CN112608901B - 一种人工抗原呈递细胞及其制备方法和应用 - Google Patents

一种人工抗原呈递细胞及其制备方法和应用 Download PDF

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CN112608901B
CN112608901B CN202011521175.9A CN202011521175A CN112608901B CN 112608901 B CN112608901 B CN 112608901B CN 202011521175 A CN202011521175 A CN 202011521175A CN 112608901 B CN112608901 B CN 112608901B
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汤朝阳
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秦乐
吴迪
冯世忠
冯嘉昆
杨乐旋
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Guangdong Zhaotai Cell Biotechnology Co ltd
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Abstract

本发明提供了一种人工抗原呈递细胞及其制备方法和应用，所述人工抗原呈递细胞表达膜蛋白MICA、IL‑12、CD19和CD86。本发明采用MICA、IL‑12、CD19和CD86的组合构建人工抗原呈递细胞进行重编程NK细胞培养，实现了短时间内培养获得大量重编程NK细胞的效果，所述重编程NK细胞肿瘤细胞毒性强，在免疫治疗领域具有重要的应用前景。

Description

一种人工抗原呈递细胞及其制备方法和应用
技术领域
本发明属于生物技术领域，涉及一种人工抗原呈递细胞及其制备方法和应用。
背景技术
近年来细胞免疫疗法在癌症治疗中展现出了巨大的潜力，被认为是最有希望攻克癌症的方法，其中，基于T细胞的免疫治疗技术受到了广泛关注，是目前的研究热点。然而，主要组织相容性复合体(MHC)极大地限制了T细胞免疫治疗的应用范围。与MHC限制的T细胞不同，NK细胞不需要进行HLA配型，因此可以作为异源性的现货细胞药品对病人进行免疫治疗，在临床上具有重要的应用前景。
研究人员也在研究T细胞的激活分化机制，尝试对其进行基因改造，获得具有增强的抗肿瘤活性的T细胞。Li等人将小鼠T细胞重编程成为NK样细胞(induced T-to-naturalkiller cells，ITNK细胞)(Li,P.et al.Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion.Science 329,85-89(2010).)，并于近年采用CRISPR/Cas9技术成功地将人T细胞重编程成为ITNK细胞。
但是NK细胞、重编程NK细胞均面临着无法持续增殖的问题。在体外实现重编程NK细胞的大规模扩增是亟待解决的关键问题。
近年来，使用基因工程技术构建的人工抗原呈递细胞(滋养层细胞)越来越多地应用于NK细胞的体外扩增。但是重编程NK细胞不同于NK细胞，其具有T细胞和NK细胞双重功能，目前尚无有效的体外扩增和活化重编程NK 细胞的方法。
发明内容
针对现有技术的不足和实际需求，本发明提供了一种人工抗原呈递细胞及其制备方法和应用，所述人工抗原呈递细胞表达多种活化重编程NK细胞的配体，显著提高了重编程NK细胞的数量和活性。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了一种人工抗原呈递细胞，所述人工抗原呈递细胞表达膜蛋白MICA、IL-12、CD19和CD86。
本发明中，将MICA、IL-12、CD19和CD86表达于K562表面构建人工抗原呈递细胞，通过与重编程NK细胞表面的受体结合用于活化重编程NK细胞，显著提高了重编程NK细胞的扩增数量和肿瘤细胞毒性。
优选地，所述IL-12通过与CD28跨膜区形成融合蛋白、表达在所述人工抗原呈递细胞表面。
优选地，所述MICA包括SEQ ID NO:1所示的氨基酸序列；
SEQ ID NO:1：
MGLGPVFLLLAGIFPFAPPGAAAEPHSLRYNLTVLSWDGSVQSGFLTEV HLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWT MPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQ WGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAAAIFVIIIFYVRCCKKKTSAAEGPELVSLQVLDQHPVGTSDHRDATQLGFQPLMSDLGSTGSTEGA。
优选地，所述IL-12包括如SEQ ID NO:2所示的氨基酸序列；
SEQ ID NO:2：
MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLL DHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMAL CLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAGGGGSMCHQQLVISWFSLVF LASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILK DQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTC GAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYY。
