CN113603787B - 一种非分泌型的人白细胞介素15及应用 - Google Patents

一种非分泌型的人白细胞介素15及应用 Download PDF

Info

Publication number
CN113603787B
CN113603787B CN202010369235.3A CN202010369235A CN113603787B CN 113603787 B CN113603787 B CN 113603787B CN 202010369235 A CN202010369235 A CN 202010369235A CN 113603787 B CN113603787 B CN 113603787B
Authority
CN
China
Prior art keywords
leu
ser
cells
glu
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010369235.3A
Other languages
English (en)
Other versions
CN113603787A (zh
Inventor
罗益华
吴春晓
赵剑锋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Shanjin Biotech Inc
Shanghai Shinegene Molecular Biotechnology Co ltd
Innocon Biomedical Technology Guangzhou Co ltd
Original Assignee
Shanghai Shanjin Biotech Inc
Shanghai Shinegene Molecular Biotechnology Co ltd
Innocon Biomedical Technology Guangzhou Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Shanjin Biotech Inc, Shanghai Shinegene Molecular Biotechnology Co ltd, Innocon Biomedical Technology Guangzhou Co ltd filed Critical Shanghai Shanjin Biotech Inc
Priority to CN202010369235.3A priority Critical patent/CN113603787B/zh
Publication of CN113603787A publication Critical patent/CN113603787A/zh
Application granted granted Critical
Publication of CN113603787B publication Critical patent/CN113603787B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • Toxicology (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种非分泌型的人白细胞介素(IL‑15)。所述的非分泌型IL‑15由信号肽，成熟的IL‑15序列，柔性链接，以及内质网驻留信号依次串联组成。本发明的非分泌型IL‑15通过使用优化的信号肽和内质网驻留信号，能在细胞内高效表达并防止IL‑15分泌到细胞外部。T细胞或NK细胞在转染了本发明的非分泌型IL‑15后，能在没有外源IL‑2或IL‑15的条件下保持杀伤力并继续增殖。在T细胞或NK细胞里同时表达本发明的非分泌型IL‑15和CAR结构后，所得的细胞能在没有外源IL‑2或IL‑15的条件下特异性地杀伤癌细胞。

Description

一种非分泌型的人白细胞介素15及应用
技术领域
本发明属于免疫治疗领域，尤其是对免疫细胞进行基因工程改造以加强对癌细胞的杀伤力。
背景技术
近年来随着免疫学研究的进一步深入，免疫疗法已成为续手术、化疗、放疗后另一种治疗癌症的重要手段。其中以嵌合抗原受体T细胞（CAR-T)为代表的细胞免疫疗法更是在治疗血液肿瘤方面取得极大的成功，但是在利用免疫细胞治疗更广泛存在的实体瘤方面，无论是新兴的CAR-T还是传统的肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte,TIL) ，都还没有突破性进展，迫切需要有新技术打破这僵局。
目前细胞免疫疗法都是先在体外激活扩增免疫细胞后再回输给患者，体外扩增免疫细胞时采用远远高于体内生理浓度的IL-2，这就导致回输的免疫细胞在体内正常IL-2环境下存在活性低，生存期短的问题(Dudley ME, Wunderlich J, et al. J Immunother.2001 Jul-Aug;24(4):363-73)，严重影响了细胞免疫疗法的疗效 (Robbins PF, DudleyME, et al. J Immunol. 2004 Dec 15; 173(12): 7125–7130)。
为克服上述的不利影响，当前在临床上的措施是在回输免疫细胞后连续几天静脉推注高剂量的IL-2以维持回输的免疫细胞的活性(Stevanović S. Helman SR, et al.Clin Cancer Res. 2019 Mar 1;25(5):1486-1493)，但这方法因为采用了高剂量的IL-2注射，副作用和风险都很大。
相比于IL-2,IL-15能更好地维持T和NK细胞的长期活性(Waldmann T, Nat RevImmunol. 2006 Aug;6(8):595-601)。已有报道采用分泌型IL-15(Zhang J, Sun R, etal. Haematologica. 2004 Mar;89(3):338-47)或膜结合型(membrane bound)的IL-15(Imamura M, Shook D, et al. Blood. 2014 Aug 14;124(7):1081-8)能维持NK细胞在低或无外源IL-2和IL-15情况下的增殖，并能增强NK细胞的杀伤力，但这类方法也有其缺陷，即分泌的或膜结合型的IL-15会激活其周围无关的非靶向免疫细胞，输入体内有可能引起严重副作用(Fehniger TA, Suzuki K, J Exp Med. 2001 Jan 15; 193(2): 219–232)。
发明内容
为提高体外扩增的免疫效应细胞在回输到体内后的活性，同时避免使用IL-2或IL-15激活非靶向细胞所带来的副作用，本发明公开了一种非分泌型的人白细胞介素15。所述的非分泌型的IL-15由优化的信号肽，成熟的人IL-15序列，柔性链接，以及内质网驻留信号依次串联组成，能在细胞内大量表达但不分泌到细胞外，有效地解决了上述问题。
