK细胞的制备方法及药物组合物及应用,通过糖代谢工程以及生物正交反应制备藕联DBCO-CD5抗体和DBCO-IL-15细胞因子的NK细胞,赋予NK细胞靶向杀伤T-ALL肿瘤的能力,同时靶向递送IL-15细胞因子,二者协同增强NK细胞杀伤T-ALL肿瘤的疗效,为治疗复发难治T-ALL提供一条新思路。
本发明的一个目的是提供一种靶向CD5的膜表面工程化NK细胞的制备方法,包括以下步骤:S1、所述NK细胞为从血液中提取单个核细胞,体外激活;S2、采用步骤S1获得的NK细胞与Ac4ManNAz溶液共培养24-72小时,获得NK-Ac4ManNAz;S3、制备DBCO-PEG4-NH2修饰的Anti-CD5抗体,通过两步法酶促DBCO-PEG4-NH2修饰抗体Fc段;S4、制备DBCO-PEG4-NHS修饰的IL-15细胞因子;S5、通过点击化学将步骤S3得到的Anti-CD5抗体和步骤S4得到的IL-15细胞因子共价偶联于步骤S2得到的NK-Ac4ManNAz,获得NK-CD5-IL-15细胞。
相比于CAR-NK或CAR-T细胞的制备,本发明通过共价键藕连抗体而非病毒载体制备具有靶向性的NK细胞,消除了采用病毒载体转导随机插入遗传序列所引发的潜在危险;其次,CAR-T在治疗复发难治T-ALL上存在T细胞功能异常、肿瘤污染、靶点重叠等问题,本发明方法所制备的NK细胞也能避免上述问题;再次,将IL-15细胞因子藕联与NK细胞,通过靶向递送有效的增强NK细胞抗肿瘤的疗效以及体内扩增能力,同时减少了大剂量细胞因子对于正常组织器官带来的损害。可以理解为,NK-CD5-IL-15细胞在赋予NK细胞靶向杀伤T-ALL肿瘤的同时,靶向递送IL-15细胞因子,二者协同增强NK细胞杀伤T-ALL以及其他CD5+肿瘤的疗效,具有极大的临床应用前景。
进一步地,所述步骤S1具体为:采集健康人外周血液,提取单个核细胞用Anti-CD16抗体、IL-2、IL-15、IL-18、IL-21细胞因子在体外激活12~15天,获得高纯度的NK细胞。
进一步地,所述步骤S2具体为:采用步骤S1获得的NK细胞,配制细胞浓度为1×107-5×107/mL的NK细胞悬液;加入浓度为10-100μM的Ac4ManNAz溶液,共培养24-72小时,获得NK-Ac4ManNAz。
进一步地,所述步骤S3具体为:取PBS溶解Anti-CD5抗体稀释至浓度0.8~1.5mg/mL,加入肽-N-糖苷酶F 6~10Unites,37℃下孵育过夜,在所述浓度及温度条件下能最大程度的去除抗体Fc段的糖苷,从而暴露与DBCO-PEG4-NH2的结合位点,超滤离心后,超滤后的液体再次溶解于0.8~1.5mL PBS,后再加入谷氨酰胺酶转氨酶6~10Unites和8~15μl浓度为80~120mM的DBCO-PEG4-NH2,上述条件下能够保证每一个抗体偶联2个DBCO-PEG4-NH2,37℃孵育过夜,超滤离心2次后,获得纯化的DBCO-CD5抗体。
DBCO-CD5抗体即DBCO-PEG4-NH2修饰的Anti-CD5抗体。Anti-CD5抗体与N-糖酰胺酶F(PNGase F)酶的作用下,抗体的Fc段Asn297位点的N-乙酰葡萄糖胺和天冬酰氨残基之间的酰胺键被切割,在谷氨酰胺转氨酶(mTG)的作用下去除糖苷的抗体与DBCO-PEG4-NH2在抗体Fc段共价结合,可以DBCO-PEG4-NH2定点修饰的Anti-CD5抗体。
进一步地,所述步骤S4具体为:将DBCO-PEG4-NHS溶解于二甲亚砜中,终浓度为80~120mM,PBS溶解IL-15细胞因子至0.8~1.2μg/mL,取8~10μl溶解后的DBCO-PEG4-NHS与IL-15细胞因子,加入0.8~1.2mL PBS中继续溶解,低温放置23~28小时,得到DBCO-IL-15混合溶液,超滤离心去除多余DBCO-PEG4-NHS,获得DBCO-IL-15。
