CN112375743A - 一种分泌型靶向her2抗原的嵌合抗原受体t细胞及其应用 - Google Patents
一种分泌型靶向her2抗原的嵌合抗原受体t细胞及其应用 Download PDFInfo
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Abstract
本发明涉及CAR‑T细胞领域,特别涉及一种分泌型靶向HER2抗原的嵌合抗原受体T细胞及其应用。利用从外周血中获得的T细胞,在含靶向实体瘤CAR基因病毒转导的过程中,同时将构建的人源化IL‑7、IL‑15基因转入T细胞,获得分泌型靶向HER2 CAR‑T细胞T细胞。内源性的IL‑7及IL‑15有促进T细胞在肿瘤组织内的存活,具有较好的应用前景。
Description
技术领域
本发明涉及CAR-T细胞领域,特别涉及一种分泌型靶向HER2抗原的嵌合抗原受体T细胞制备及应用
背景技术
过继免疫治疗是将自身或异体的免疫细胞或免疫因子在体外扩增后,回输给免疫功能低下者如肿瘤患者,使其获得抗肿瘤免疫力的过程。主要分为过继性免疫因子治疗和过继免疫细胞治疗(adoptive cell transfer therapy,ACT),CAR-T细胞疗法属于过继性细胞免疫疗法的范畴。
CAR-T疗法,全称嵌合抗原受体T细胞免疫疗法。是一种以患者自身T细胞为基础的治疗,通过白细胞分离术收集患者的外周血单核细胞(PBMC细胞),再分离出特定的T细胞亚群,如CD4+、CD8+、CD25+或CD62L+T细胞。分离出的T细胞起始亚群需要持续和充分的激活才能实现体外扩增,这就需要通过T细胞受体信号和CD28、4-1BB或OX40信号等共刺激信号产生主要的特异性信号,从而激活T细胞。再通过电穿孔、慢病毒或逆转录病毒载体,将CAR基因导入激活的T细胞中进行基因修饰,从而得到可表达CAR基因的CAR-T细胞。简单来说,就是从癌症病人身上分离出免疫T细胞,利用基因工程技术的手段为T细胞引入一个能够识别肿瘤细胞并同时激活T细胞的嵌合抗原受体CAR,然后将扩增好的CAR-T细胞回输到病人体内。
嵌合抗原受体(Chimeric antigen receptor)T细胞技术是不受免疫系统MHC的限制性,通过基因改造技术能够对肿瘤细胞特异靶向,从而对肿瘤进行杀伤的一种发展迅速的细胞治疗技术。自2017年FDA批准了诺华公司的Tisagenlecleucel以及Kite的Axicabtegene Ciloleucel两种CAR-T细胞产品上市以来,其分别用于治疗儿童和年轻人B细胞急性淋巴性白血病以及成人复发性或难治性大B细胞淋巴瘤,CAR-T免疫疗法在针对血液瘤涉及急性B淋巴细胞白血病、非霍奇金氏淋巴瘤以及多发性骨髓瘤等的治疗方面取得了显著成效。并且美国医保与医助服务中心(Centers for Medicare and MedicaidServices,CMS)于2019年2月15日正式发布拟议决定备忘录:批准CAR-T细胞治疗正式纳入医保。这使得CAR-T疗法能够更快速的运用于临床,为患者带来福音。
据《2018年全球癌症统计数据》显示中国癌症的发病人数及死亡人数均为世界第一。肺癌、乳腺癌以及结直肠癌为全球发病率最高的三种癌症,而死亡率最高的则分别为肺癌、结直肠癌和胃癌。尽管CAR-T疗法在血液瘤中取得显著的进展,而其针对实体瘤的治疗仍有待解决。
表皮生长因子受体2(human epidermal growth factor receptor 2,HER2/ErbB2)属于表皮生长因子受体家族的一种蛋白,在细胞生长,发育和分化过程中起着重要作用,HER2蛋白过量表达会导致细胞功能紊乱并与肿瘤的发生有着密切的关系。在上皮细胞癌症中,HER2高表达的细胞表现出了增强的迁移性和浸润性,患者预后差且容易复发。近年来基于抗体的肿瘤靶向治疗取得了一定的进步,如靶向HER2的曲妥珠单抗。尽管曲妥珠单抗在一线治疗中取得了部分进展,但由于抗体在体内易代谢,细胞和组织中渗透性差等自身性质,再加之肿瘤组织微环境的影响等,使得该靶向疗法仍然存在局限性。
CAR-T细胞在体内能识别靶细胞,在杀伤的同时释放大量细胞因子,改变肿瘤微环境。但肿瘤微环境确常常能抑制CAR-T细胞的活性,使其处于“静息”状态或者诱导CAR-T细胞凋亡。目前IL-2已经是公认的促进T细胞增殖的细胞因子,但是其本身对体内存在的resting naive T细胞及记忆T细胞的促增殖作用有限且在体内应用具有风险。而IL-7可通过Jak-STAT通路及pI3K-AKt通路一方面抑制细胞凋亡,提高细胞存活;另一方面诱导周期蛋白的表达,促进细胞周期进展,从而促进naive T细胞及记忆T细胞的增殖。IL-15与其受体结合后主要通过JAK1/STAT3、JAK3/STAT5以及Ras/MAPK三条信号通路发挥作用,通过增生信号的增强以及凋亡信号的减弱,从而增加CD8+T细胞的免疫应答。IL-7/15均对CAR-T细胞的存活具有促进作用。
发明内容
发明目的
技术方案
一种分泌型靶向HER2抗原的嵌合抗原受体T细胞,其特征在于:
CAR-P2A-IL-7-P2A-IL-15;
其中,P2A的氨基酸序列为ATNFSLLKQAGDVEENPGP;
IL-7氨基酸序列为
MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH;
IL-15的氨基酸序列为
MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS。