IL-15基因本身通过选择性剪切（alternative splicing）形成一长一短2种信号肽，其中较短的信号肽所生成的IL-15只有通过与IL-15的alpha受体结合才能分泌到细胞外(Bergamaschi C，Jalah R， et al. J Immunol. 2009 Sep 1;183(5):3064-72)。而较长信号肽的因其本身的特殊序列，造成IL-15只有很低的分泌水平(Bamford RN, DeFilippisAP，et al. J Immunol. 1998 May 1;160(9):4418-26)。本发明通过利用EB病毒的BARF1的信号肽来替换IL-15自身的信号肽，大幅提高了分泌型IL-15的表达量。
为避免IL-15分泌到细胞外激活其周围无关的非靶向免疫细胞，本发明在成熟的人白细胞介素15序列的C-末端添加内质网驻留信号(氨基酸序列为KDEL)。成熟的IL-15序列与内质网驻留信号之间以一小段柔性链接(氨基酸序列为GSGS)相连，用以克服空间位阻使得IL-15能自由旋转并与其受体结合发挥IL-15的免疫活性。
本发明的有益效果是：以上述方法优化过的的非分泌型的IL-15能在细胞内高效表达并防止IL-15分泌到细胞外部。T淋巴细胞或NK细胞在转导了本发明的非分泌型IL-15后，能在没有外源IL-2或IL-15的条件下保持杀伤活性并继续增殖。将优化过的非分泌型IL-15与嵌合抗原受体(CAR)同时在T淋巴细胞或NK细胞内表达，所得的细胞能在没有外源的IL-2或IL-15环境下有效杀伤CAR所识别的癌细胞。
附图说明
图1是非分泌型的人白细胞介素15的结构示意图。
图2是转导了不同信号肽的非分泌型的人IL-15的T细胞对其靶细胞LCL的杀伤效果。BARF1S-IL15-ER，IL2S-lL5-ER和IL15S-IL15-ER分别为使用BARF1,IL-2和IL-15信号肽的非分泌型IL-15，None为未转导的T细胞。
图3是转导了不同信号肽的非分泌型的人IL-15的NK细胞对其靶细胞K562的杀伤效果。BARF1S-IL15-ER，IL2S-lL5-ER和IL15S-IL15-ER分别为使用BARF1,IL-2和IL-15信号肽的非分泌型IL-15，None为未转导的NK细胞。
图4是GPC3特异性CAR-NK细胞对HepG2细胞在体外增殖的影响。NK为未转导CAR结构的NK细胞，CAR-NK为转染了GPC3特异性CAR结构的NK细胞，CAR-IL15-NK为转导了能同时表达GPC3特异性CAR结构和使用BARF1信号肽的非分泌型人IL-15的NK细胞。
图5是GPC3特异性CAR-T细胞对HepG2细胞在体外增殖的影响。T为未转导CAR结构的T细胞，CAR-T为转染了GPC3特异性CAR结构的T细胞，CAR-IL15-T为转导了能同时表达GPC3特异性CAR结构和使用BARF1信号肽的非分泌型人IL-15的T细胞。
具体实施方式
下面用实例具体说明本发明，但本发明并不以此为限。在下面的具体步骤里未注明具体条件的实验方法，则按照常规条件或制造厂商所建议的条件进行。
一、BARF1的信号肽能支持人IL-15高表达。
（1）含不同信号肽的IL-15基因的制备。通过基因合成技术直接合成具有IL-15，IL-2和EB病毒的BARF1信号肽的IL-15基因（氨基酸编码分别为SEQ ID NO:5，7和9，对应的基因编码序列分别为SEQ ID NO:6，8和10)。
（2）将上述合成的IL-15基因插入Clontech公司的pLVX-IRES-ZsGreen1质粒，所得质粒用Invitrogen公司的Lipofectamine 2000转染293T细胞，24小时后取上清液，用Abcam公司的人IL-15 ELISA试剂盒测量其中的IL-15浓度。结果如表1所示，使用IL-15自身信号肽的IL-15表达量很低，改用IL-2的信号肽后表达量提高了约6倍，而使用EB病毒BARF1基因信号肽的IL-15表达量最高，比用IL-15本身信号肽的提高了10倍以上。
表1. 不同信号肽对人白细胞介素15的表达量的影响
Figure 632748DEST_PATH_IMAGE002
二、内质网驻留信号能防止人IL-15的分泌。
（1）含有相同内质网驻留信号，但不同信号肽的IL-15基因的制备。按图1所示，通过基因合成技术直接合成具有IL-15、IL-2或EB病毒的BARF1信号肽，以及相同的柔性链接和内质网驻留信号的IL-15基因（氨基酸编码分别为SEQ ID NO:1，11和13，对应的基因编码序列分别为SEQ ID NO:2，12和14)。
（2）将上述合成的非分泌型IL-15基因插入pLVX-IRES-ZsGreen1，所得质粒用Lipofectamine 2000转染293T细胞，24小时后取上清液，用人IL-15 ELISA试剂盒测量其中的IL-15浓度。转染了三种不同质粒的细胞分泌到上清液的IL-15浓度均小于5 pg/ml，这证明了内质网驻留信号能有效防止IL-15分泌到细胞外。
三、非分泌型的人IL-15能维持T细胞在无外源IL-2或IL-15条件下的活力。
（1）表达非分泌型IL-15慢病毒的制备。将pMD2G和psPAX和上述表达非分泌型IL-15的质粒混合后用Lipofectamine 2000转染293T细胞，48小时后收集含慢病毒的上清液，先低速离心去细胞碎片。再用0.45µm滤膜过滤病毒上清液。过滤液按照4:1的比例加入5×PEG-it病毒浓缩液。4˚C静置过夜后，离心弃上清，用含5% FBS的DMEM培养液重悬病毒团块。取少量用于滴度测定，剩余的病毒分装后置于-80˚C保存。
（2）T细胞的制备。抽取志愿者50毫升外周血，用Ficoll-Plaque PLUS淋巴细胞分离液通过密度梯度离心分离出单个核细胞（PBMC），用PBS洗涤2次后与40 Gy的伽马射线辐照后的B淋巴母细胞(LCL)以10:1的比例于10% FBS的RPMI 1640培养液里共培养，12天取所得细胞与辐照后的LCL以2:1的比例共培养，同时加入IL-2至10 IU/ml进行增殖。7天后再将所得细胞与辐照后的LCL以2:1的比例在含10 IU/ml IL-2的培养液里继续共培养7天，即得LCL特异性的T细胞。
（3）T细胞的转导。将T细胞重悬在含10 IU/ml IL-2的RPMI 1640培养液中，以MOI=5的比例加入表达不同非分泌型IL-15的慢病毒，以含IL-2的培养液继续培养1天即得到表达不同非分泌型IL-15的T细胞。
（4）表达非分泌型IL-15的T细胞的增殖能力。将上述3种表达不同非分泌型IL-15的T细胞与辐照后的LCL以2:1的比例在无外源IL-2的条件下共培养，7天后测所得的细胞数。未转导的T细胞和转导了使用IL-15自身信号肽的非分泌型IL-15的T细胞都只有少量的增殖，转导了IL-2信号肽的非分泌型IL-15的T细胞数目为起始细胞数的2.4倍，而转导了使用BARF1信号肽的非分泌性IL-15的增殖倍数最高，达到起始细胞数的4.1倍，这表明使用BARF1信号肽的IL-15能更好地支持T细胞在没有外源IL-2或IL-15条件下的增殖。