所述DBCO-IL-15即DBCO-PEG4-NHS修饰的IL-15细胞因子,所述低温优选为4~6℃。
进一步地,所述步骤S5具体为:采用步骤S3中获得的DBCO-CD5抗体配置成浓度为0.1-1mg/mL的Anti-CD5抗体溶液;采用步骤S4中获得的DBCO-IL-15配置成浓度为20-200ng/mL的IL-15细胞因子溶液;将所述Anti-CD5抗体溶液、IL-15细胞因子溶液和步骤S2中获得的NK-Ac4ManNAz低温下共孵育0.5-2h,离心后弃去上清,用如培养基重悬获得NK-CD5-IL-15细胞。所述低温优选为4~6℃,有利于DBCO-CD5抗体和DBCO-IL-15细胞因子与NK-Ac4ManNAz偶联。
进一步地,在步骤S2中,Ac4ManNAz溶液的浓度为50μM,培养时间为48小时。
进一步地,在步骤S5中,采用步骤S3中获得的DBCO-CD5抗体配置成浓度为0.1-0.5mg/mL的Anti-CD5抗体溶液;采用步骤S4中获得的DBCO-IL-15配置成浓度为20-50ng/mL的IL-15细胞因子溶液,孵育时间为1h。
本发明的另一目的在于提供一种药物组合物,所述药物组合物包含上述制备方法制备的膜表面工程化NK细胞以及任选的药学上可接受的辅料。
本发明的另一目的在于提供一种上述制备方法所制备的膜表面工程化NK细胞的应用,用于治疗或辅助治疗CD5+肿瘤。
与现有技术相比,本发明的有益效果为:相比于CAR-NK或CAR-T细胞的制备,本发明通过共价键藕连抗体而非病毒载体制备具有靶向性的NK细胞,消除了采用病毒载体转导随机插入遗传序列所引发的潜在危险;其次,CAR-T在治疗复发难治T-ALL上存在T细胞功能异常、肿瘤污染、靶点重叠等问题,本发明方法所制备的NK细胞也能避免上述问题;再次,将IL-15细胞因子藕联与NK细胞,通过靶向递送有效的增强NK细胞抗肿瘤的疗效以及体内扩增能力,同时减少了大剂量细胞因子对于正常组织器官带来的损害。可以理解为,NK-CD5-IL-15细胞在赋予NK细胞靶向杀伤T-ALL肿瘤的同时,靶向递送IL-15细胞因子,使靶向CD5的NK细胞具有体内扩增能力,二者协同增强NK细胞杀伤T-ALL以及其他CD5+肿瘤的疗效,具有极大的临床应用前景。
附图说明
图1显示:体外扩增NK细胞(CD3-CD56+)纯度检测图。
图2显示:NK-Ac4ManNAz细胞检测流式图。
图3显示:NK-Ac4ManNAz细胞检测免疫荧光图。
图4显示:NK-CD5-IL-15的流式图。
图5显示:NK-CD5-IL-15的免疫荧光检测情况。
图6显示:NK-CD5-IL-15细胞杀伤CD5阳性Jurka肿瘤的疗效。
图7显示:NK-CD5-IL-15杀伤Jurka肿瘤细胞后上清中TNF-α和IFN-γ的水平。
图8显示:NK-CD5-IL-15与NK细胞杀伤Jurka肿瘤细胞后胞内Perforin、IFN-γ的表达情况。
图9显示:NK-CD5-IL-15细胞中CD56+bright和CD56+dim亚群的细胞功能的变化。
如图10显示:NK-CD5-IL-15的制备示意图。
具体实施方式
本发明附图仅用于示例性说明,不能理解为对本发明的限制。为了更好说明以下实施例,附图某些部件会有省略、放大或缩小,并不代表实际产品的尺寸;对于本领域技术人员来说,附图中某些公知结构及其说明可能省略是可以理解的。
实施例1
NK-CD5-IL-15细胞制备包括以下步骤:
1.1由外周血的单个核细胞经体外扩增培养得到NK细胞,如图1-A流式检测图所示,所述NK细胞(CD3-CD56+)纯度可达90%,三例外周血NK细胞培养的统计图如图1-B所示;
1.2激活后的NK细胞,调整细胞浓度为1×10 7-5×10 7/mL的NK细胞悬液,加入浓度为10-100μM的叠氮修饰的甘露糖(Ac4ManNAz,AAM)共培养24-72hr,获得膜表面表达叠氮基团的NK细胞(NK-Ac4ManNAz,NK-AAM);
1.3检测NK-Ac4ManNAz细胞的制备情况,重悬细胞后加入DBCO-CY5,4℃孵育30min后制备NK-Ac4ManNAz-DBCO-CY5细胞;
1.4通过流式细胞仪检测NK-Ac4ManNAz-DBCO-CY5细胞,结果如图2中所示;通过共聚焦荧光显微镜检测NK-Ac4ManNAz-DBCO-CY5细胞,结果如图3中所示。
1.5制备DBCO-PEG4-NH2修饰的Anti-CD5抗体,通过两步法酶促DBCO-PEG4-NH2修饰抗体Fc段,具体步骤如下:取PBS溶解Anti-CD5抗体稀释至浓度1mg/mL(Kamiya,型号MC-313),加入肽-N糖苷酶F 6U,37℃过夜,超滤离心后(100KD规格超滤管),超滤后的液体再次溶解于1mL PBS,后再加入谷氨酰胺酶转氨酶6U,DBCO-PEG4-NH2 10μl(浓度为100mM),37℃过夜后,超滤离心去除多余物质(100KD规格超滤管);
1.6制备DBCO-PEG4-NHS修饰的IL-15细胞因子,DBCO-PEG4-NHS末端修饰的IL-15的制备方法:将DBCO-PEG4-NHS溶于二甲亚砜中,终浓度为100mM,PBS溶解的IL-15细胞因子至1μg/mL(ACROBiosystems,GMP-L15H13),取10μl DBCO-PEG4-NHS与IL-15细胞因子(1μg/mL)在1mL PBS中溶解,4℃放置24hr得到DBCO-IL-15混合溶液,超滤离心去除多余DBCO-PEG4-NHS(细胞因子选择3KD规格)。
1.7将得到的NK-Ac4ManNAz细胞与人工合成的浓度为0.1-1mg/mL的DBCO-PEG4-NH2修饰的Anti-CD5抗体以及浓度为20-200ng/mL的DBCO-PEG4-NHS修饰的IL-15细胞因子在4℃共孵育0.5-2hr,获得所述NK-CD5-IL-15工程化自然杀伤细胞。
实施例2
NK-CD5-IL-15细胞流式以及免疫荧光的检测情况:
按照实施例1制备方法制备NK-CD5-IL-15细胞,通过流式细胞仪检测NK-CD5-IL-15细胞的荧光如图4中所示,NK-CD5-IL-15细胞比例达到100%,通过共聚焦荧光显微镜观察NK-CD5-IL-15细胞的结果如图5中所示。
实施例3
NK-CD5-IL-15细胞体外杀伤活性检测,步骤如下:
1.1按照实施方案1中制备NK-CD5-IL-15细胞5×106个/mL,制备对照NK细胞、NK-Ac4ManNAz细胞5×106个/mL;
1.2取对数生长期的Jurka-luciferase细胞,制备单细胞悬液至5×10 5个/mL的细胞悬液;
1.3取Jurka-luciferase肿瘤细胞在96孔板中铺板,每孔100μL,按照效靶比10:1、5:1、2.5:1、0:1分别加入NK、NK-Ac4ManNAz、NK联合游离Anti-CD5和IL-15、NK-CD5-IL-15,同时设置未加入免疫细胞的培养基作为空白对照组,每组3个复孔,置入37℃含5% CO2培养箱中静置培养18-24hr;
1.4效应细胞与肿瘤细胞共培养18-24hr后在96孔板中加入荧光素酶,通过酶标仪检测荧光强度,杀伤率=[1-(对照组荧光强度-效应细胞荧光强度)/对照组荧光强度]×100%;