所述的一种分泌型靶向HER2抗原的嵌合抗原受体T细胞制备方法,其特征在于:
(1)PCR扩增CAR,IL-7和IL-15DNA片段,再通过Gibson assembly法首先将IL-7、IL-15、CAR三个基因片段通过P2A连接得到目的片段并克隆至表达载体上,形成表达质粒;
(2)将表达质粒与慢病毒包装系统的辅助质粒pLp1、pLp2、pLp/VSVG构建慢病毒质粒anti-HER2 CAR-IL-7/15;
(3)将慢病毒质粒anti-HER2 CAR-IL-7/15转染T细胞获得分泌型靶向HER2抗原的嵌合抗原受体T细胞;慢病毒为anti-HER2 CAR-IL-7/15,慢病毒MOI为5-20。
所述的方法,其特征在于步骤(1)中PCR采用的引物为:
引物1:GGATCTATTTGGCCTGATAAATGTTCCATGAAACTTTTAGGT
引物2:
TTTAAACTCATAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCGTGTTCTTTAGTGCCCATCAAAA
引物3:CCCTGGACCTATGAGAATTTCGAAACCACATTTG
引物4:
TTCTGTACATAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCAGAAGTGTTGATGAACATTTGGAC
引物5:CCCTGGACCTATGTACAGAATGCAGCTGCTGTC
引物6:AGAACTAGTCTCGAGGAATTTCACCTAGGGGGGAGGGC
其中:引物1和引物2与CAR序列为模板扩增,引物3和引物4引物扩增IL-7,引物5和引物6引物扩增IL-15,3段扩增序列之间含有15bp的相同的序列的重叠片段,再使用IN-FUSION酶反应30min,使各个序列重叠片段连接,再通过相同的方法将目的片段连接至线性化的表达载体上。
步骤(3)所述T细胞在病毒转染过程中,将包括共表达CAR和IL-7/15的慢病毒转导入T细胞;其具体步骤为:
a、采集外周血,经密度梯度分离或者单采获得外周血单个核细胞,分选后获得纯化的T细胞;所述的分选采用磁珠阴性分选法将T细胞从PBMC中分离富集;采用anti-CD3/anti-CD28抗体偶联的磁珠进行激活,激活时采用的磁珠与T细胞的加入比例为1:3~3:1。
b、T细胞激活后,取出慢病毒进行转导,随即吸弃病毒上清,更换培养液继续培养;
c、将步骤b转导后的T细胞进行流式分析,培养后即得到分泌型靶向HER2抗原的嵌合抗原受体T细胞
步骤a T细胞的完全培养基为RPMI-1640基础培养基,包括10%的胎牛血清,1%的青霉素-链霉素双抗以及500IU/mL的IL-2。
所述的嵌合抗原受体T细胞在制备HER2阳性肿瘤药物中的应用。
附图说明
图1为一种分泌型靶向HER2抗原的嵌合抗原受体结构图;
图2为CAR-T细胞流式分析图;
图3为小鼠肿瘤组织T细胞浸润图;
图4为实施例5的实验结果。
具体实施方式
为了进一步阐述本发明所用的技术手段,以下通过具体实施方式来进一步解释本发明的技术方案,但本发明并非局限在实施例范围内。
本发明提供了一种将包括CAR和IL-7/IL-15慢病毒同时转导入T细胞的嵌合抗原受体T细胞的制备方法,包括纯化T细胞的获取、慢病毒的制备、嵌合抗原受体T细胞的获取、分泌型靶向HER2 CAR-T细胞的特性检测。
实施例1纯化T细胞的获取
用EDTA抗凝管取健康人的外周血,然后加入等体积的DPBS进行稀释。之后以稀释后的外周血与淋巴细胞分离液体积比为3:2的比例,加入淋巴细胞分离液,400g离心40min;从上至下细胞可分为4层,吸取第二层的白膜层,经DPBS洗涤后即可获得外周血单个核细胞,然后依据美天旎的Pan T Cell Isolation Kit说明书操作即可获取纯化的T细胞。
将纯化的T细胞计数后利用完全培养基调整细胞浓度为100万/mL,根据磁珠与细胞比为1:1~3:1的比例加入anti-CD3/CD28偶联的磁珠,然后将T细胞活化。
实施例2构建表达质粒
通过基因合成的方法和PCR技术体外扩增CAR,IL-7和IL-15 DNA片段,再通过Gibson assembly法首先将IL-7、IL-15、CAR三个基因片段通过P2A连接得到目的片段并克隆至表达载体上,形成表达质粒(图1)。
具体方法如下:
设计如下6条引物:
引物1:GGATCTATTTGGCCTGATAAATGTTCCATGAAACTTTTAGGT
引物2:
TTTAAACTCATAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCGTGTTCTTTAGTGCCCATCAAAA
引物3:CCCTGGACCTATGAGAATTTCGAAACCACATTTG
引物4:
TTCTGTACATAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCAGAAGTGTTGATGAACATTTGGAC
引物5:CCCTGGACCTATGTACAGAATGCAGCTGCTGTC
引物6:AGAACTAGTCTCGAGGAATTTCACCTAGGGGGGAGGGC