表2
信号肽 未转染 IL-15 IL-2 BARF1
7天后T细胞扩增倍数 1.3 1.2 2.4 4.1
（5）表达非分泌型IL-15的T细胞的特异性杀伤力。将上述表达非分泌型IL-15的T细胞在无IL-2的培养液里继续培养，1天后用PerkinElmer公司的DELFIA细胞毒性试剂盒测试T细胞的杀伤力，其中表达EB病毒抗原的LCL作为靶细胞，T细胞作为效应细胞，以40:1，20:1，10:1，5:1和2.5:1的效靶比将T细胞和LCL共培养3小时后测特异性杀伤力。结果如图2所示，转导了使用IL-15信号肽的非分泌型IL-15的T细胞对LCL的杀伤力很低，即使在40:1的效靶比下也不到30%，其整体杀伤曲线和未转染的T细胞很类似；而转导了使用BARF1信号肽的非分泌型IL-15的T细胞对LCL的杀伤力最高，在各个效靶比下的杀伤力都比使用IL-2信号肽的高一倍以上。这表明使用BARF1信号肽的非分泌型IL-15能更好地维持T细胞在无外源IL-2或IL-15条件下的杀伤力。
四、非分泌型的人IL-15能维持NK细胞在无外源IL-2或IL-15条件下的杀伤力。
（1）NK细胞的制备。抽取志愿者50毫升外周血，用Ficoll-Plaque PLUS淋巴细胞分离液通过密度梯度离心分离出单个核细胞（PBMC），PBS洗涤2次后次后用Miltenyi公司的CD3 Microbeads清除其中的T淋巴细胞，再与40 Gy的伽马射线辐照后的B淋巴母细胞(LCL)以1:1的比例于含10 IU/ml IL-2的RPMI 1640培养液里进行激活和增殖，7天取所得细胞与辐照后的LCL以1:1的比例继续共培养， 7天后即得NK细胞。
（2）NK细胞的转导。将所得NK细胞重悬在含10 IU/ml IL-2的RPMI 1640培养液中，以MOI=10的比例加入表达不同非分泌型IL-15培养1天即得到表达不同非分泌型IL-15的NK细胞。
（3）表达非分泌型IL-15的NK细胞的特异性杀伤力。将上述表达非分泌型IL-15的NK细胞在无IL-2的培养液里继续培养，1天后用PerkinElmer公司的DELFIA细胞毒性试剂盒测试不同NK细胞的杀伤力，其中K562细胞作为靶细胞，NK细胞作为效应细胞，以40:1，20:1，10:1，5:1和2.5:1的效靶比将NK细胞和K562细胞共培养3小时后测特异性杀伤力。结果如图3所示，未转导的NK细胞和转导了使用IL-15信号肽的非分泌型IL-15的NK细胞对LCL的杀伤力都很低，在不同效靶比下杀伤力都小于20%；而转导了使用BARF1信号肽的非分泌型IL-15能大幅度提高NK细胞对K562的杀伤力，在各个效靶比下的杀伤力都比使用IL-2信号肽的高。这表明使用BARF1信号肽的非分泌型IL-15能更好地维持NK细胞的杀伤力。
五、非分泌型的人IL-15能维持维持CAR-NK细胞在无外源IL-2或IL-15环境下的杀伤力。
（1）表达GPC3特异性CAR慢病毒的制备。通过基因合成技术直接合成GPC3特异性的CAR结构以及能同时表达GPC3特异性CAR结构和使用BARF1信号肽的非分泌型IL-15结构（氨基酸编码分别为SEQ ID NO:15和3，对应的基因编码序列分别为SEQ ID NO:16和4)。将上述合成的基因分别插入pLVX-IRES-ZsGreen1，再与pMD2G和psPAX质粒混合后用Lipofectamine 2000转染293T细胞，48小时后收集含慢病毒的上清液，先低速离心去细胞碎片。再用0.45µm滤膜过滤病毒上清液。过滤液按照4:1的比例加入5×PEG-it病毒浓缩液。4˚C静置过夜后，离心弃上清，用含5% FBS的DMEM培养液重悬病毒团块。取少量用于滴度测定，剩余的病毒分装后置于-80˚C保存。
（2）GPC3特异性CAR-NK细胞的制备。将NK92细胞重悬在含30 IU/ml IL-2的RPMI1640培养液中，以MOI=10的比例加入上述两种表达GPC3-CAR结构的慢病毒，继续培养一天后即得到表达GPC3-CAR的CAR-NK细胞以及同时表达GPC3-CAR结构和使用BARF1信号肽的非分泌型IL-15结构的CAR-IL15-NK细胞。
（3）GPC3特异性CAR-NK细胞对GPC3阳性癌细胞的体外增殖的抑制。将GPC3特异性的CAR-NK和CAR-IL15-NK细胞与GPC3阳性的HepG2肝癌细胞以1:1的比例共培养，4天后测所得的HepG2细胞数目并与共培养起始时的细胞数目比较。结果如图4所示，在没有添加IL-2的条件下，CAR-NK只能稍微减慢HepG2细胞的增殖，HepG2细胞数目增加了近2倍；而在添加30 IU/ml的IL-2的条件下，HepG2的细胞数目降至起始时的30%，说明CAR-NK需要IL-2来维持其杀伤力。但CAR-IL15-NK对HepG2体外增殖的抑制不需要在培养液里额外添加IL-2，无论是否添加IL-2，共培养4天后HepG2的细胞数目都降到起始时的20%以下，这表明在无外源的IL-2或IL-15条件下，使用BARF1信号肽的非分泌型IL-15能维持表达CAR结构的NK细胞对其靶细胞的杀伤力。
六、非分泌型的人IL-15能维持维持CAR-T细胞在无外源IL-2或IL-15环境下的杀伤力。
（1）T细胞的制备。抽取志愿者50毫升外周血，用Ficoll-Plaque PLUS淋巴细胞分离液通过密度梯度离心分离出单个核细胞（PBMC），用PBS洗涤2次后与40 Gy的伽马射线辐照后的B淋巴母细胞(LCL)以10:1的比例于10% FBS的RPMI 1640培养液里共培养，12天取所得细胞与辐照后的LCL以2:1的比例共培养，同时加入IL-2至10 IU/ml进行增殖。7天后再将所得细胞与辐照后的LCL以2:1的比例在含10 IU/ml IL-2的培养液里继续共培养7天，即得LCL特异性的T细胞。
（2）GPC3特异性CAR-T细胞的制备。将上述LCL特异性的T细胞重悬在含10 IU/mlIL-2的RPMI 1640培养液中，以MOI=5的比例加入上述两种表达GPC3-CAR结构的慢病毒，继续培养一天后即得到表达GPC3-CAR的CAR-T细胞以及同时表达GPC3-CAR结构和使用BARF1信号肽的非分泌型IL-15结构的CAR-IL15-T细胞。
（3）GPC3特异性CAR-T细胞对GPC3阳性癌细胞的体外增殖的抑制。将GPC3特异性的CAR-T和CAR-IL15-T细胞与GPC3阳性的HepG2肝癌细胞以1:1的比例共培养，4天后测所得的HepG2细胞数目并与共培养起始时的细胞数目比较。结果如图5所示，在没有添加IL-2的条件下，HepG2与CAR-T共培养后细胞数目比起始时仍有增加但比与未转导的T细胞共培养的对照组要少，在添加10 IU/ml的IL-2后，HepG2的细胞数目降至起始时的30%以下，说明IL-2能增强CAR-T的杀伤力。但CAR-IL15-T对HepG2体外增殖的抑制不需要在培养液里额外添加IL-2，共培养4天后，无论是否添加IL-2，HepG2的细胞数目都降到起始时的30%以下，这表明同时表达非分泌型IL-15能维持CAR-T细胞在无外源IL-2或IL-15的条件下杀伤力。