1.5NK-CD5-IL-15体外杀伤Jurka-luciferase的比例比NK、NK-Ac4ManNAz和NK+Anti-CD5+IL-15组更高,这提示该方法有效的增强了NK-CD5-IL-15细胞对于Jurka-luciferase的杀伤能力,结果详见图6;
实施例4
CBA法检测NK-CD5-IL-15细胞杀伤肿瘤细胞后上清中炎症因子的水平,步骤如下:
4.1取Jurka-luciferase肿瘤细胞在96孔板中铺板,每孔100μL,按照效靶比10:1分别加入NK、NK-Ac4ManNAz、NK联合游离Anti-CD5和IL-15、NK-CD5-IL-15,每组3个复孔,置入37℃含5% CO2培养箱中静置培养18-24hr;
4.2效应细胞与肿瘤细胞共培养18-24hr后离400g离心5min,取上清检测TNF-α、IFN-γ。具体操作方法为:涡旋混匀混合好的捕获微球,每个实验管都加入50μL,标准品管中每管加入50μL梯度稀释好的标准品,样本管中每管加入50μL待测样本,所有实验管中都加入50μL人细胞因子PE检测试剂,所有实验管室温避光孵育3hr;孵育完成后每管加入1mL洗液,200g离心5min。小心吸去上清,每管加200μL洗液,重悬微球,上机检测;
4.3CBA法检测NK-CD5-IL-15细胞与NK、NK-Ac4ManNAz和NK+Anti-CD5+IL-15细胞杀伤Jurka-luciferase后细胞上清中TNF-α、IFN-γ的水平,结果显示NK+Anti-CD5+IL-15和NK-CD5-IL-15组杀伤肿瘤细胞后能分泌更高水平的TNF-α、IFN-γ,详见图7。
实施例5
检测NK-CD5-IL-15细胞杀伤肿瘤细胞后细胞功能的变化情况,步骤如下:
1.1按照实施方案1中制备NK、NK-Ac4ManNAz、NK+Anti-CD5、NK-CD5-IL-15细胞5×106个/mL;
1.2取对数生长期的Jurka-luciferase细胞,制备单细胞悬液至1×10 6个/mL的细胞悬液;
1.3将不同组别NK细胞与Jurka-luciferase肿瘤细胞按照效靶比5:1共培养,同时在培养基中加入蛋白转运酶抑制剂BFA/Monensin 1μg/mL,每组3个复孔,置入37℃含5%CO2培养箱中静置培养6hr,然后对不同组别NK细胞进行破膜、固定,离心重悬后加入IFN-γ和Perforin的流式抗体,4℃孵育30min,洗去多余的抗体,用流式分析仪分析NK细胞IFN-γ和Perforin表达情况。
1.4如图8所示,与NK、NK-Ac4ManNAz、NK+Anti-CD5+IL-15组相比,NK-CD5-IL-15细胞与肿瘤细胞共培养6hr后表达IFN-γ和Perforin的细胞比例明显升高,说明NK细胞藕联了DBCO-CD5抗体和DBCO-IL-15细胞因子后对于CD5阳性的细胞具有更强的靶向杀伤能力。
1.5如图9所示,与NK+Anti-CD5+IL-15组相比,NK-CD5-IL-15细胞CD56+bright细胞中能够分泌IFN-γ和Perforin的细胞比例更高,这提示通过点击化学将Anti-CD5和IL-15偶联于NK细胞上,或可以特异性的增强CD56+bright细胞的功能;
显然,本发明的上述实施例仅仅是为清楚地说明本发明技术方案所作的举例,而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (10)
1.一种靶向CD5的膜表面工程化NK细胞的制备方法,其特征在于,包括以下步骤:
S1、所述NK细胞为从血液中提取单个核细胞,体外激活;
S2、采用步骤S1获得的NK细胞与Ac4ManNAz溶液共培养24-72小时,获得NK-Ac4ManNAz;