优选地，所述CD28跨膜区包括SEQ ID NO:3所示的氨基酸序列；
SEQ ID NO:3：
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGV LACYSLLVTVAFIIFWV。
优选地，所述CD19包括SEQ ID NO:4所示的氨基酸序列；
SEQ ID NO:4：
MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQL TWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPS EKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDS VSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVG ILHL。
优选地，所述CD86包括SEQ ID NO:5所示的氨基酸序列；
SEQ ID NO:5：
MGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVF WQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHG YPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMT IFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCD KSDTCF。
第二方面，本发明提供了一种表达载体，所述表达载体包括MICA编码基因、IL-12和CD28跨膜区融合蛋白编码基因、CD19编码基因和CD86编码基因。
优选地，所述表达载体包括病毒载体。
优选地，所述病毒载体包括慢病毒载体、逆转录病毒载体或腺相关病毒载体中的任意一种，优选为慢病毒载体。
第三方面，本发明提供了一种重组慢病毒，所述重组慢病毒采用第二方面所述的表达载体与包装辅助质粒共转染哺乳细胞制备得到。
优选地，所述哺乳细胞包括293细胞、293T细胞或293F细胞中的任意一种或至少两种的组合。
第四方面，本发明提供了一种第一方面所述的人工抗原呈递细胞的制备方法，所述方法包括将第三方面所述的重组慢病毒导入K562细胞，利用抗生素和流式细胞仪筛选阳性克隆的步骤。
第五方面，本发明提供了一种培养基，所述培养基包括第一方面所述的人工抗原呈递细胞。
优选地，所述培养基的培养液包括Eagle培养液、RPMI-1640培养基或 Ham’s F-10中的任意一种或至少两种的组合。
第六方面，本发明提供了一种重编程NK细胞的培养方法，所述培养方法包括采用第五方面所述的培养基培养重编程NK细胞的步骤。
优选地，所述培养方法还包括对重编程NK细胞进行辐照的步骤。
优选地，所述辐照的剂量为50～500Gy，例如可以是50Gy、100Gy、150Gy、 200Gy、250Gy、300Gy、350Gy、400Gy、450Gy或500Gy。
优选地，所述重编程NK细胞的制备方法为将靶向Bcl11b基因的 CRISPR/Cas9质粒电转入活化的T细胞制备得到。
第七方面，本发明提供了一种重编程NK细胞，所述重编程NK细胞采用第六方面所述的培养方法培养得到。
与现有技术相比，本发明具有如下有益效果：
(1)本发明采用MICA、IL-12、CD19和CD86的组合构建人工抗原呈递细胞进行重编程NK细胞培养，所述人工抗原呈递细胞对重编程NK细胞具有显著的促扩增和活化作用，培养2周后，重编程NK细胞数量增长约100倍；
(2)本发明的重编程NK细胞相较于T细胞和NK细胞杀瘤能力强，在肿瘤靶细胞远多于重编程NK细胞的情况下，也具有显著的肿瘤细胞毒性，在免疫治疗领域具有应用前景。
附图说明
图1为重编程NK细胞与人工抗原呈递细胞共培养后的体外扩增能力；
图2为重编程NK细胞的肿瘤细胞毒性；
图3为重编程NK细胞的IFN-γ分泌能力。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1重编程NK细胞的构建
(1)根据Bcl11b基因设计crRNA(SEQ ID NO:6)和tracrRNA(SEQ ID NO:7)，进行基因合成，将crRNA、tracrRNA和Cas9蛋白按照1:2:2的比例混匀，采用OPTI-MEMTM配制得到浓度为40μM RNP混合溶液，待用；
SEQ ID NO:6：gaagcagtgtggcggcagctgttttagagcta；
SEQ ID NO:7：tagcaagttaaaataaggctagtcatttatcacattgaaaatctggcaccgagtcggtg；
(2)采用Ficoll密度梯度离心试剂盒(GE公司)从全血中分离外周血单个核细胞(PBMC)，去除红细胞后，再利用MACS Pan-T磁珠分选出T细胞，用T细胞培养基稀释至细胞浓度为2.5×106个/mL；将4mL T细胞接种于培养皿中，加入100ng/mL抗人CD3抗体和100ng/mL抗人CD28抗体的组合进行活化；
(3)3天后，取出悬浮的细胞进行计数，300×g离心10min，用10mL Opti-MEM重悬细胞沉淀，300×g离心10min，将细胞沉淀重悬于100μL Opti-MEM中，加入浓度为40μM的RNP混合溶液，混匀后转移至电Lonza电击杯，将电击杯置于Lonza 4D-NucleofectorTM X Unit(单电击杯模块中)，进行电转，设定电转的电压为800V，时间为4ms；