引物1和引物2与CAR序列为模板扩增,引物3和引物4引物扩增IL-7,引物5和引物6引物扩增IL-15,3段扩增序列之间含有15bp的相同的序列的重叠片段。再使用IN-FUSION酶反应(ClontechSnap Assembly Master Mix,购自Takara)30min,使各个序列重叠片段连接,再通过相同的方法将目的片段连接至线性化的表达载体上。
实施例3构建慢病毒质粒anti-HER2 CAR-IL-7/15
将表达质粒以及慢病毒包装系统的辅助质粒pLp1、pLp2、pLp/VSVG经大肠杆菌扩增之后,利用无内毒素质粒提取试剂盒提取质粒用于病毒包装,形成慢病毒质粒anti-HER2CAR-IL-7/15。在病毒包装前一天将1000-2000万293FT细胞铺至T75细胞培养瓶中,当细胞长至80%密度时进行病毒包装。在包装前将细胞用预热无双抗的FreeStyleTM 293表达培养基换液。在lipo2000促转染试剂的作用下进行IL-7、IL-15及CAR三种慢病毒的包装。转染后6h后更换培养液。培养48h后收集病毒上清,经0.45μm的滤膜过滤后于-80℃冰箱分装保存并对病毒滴度进行检测。
实施例4分泌型靶向HER2 CAR-T细胞的获取
从-80℃冰箱取出冻存的慢病毒质粒anti-HER2 CAR-IL-7/15,于4℃冰浴中融化。从培养箱取出活化的T细胞进行细胞计数,320g离心5min,弃上清。将慢病毒以MOI均为15计算所需的病毒液体积。将病毒液与促转染试剂室温孵育5min后,重悬细胞沉淀,以2000-3000rpm的转速室温离心1-3h。离心结束后,吸弃上清,用培养液重悬细胞沉淀后继续于培养箱培养。在转导后48h,检测病毒的转导效率,结果见图3。
实施例5分泌型靶向HER2 CAR-T细胞的特性检测
选取NOD/SCID鼠,皮下接种HER2阳性乳腺癌细胞,待肿瘤体积增大为100mm3时尾静脉注射分泌型靶向HER2 CAR-T细胞。21天剥离小鼠肿瘤组织,多聚甲醛固定后切片,使用CD3抗体进行免疫组化染色。由图3可知,在小鼠肿瘤组织中能检测到阳性T细胞。
实施例6分泌型靶向HER2 CAR-T的活性测试
取制备的T细胞,HER2 CAR-T和IL-7/15HER2 CAR-T细胞,与HER2阳性细胞按效靶比1:1,5:1和10:1共孵育16h,离心取上清,检测LDH含量,计算杀伤率,见图4所示,随着CAR效靶升高,其杀伤率大幅提高,同时IL-7/15HER2 CAR-T细胞优于HER2 CAR-T。【参考文献:Yanjing,Song,Chuan,et al.Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro andxenotransplanted tumors in vivo.[J].Volume 9,Issue 10.】
综上所述,本发明公开了一种分泌型靶向HER2 CAR-T细胞的制备及其应用。在CAR慢病毒转导的过程中,同时将携带IL-7基因及IL-15基因转导入细胞,其中慢病毒共转导过程中MOI对转导效率有极大的影响。通过大量实验摸索,我们得到了适宜的MOI,从而使表达三种基因的慢病毒同时转导入细胞成为可能。并且在小鼠肿瘤体内实验中检测到阳性T细胞,该方法具有较好的应用前景。
申请人声明,以上对本发明中涉及的技术手段及方案进行了详细阐述,但是其只是作为范例,本发明的保护范围并不局限于上述具体实例。所属技术领域的人员对本发明所做的任何等同变换或改进,均包括在本发明的保护范围及公开范围内。
序列表
<110> 山东省医学科学院附属医院 , 江苏艾洛特生物科技有限公司 ,江苏艾洛特医药研究院有限公司
<120> 一种分泌型靶向HER2抗原的嵌合抗原受体T细胞及其应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> PRT
<213> P2A(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 2
<211> 177
<212> PRT
<213> IL-7(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile
1 5 10 15
Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys
20 25 30
Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu
35 40 45
Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe
50 55 60
Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe
65 70 75 80
Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser
85 90 95
Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr
100 105 110
Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala
115 120 125
Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu
130 135 140
Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu
145 150 155 160
Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu
165 170 175
His
<210> 3
<211> 162
<212> PRT
<213> IL-15(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr
1 5 10 15
Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His
20 25 30
Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala
35 40 45
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
50 55 60
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
65 70 75 80
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
85 90 95
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
100 105 110
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
115 120 125
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
130 135 140
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
145 150 155 160
Thr Ser
<210> 4
<211> 42
<212> DNA
<213> 引物1(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
ggatctattt ggcctgataa atgttccatg aaacttttag gt 42
<210> 5
<211> 91
<212> DNA
<213> 引物2(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
tttaaactca taggtccagg gttctcctcc acgtctccag cctgcttcag caggctgaag 60
ttagtagcgt gttctttagt gcccatcaaa a 91
<210> 6
<211> 34
<212> DNA
<213> 引物3(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
ccctggacct atgagaattt cgaaaccaca tttg 34
<210> 7
<211> 91
<212> DNA
<213> 引物4(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
ttctgtacat aggtccaggg ttctcctcca cgtctccagc ctgcttcagc aggctgaagt 60
tagtagcaga agtgttgatg aacatttgga c 91
<210> 8
<211> 33
<212> DNA
<213> 引物5(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
ccctggacct atgtacagaa tgcagctgct gtc 33
<210> 9
<211> 38
<212> DNA
<213> 引物6(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
agaactagtc tcgaggaatt tcacctaggg gggagggc 38
Claims (4)
1.一种分泌型靶向HER2抗原的嵌合抗原受体T细胞,其特征在于:该T细胞含有序列CAR-P2A-IL-7-P2A-IL-15;
其中,P2A的氨基酸序列为ATNFSLLKQAGDVEENPGP;
IL-7氨基酸序列为MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH;
IL-15的氨基酸序列为MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS。
2.根据权利要求1所述的一种分泌型靶向HER2抗原的嵌合抗原受体T细胞制备方法,其特征在于:
(1)PCR扩增CAR,IL-7和IL-15DNA片段,再通过Gibson assembly法首先将IL-7、IL-15、CAR三个基因片段通过P2A连接得到目的片段并克隆至表达载体上,形成表达质粒;
(2)将表达质粒与慢病毒包装系统的辅助质粒pLp1、pLp2、pLp/VSVG构建慢病毒质粒;
(3)将慢病毒质粒转染T细胞获得分泌型靶向HER2抗原的嵌合抗原受体T细胞。
3.根据权利要求2所述的方法,其特征在于步骤(1)中PCR采用的引物为:
引物1:GGATCTATTTGGCCTGATAAATGTTCCATGAAACTTTTAGGT
引物2:
TTTAAACTCATAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCGTGTTCTTTAGTGCCCATCAAAA
引物3:CCCTGGACCTATGAGAATTTCGAAACCACATTTG
引物4:
TTCTGTACATAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAG CAGGCTGAAGTTAGTAGCAGAAGTGTTGATGAACATTTGGAC
引物5:CCCTGGACCTATGTACAGAATGCAGCTGCTGTC
引物6:AGAACTAGTCTCGAGGAATTTCACCTAGGGGGGAGGGC
其中:引物1和引物2与CAR序列为模板扩增,引物3和引物4引物扩增IL-7,引物5和引物6引物扩增IL-15,3段扩增序列之间含有15bp的相同的序列的重叠片段,再使用IN-FUSION酶反应30min,使各个序列重叠片段连接,再通过相同的方法将目的片段连接至线性化的表达载体上。
4.根据权利要求1的嵌合抗原受体T细胞在制备HER2阳性肿瘤药物中的应用。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114410689A (zh) * | 2022-03-29 | 2022-04-29 | 北京循生生物医学研究有限公司 | 一种增强肿瘤浸润淋巴细胞杀伤力的制备方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108884164A (zh) * | 2016-02-25 | 2018-11-23 | 细胞医学瑞士公司 | 用于免疫疗法的经修饰细胞 |
CN109722420A (zh) * | 2019-03-15 | 2019-05-07 | 江苏艾洛特医药研究院有限公司 | 一种改良嵌合抗原受体t细胞的制备及其应用 |
CN110684739A (zh) * | 2019-11-11 | 2020-01-14 | 深圳市体内生物医药科技有限公司 | 一种嵌合抗原受体t细胞及其应用 |
-
2020
- 2020-11-20 CN CN202011306921.2A patent/CN112375743A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108884164A (zh) * | 2016-02-25 | 2018-11-23 | 细胞医学瑞士公司 | 用于免疫疗法的经修饰细胞 |
CN109722420A (zh) * | 2019-03-15 | 2019-05-07 | 江苏艾洛特医药研究院有限公司 | 一种改良嵌合抗原受体t细胞的制备及其应用 |
CN110684739A (zh) * | 2019-11-11 | 2020-01-14 | 深圳市体内生物医药科技有限公司 | 一种嵌合抗原受体t细胞及其应用 |
Non-Patent Citations (7)
Title |
---|
GIEDRE KRENCIUTE: "Transgenic Expression of IL15 Improves Antiglioma Activity of IL13Rα2-CAR T Cells but Results in Antigen Loss Variants", 《CANCER IMMUNOL RES》 * |
GIEDRE KRENCIUTE: "Transgenic Expression of IL15 Improves Antiglioma Activity of IL13Rα2-CAR T Cells but Results in Antigen Loss Variants", 《CANCER IMMUNOL RES》, vol. 5, no. 7, 26 May 2017 (2017-05-26), pages 571 - 581 * |
文萍: "靶向EpCAM的嵌合抗原受体修饰的T细胞的构建及其对结肠癌细胞的杀伤研究", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
文萍: "靶向EpCAM的嵌合抗原受体修饰的T细胞的构建及其对结肠癌细胞的杀伤研究", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》, 15 January 2018 (2018-01-15), pages 54 - 55 * |
王泽凡: "CAR-T在实体瘤中的研究进展", 《中国肿瘤》 * |
王泽凡: "CAR-T在实体瘤中的研究进展", 《中国肿瘤》, vol. 28, no. 9, 31 December 2019 (2019-12-31), pages 701 * |
郑全辉: "《肿瘤免疫学研究进展》", 31 December 2018, 上海交通大学出版社, pages: 258 - 259 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114410689A (zh) * | 2022-03-29 | 2022-04-29 | 北京循生生物医学研究有限公司 | 一种增强肿瘤浸润淋巴细胞杀伤力的制备方法 |
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