序列表
<110> 英诺康生物医药科技（广州）有限公司
上海闪锦生物科技有限公司
上海闪晶分子生物科技有限公司
<120> 一种非分泌型的人白细胞介素15及应用
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 142
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Arg Phe Ile Ala Gln Leu Leu Leu Leu Ala Ser Cys Val Ala
1 5 10 15
Ala Gly Gln Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile
20 25 30
Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu
35 40 45
Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
50 55 60
Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His
65 70 75 80
Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
85 90 95
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu
100 105 110
Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln
115 120 125
Met Phe Ile Asn Thr Ser Gly Ser Gly Ser Lys Asp Glu Leu
130 135 140
<210> 2
<211> 429
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggccaggt tcatcgctca gctcctcctg ttggcctcct gtgtggccgc cggccaggct 60
aactgggtga acgtgatcag cgacctgaag aagatcgagg acctgatcca gagcatgcac 120
atcgacgcca ccctgtacac cgagagcgac gtgcacccca gctgcaaggt gaccgccatg 180
aagtgcttcc tgctggagct gcaggtgatc agcctggaga gcggcgacgc cagcatccac 240
gacaccgtgg agaacctgat catcctggcc aacaacagcc tgagcagcaa cggcaacgtg 300
accgagagcg gctgcaagga gtgcgaggag ctggaggaga agaacatcaa ggagttcctg 360
cagagcttcg tgcacatcgt gcagatgttc atcaacacca gcggcagcgg cagcaaggac 420
gagctgtga 429
<210> 3
<211> 878
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
20 25 30
Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
35 40 45
Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln
50 55 60
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg
65 70 75 80
Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
100 105 110
Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr
115 120 125
Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
145 150 155 160
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln
180 185 190
Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser
210 215 220
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr
305 310 315 320
Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val
325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn
420 425 430
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
435 440 445
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
465 470 475 480
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ala Ser Gly Ser Gly
485 490 495
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
500 505 510
Pro Gly Pro Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His
515 520 525
Ile Val Leu Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln
530 535 540
Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val
545 550 555 560
Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys
565 570 575
Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
580 585 590
Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu
595 600 605
Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His
610 615 620
Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu
625 630 635 640
Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp
645 650 655
Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
660 665 670
Pro Asp Leu Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu
675 680 685
Pro Pro Leu Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr
690 695 700
Arg Val Asn Arg Gln Gln Lys Leu Ser Ser Gly Ser Gly Ala Thr Asn
705 710 715 720
Phe Ser Leu Leu Lys Leu Ala Gly Asp Val Glu Leu Asn Pro Gly Pro
725 730 735
Met Ala Arg Phe Ile Ala Gln Leu Leu Leu Leu Ala Ser Cys Val Ala
740 745 750
Ala Gly Gln Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile
755 760 765
Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu
770 775 780
Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
785 790 795 800
Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His
805 810 815
Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
820 825 830
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu
835 840 845
Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln
850 855 860
Met Phe Ile Asn Thr Ser Gly Ser Gly Ser Lys Asp Glu Leu
865 870 875
<210> 4
<211> 2637
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggatgttg tgatgactca gtctccactc tccctgcccg tcacccctgg agagccggcc 120
tccatctcct gcagatctag tcagagcctt gtacacagta atgccaacac ctatttacat 180
tggtacctgc agaagccagg gcagtctcca cagctcctga tctataaagt ttccaaccga 240
ttttctgggg tccctgacag gttcagtggc agtggatcag gcacagattt tacactgaaa 300
atcagcagag tggaggctga ggatgttggg gtttattact gctctcaaaa tacacatgtt 360
cctcctacgt ttggccaggg gaccaagctg gagatcaaac gtggtggagg cggttcaggc 420
ggaggtggct ctggcggtgg cggatcgcag gtgcagctgg tgcagtctgg agctgaggtg 480
aagaagcctg gggcctcagt gaaggtctcc tgcaaggctt ctggatacac cttcaccgac 540
tatgaaatgc actgggtgcg acaggcccct ggacaagggc ttgagtggat gggagctctt 600
gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcagagt cacgctgacc 660
gcggacgaat ccacgagcac agcctacatg gagctgagca gcctgagatc tgaggacacg 720
gccgtgtatt actgtacaag attctactcc tatacttact ggggccaggg aaccctggtc 780
accgtctcct caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg agccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatatc atctccttct tcctcgccct cacctccacc 960
gccctcctct tcctcctctt cttcctgacc ctgagattct ccgttgttaa gcggggcaga 1020
aagaaactgc tgtacatttt caagcagccc ttcatgcgcc ccgtccagac cacccaggaa 1080
gaggacggct gctcctgccg gtttcccgaa gaggaggagg gcgggtgcga gctgagagtg 1140
aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1200
gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260
cctgagatgg ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgcgctagcg gcagcggagc cacgaacttc 1500
tctctgttaa agcaagcagg agatgttgaa gaaaaccccg ggcctatggg tcgggggctg 1560
ctcaggggcc tgtggccgct gcacatcgtc ctgtggacgc gtatcgccag caccatcccc 1620
ccccacgtgc agaagagcgt gaacaacgac atgatcgtga ccgacaacaa cggcgccgtg 1680
aagttccccc agctgtgcaa gttctgcgac gtgcgcttca gcacctgcga caaccagaag 1740