(4)将电转后的细胞采用含有1ng/mL IL-12、1ng/mL IL-15和1ng/mL IL-18的T551-H3完全培养基培养72h，低速离心细胞，更换新鲜的T551-H3 完全培养基继续培养12h，得到重编程NK细胞。
实施例2人工抗原呈递细胞的构建
(1)人工合成T2A连接的含有MICA编码基因、IL-12和CD28跨膜区、 CD19和CD86的编码基因的核酸分子，并在两端分别添加Pme1和Spe1酶切位点及其保护碱基；
利用限制性内切酶Pme1和Spe1对核酸分子进行双酶切，37℃水浴中孵育 30min，使用1.5％琼脂凝胶电泳回收获得含有粘性末端的酶切产物；
将酶切产物连接入经Pme1和Spe1双酶切的线性化pWPXLd-eGFP质粒(含粘性末端)中，连接反应在T4 DNA聚合酶(Invitrogent公司)的参与下进行，得到慢病毒载体。
(2)采用293T细胞制备重组慢病毒，当293T细胞铺100mm培养皿板底达80～90％时，进行慢病毒包装：病毒包装前2h，更换培养基为含1％胎牛血清的DMEM，加入量为6mL/100mm培养皿；
配制如表1所示的质粒混合液，pWPXLd-表达质粒包括含有 MICA-IL-12-CD19-CD86编码基因的慢病毒载体、含有MICA编码基因的慢病毒载体、含有IL-12编码基因的慢病毒载体、含有CD19编码基因的慢病毒载体或含有CD86编码基因的慢病毒载体，pWPXLd-eGFP质粒为不含有外源编码基因的空载体；
表1
将36μg PEI加至另一500μL opti-MEM培养基内，混匀，室温静置5min；将表1所示的质粒混合液与PEI混合，吹打混匀，室温静置25～30min；将上述混合液逐滴加至培养在100mm培养皿中的293T细胞上；
培养6h后，更换培养基为含1％胎牛血清的DMEM，加入量为7mL/100mm 培养皿；包装后24h、48h和72h，收取病毒上清，同时向293T细胞补加培养基，加入量为7mL/100mm培养皿；1000g离心10min，0.45μm滤器过滤，得到表达外源基因的重组慢病毒或空白对照eGFP慢病毒，4℃保存待用。
(3)将野生型K562细胞重悬于10mL Opti-MEM中，300×g离心10min，将细胞沉淀重悬于100μL缓冲液中，加入步骤(2)的重组慢病毒，并加入8μg/mL polybrene和300IU/mLIL-2，置于37℃、5％CO2培养箱培养；24h后，300g 离心5min，去上清，用含300IU/mL IL-2的新鲜培养基重悬细沉淀，即得人工抗原呈递细胞。
实施例3重编程NK细胞的体外扩增
本实施例将重编程NK细胞培养于含有10％胎牛血清的RPMI-1640培养基中，培养24小时后进行细胞计数，调整细胞浓度为2×106个/mL，在第0天向培养基中加入等比例的人工抗原呈递细胞，随后采用含2×106个/mL人工抗原呈递细胞、10％胎牛血清和50U/mL的IL-2的RPMI-1640培养基继续培养14天， 37℃、5％CO2培养箱中500Gy辐照条件下共培养。
如图1所示，重编程NK细胞与MICA-IL-12-CD19-CD86人工抗原呈递细胞共培养后，细胞数量明显增加，培养2周后，重编程NK细胞数量增长约100 倍，明显高于对照组。
实施例4重编程NK细胞的体外杀伤
将实施例3的培养14天的重编程NK细胞、T细胞和NK细胞在不同的E:T 比例下分别与5×103个黑色素瘤细胞A375共培养于U型96孔板中，每组实验重复3次；
经过18小时的共培养后，向96孔板中加入100μL/孔的荧光素酶底物(1×)，将细胞重悬混匀，立即通过多功能酶标仪测定RLU(relative light unit)，测定时间为1秒，利用荧光素酶(Luciferase)定量杀伤效率评估方法，体外比较不同重编程NK细胞对A375的杀伤作用，杀伤比例计算公式如下：
100％×(对照孔读数-实验孔读数)/对照孔读数(不加细胞的空白组读数可以忽略)
结果如图2所示，重编程NK细胞的杀伤效率高于T细胞和NK细胞，而与MICA-IL-12-CD19-CD86-K562细胞共培养获得的重编程NK细胞的杀伤效率更高，在E:T很小的情况下，即肿瘤细胞数量远大于重编程NK细胞数量，重编程NK细胞也具有较强的肿瘤杀伤作用。
实施例5重编程NK细胞对IFN-γ的分泌
分别将不同培养条件获得的重编程NK细胞、T细胞和NK细胞在E:T＝1:1 的比例下与5×103个黑色素瘤细胞A375共培养于24孔板中，于孵箱中共培养 12h；采用IFN-γELISA检测试剂盒对共培养上清进行检测。
如图3所示，重编程NK细胞分泌IFN-γ的水平高于T细胞和NK细胞，而与MICA-IL-12-CD19-CD86-K562细胞共培养获得的重编程NK细胞分泌 IFN-γ的水平最高。
综上所述，本发明采用表达膜固定MICA、IL-12、CD19和CD86的人工抗原呈递细胞与重编程NK细胞共培养，得到的重编程NK细胞保留有T细胞和 NK细胞的固有属性，同时具有增强的杀瘤能力和稳定的杀瘤效果，通过分泌 IFN-γ实现对肿瘤细胞的杀伤作用。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 广东昭泰体内生物医药科技有限公司
<120> 一种人工抗原呈递细胞及其制备方法和应用
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tagcaagtta aaataaggct agtcatttat cacattgaaa atctggcacc gagtcggtg 59