S3、制备DBCO-PEG4-NH2修饰的Anti-CD5抗体,通过两步法酶促DBCO-PEG4-NH2修饰抗体Fc段;
S4、制备DBCO-PEG4-NHS修饰的IL-15细胞因子;
S5、通过点击化学将步骤S3得到的Anti-CD5抗体和步骤S4得到的IL-15细胞因子共价偶联于步骤S2得到的NK-Ac4ManNAz,获得NK-CD5-IL-15细胞。
2.根据权利要求1所述的靶向CD5的膜表面工程化NK细胞的制备方法,其特征在于,所述步骤S1具体为:采集健康人外周血液,提取单个核细胞用Anti-CD16抗体、IL-2、IL-15、IL-18、IL-21细胞因子在体外激活12~15天此处,获得高纯度的NK细胞。
3.根据权利要求1所述的靶向CD5的膜表面工程化NK细胞的制备方法,其特征在于,所述步骤S2具体为:采用步骤S1获得的NK细胞,配制细胞浓度为1×107-5×107/mL的NK细胞悬液;加入浓度为10-100μM的Ac4ManNAz溶液,共培养24-72小时,获得NK-Ac4ManNAz。
4.根据权利要求1所述的靶向CD5的膜表面工程化NK细胞的制备方法,其特征在于,所述步骤S3具体为:取PBS溶解Anti-CD5抗体稀释至浓度0.8~1.5mg/mL,加入肽-N-糖苷酶F6~10Unites,37℃过夜,超滤离心后,超滤后的液体再次溶解于0.8~1.5mL PBS,后再加入谷氨酰胺酶转氨酶6~10Unites和8~15μl浓度为80~120mM的DBCO-PEG4-NH2,37℃过夜后,超滤离心去除多余物质,获得DBCO-CD5抗体。
5.根据权利要求1所述的靶向CD5的膜表面工程化NK细胞的制备方法,其特征在于,所述步骤S4具体为:将DBCO-PEG4-NHS溶解于二甲亚砜中,终浓度为80~120mM,PBS溶解IL-15细胞因子至0.8~1.2μg/mL,取8~10μl溶解后的DBCO-PEG4-NHS与IL-15细胞因子,加入0.8~1.2mL PBS中继续溶解,低温放置23~28小时,得到DBCO-IL-15混合溶液,超滤离心去除多余DBCO-PEG4-NHS,获得DBCO-IL-15。
6.根据权利要求5所述的靶向CD5的膜表面工程化NK细胞的制备方法,其特征在于,所述步骤S5具体为:采用步骤S3中获得的DBCO-CD5抗体配置成浓度为0.1-1mg/mL的Anti-CD5抗体溶液;采用步骤S4中获得的DBCO-IL-15配置成浓度为20-200ng/mL的IL-15细胞因子溶液;将所述Anti-CD5抗体溶液、IL-15细胞因子溶液和步骤S2中获得的NK-Ac4ManNAz低温共孵育0.5-2h,获得NK-CD5-IL-15细胞。
7.根据权利要求3所述的靶向CD5的膜表面工程化NK细胞的制备方法,其特征在于,在步骤S2中,Ac4ManNAz溶液的浓度为50μM,培养时间为48小时。
8.根据权利要求6所述的靶向CD5的膜表面工程化NK细胞的制备方法,其特征在于,在步骤S5中,采用步骤S3中获得的DBCO-CD5抗体配置成浓度为0.1-0.5mg/mL的Anti-CD5抗体溶液;采用步骤S4中获得的DBCO-IL-15配置成浓度为20-50ng/mL的IL-15细胞因子溶液,孵育时间为1h。
9.一种药物组合物,其特征在于,所述药物组合物包含权利要求1~8任一所述制备方法制备的膜表面工程化NK细胞以及任选的药学上可接受的辅料。
10.一种如权利要求1~8中任一项所述的制备方法所制备的膜表面工程化NK细胞的应用,其特征在于,用于治疗或辅助治疗CD5+肿瘤。
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