agctgcatga gcaactgcag catcaccagc atctgcgaga agccccagga ggtgtgcgtg 1800
gccgtgtggc gcaagaacga cgagaacatc accctggaga ccgtgtgcca cgaccccaag 1860
ctgccctacc acgacttcat cctggaggac gccgccagcc ccaagtgcat catgaaggag 1920
aagaagaagc ccggcgagac cttcttcatg tgcagctgca gcagcgacga gtgcaacgac 1980
aacatcatct tcagcgagga gtacaacacc agcaaccccg acctgctgct ggtgatcttc 2040
caggtgaccg gcatcagcct gctgcccccc ctgggcgtgg ccatcagcgt gatcatcatc 2100
ttctactgct accgcgtgaa ccgccagcag aagctgagca gcggcagcgg tgccacaaat 2160
ttttctttgt tgaagttagc aggggatgtt gaacttaacc ccggcccaat ggccaggttc 2220
atcgctcagc tcctcctgtt ggcctcctgt gtggccgccg gccaggctaa ctgggtgaac 2280
gtgatcagcg acctgaagaa gatcgaggac ctgatccaga gcatgcacat cgacgccacc 2340
ctgtacaccg agagcgacgt gcaccccagc tgcaaggtga ccgccatgaa gtgcttcctg 2400
ctggagctgc aggtgatcag cctggagagc ggcgacgcca gcatccacga caccgtggag 2460
aacctgatca tcctggccaa caacagcctg agcagcaacg gcaacgtgac cgagagcggc 2520
tgcaaggagt gcgaggagct ggaggagaag aacatcaagg agttcctgca gagcttcgtg 2580
cacatcgtgc agatgttcat caacaccagc ggcagcggca gcaaggacga gctgtga 2637
<210> 5
<211> 162
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
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> 6
<211> 489
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgagaattt cgaaaccaca tttgagaagt atttccatcc agtgctactt gtgtttactt 60
ctaaacagtc attttctaac tgaagctggc attcatgtct tcattttggg ctgtttcagt 120
gcagggcttc ctaaaacaga agccaactgg gtgaacgtga tcagcgacct gaagaagatc 180
gaggacctga tccagagcat gcacatcgac gccaccctgt acaccgagag cgacgtgcac 240
cccagctgca aggtgaccgc catgaagtgc ttcctgctgg agctgcaggt gatcagcctg 300
gagagcggcg acgccagcat ccacgacacc gtggagaacc tgatcatcct ggccaacaac 360
agcctgagca gcaacggcaa cgtgaccgag agcggctgca aggagtgcga ggagctggag 420
gagaagaaca tcaaggagtt cctgcagagc ttcgtgcaca tcgtgcagat gttcatcaac 480
accagctga 489
<210> 7
<211> 134
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile
20 25 30
Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu
35 40 45
Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
50 55 60
Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His
65 70 75 80
Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
85 90 95
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu
100 105 110
Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln
115 120 125
Met Phe Ile Asn Thr Ser
130
<210> 8
<211> 405
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60
aactgggtga acgtgatcag cgacctgaag aagatcgagg acctgatcca gagcatgcac 120
atcgacgcca ccctgtacac cgagagcgac gtgcacccca gctgcaaggt gaccgccatg 180
aagtgcttcc tgctggagct gcaggtgatc agcctggaga gcggcgacgc cagcatccac 240
gacaccgtgg agaacctgat catcctggcc aacaacagcc tgagcagcaa cggcaacgtg 300
accgagagcg gctgcaagga gtgcgaggag ctggaggaga agaacatcaa ggagttcctg 360
cagagcttcg tgcacatcgt gcagatgttc atcaacacca gctga 405
<210> 9
<211> 134
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Ala Arg Phe Ile Ala Gln Leu Leu Leu Leu Ala Ser Cys Val Ala
1 5 10 15