Claims (6)

1.一种人工抗原呈递细胞，其特征在于，所述人工抗原呈递细胞表达膜蛋白MICA-IL-12-CD19-CD86；
所述IL-12与CD28跨膜区融合表达在所述人工抗原呈递细胞表面；
所述MICA的氨基酸序列如SEQ ID NO:1所示；
所述IL-12的氨基酸序列如SEQ ID NO:2所示；
所述CD28跨膜区的氨基酸序列如SEQ ID NO:3所示；
所述CD19的氨基酸序列如SEQ ID NO:4所示；
所述CD86的氨基酸序列如SEQ ID NO:5所示。
2.一种表达载体，其特征在于，所述表达载体用于制备如权利要求1所述的人工抗原递呈细胞；
所述表达载体包括MICA编码基因、IL-12编码基因、CD28跨膜区的编码基因、CD19编码基因和CD86编码基因；
所述MICA的氨基酸序列如SEQ ID NO:1所示，所述IL-12的氨基酸序列如SEQ ID NO:2所示，所述CD28跨膜区的氨基酸序列如SEQ ID NO:3所示，所述CD19的氨基酸序列如SEQ IDNO:4所示，所述CD86的氨基酸序列如SEQ ID NO:5所示；所述表达载体为慢病毒载体。
3.一种重组慢病毒，其特征在于，所述重组慢病毒采用权利要求2所述的表达载体与包装辅助质粒共转染哺乳细胞制备得到，所述哺乳细胞为293T细胞。
4.一种如权利要求1所述的人工抗原呈递细胞的制备方法，其特征在于，所述方法包括将权利要求3所述的重组慢病毒导入K562细胞，利用抗生素和流式细胞仪筛选阳性克隆的步骤。
5.一种培养基，其特征在于，所述培养基包括权利要求1所述的人工抗原呈递细胞；所述培养基还包括50U/mL IL-2；所述培养基中还包括10％胎牛血清；所述培养基的培养液为RPMI-1640培养基。
6.一种重编程NK细胞的培养方法，其特征在于，所述培养方法包括采用权利要求5所述的培养基来培养重编程NK细胞的步骤；
所述培养方法还包括对重编程NK细胞进行辐照的步骤；
所述辐照的剂量为500Gy；
所述重编程NK细胞的制备方法为将靶向Bcl11b基因的CRISPR/Cas9质粒电转入活化的T细胞制备得到，crRNA的核苷酸序列如SEQ ID NO:6所示，tracrRNA的核苷酸序列如SEQ IDNO:7所示。
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