Ala Gly Gln Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile
20 25 30
Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu
35 40 45
Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
50 55 60
Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His
65 70 75 80
Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
85 90 95
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu
100 105 110
Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln
115 120 125
Met Phe Ile Asn Thr Ser
130
<210> 10
<211> 405
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggccaggt tcatcgctca gctcctcctg ttggcctcct gtgtggccgc cggccaggct 60
aactgggtga acgtgatcag cgacctgaag aagatcgagg acctgatcca gagcatgcac 120
atcgacgcca ccctgtacac cgagagcgac gtgcacccca gctgcaaggt gaccgccatg 180
aagtgcttcc tgctggagct gcaggtgatc agcctggaga gcggcgacgc cagcatccac 240
gacaccgtgg agaacctgat catcctggcc aacaacagcc tgagcagcaa cggcaacgtg 300
accgagagcg gctgcaagga gtgcgaggag ctggaggaga agaacatcaa ggagttcctg 360
cagagcttcg tgcacatcgt gcagatgttc atcaacacca gctga 405
<210> 11
<211> 170
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
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 Gly Ser Gly Ser Lys Asp Glu Leu
165 170
<210> 12
<211> 513
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgagaattt cgaaaccaca tttgagaagt atttccatcc agtgctactt gtgtttactt 60
ctaaacagtc attttctaac tgaagctggc attcatgtct tcattttggg ctgtttcagt 120
gcagggcttc ctaaaacaga agccaactgg gtgaacgtga tcagcgacct gaagaagatc 180
gaggacctga tccagagcat gcacatcgac gccaccctgt acaccgagag cgacgtgcac 240
cccagctgca aggtgaccgc catgaagtgc ttcctgctgg agctgcaggt gatcagcctg 300
gagagcggcg acgccagcat ccacgacacc gtggagaacc tgatcatcct ggccaacaac 360
agcctgagca gcaacggcaa cgtgaccgag agcggctgca aggagtgcga ggagctggag 420
gagaagaaca tcaaggagtt cctgcagagc ttcgtgcaca tcgtgcagat gttcatcaac 480
accagcggca gcggcagcaa ggacgagctg tga 513
<210> 13
<211> 142
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile
20 25 30
Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu
35 40 45
Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
50 55 60
Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His
65 70 75 80
Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser
85 90 95
Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu
100 105 110
Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln
115 120 125
Met Phe Ile Asn Thr Ser Gly Ser Gly Ser Lys Asp Glu Leu
130 135 140
<210> 14
<211> 429
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60
aactgggtga acgtgatcag cgacctgaag aagatcgagg acctgatcca gagcatgcac 120
atcgacgcca ccctgtacac cgagagcgac gtgcacccca gctgcaaggt gaccgccatg 180
aagtgcttcc tgctggagct gcaggtgatc agcctggaga gcggcgacgc cagcatccac 240
gacaccgtgg agaacctgat catcctggcc aacaacagcc tgagcagcaa cggcaacgtg 300
accgagagcg gctgcaagga gtgcgaggag ctggaggaga agaacatcaa ggagttcctg 360
cagagcttcg tgcacatcgt gcagatgttc atcaacacca gcggcagcgg cagcaaggac 420
gagctgtga 429
<210> 15
<211> 491
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
20 25 30
Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
35 40 45
Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln
50 55 60
Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg
65 70 75 80
Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
100 105 110
Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr
115 120 125
Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
145 150 155 160
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
165 170 175
Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln
180 185 190
Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
195 200 205
Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser
210 215 220
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
225 230 235 240
Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln
245 250 255
Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr
305 310 315 320
Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val
325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn
420 425 430
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
435 440 445
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
465 470 475 480
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 16
<211> 1476
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggatgttg tgatgactca gtctccactc tccctgcccg tcacccctgg agagccggcc 120
tccatctcct gcagatctag tcagagcctt gtacacagta atgccaacac ctatttacat 180
tggtacctgc agaagccagg gcagtctcca cagctcctga tctataaagt ttccaaccga 240
ttttctgggg tccctgacag gttcagtggc agtggatcag gcacagattt tacactgaaa 300
atcagcagag tggaggctga ggatgttggg gtttattact gctctcaaaa tacacatgtt 360
cctcctacgt ttggccaggg gaccaagctg gagatcaaac gtggtggagg cggttcaggc 420
ggaggtggct ctggcggtgg cggatcgcag gtgcagctgg tgcagtctgg agctgaggtg 480
aagaagcctg gggcctcagt gaaggtctcc tgcaaggctt ctggatacac cttcaccgac 540
tatgaaatgc actgggtgcg acaggcccct ggacaagggc ttgagtggat gggagctctt 600
gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcagagt cacgctgacc 660
gcggacgaat ccacgagcac agcctacatg gagctgagca gcctgagatc tgaggacacg 720
gccgtgtatt actgtacaag attctactcc tatacttact ggggccaggg aaccctggtc 780
accgtctcct caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg agccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatatc atctccttct tcctcgccct cacctccacc 960
gccctcctct tcctcctctt cttcctgacc ctgagattct ccgttgttaa gcggggcaga 1020
aagaaactgc tgtacatttt caagcagccc ttcatgcgcc ccgtccagac cacccaggaa 1080
gaggacggct gctcctgccg gtttcccgaa gaggaggagg gcgggtgcga gctgagagtg 1140
aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1200
gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260
cctgagatgg ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa 1320
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440
gacgcccttc acatgcaggc cctgccccct cgctag 1476

Claims (5)

1.一种非分泌型的人白细胞介素15，由信号肽、成熟的人白细胞介素15序列、柔性链接以及内质网驻留信号依次串联组成，其特征在于所述的非分泌型的人白细胞介素15的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的非分泌型的人白细胞介素15，其特征在于其编码基因序列如SEQ ID NO:2所示。
3.一种能同时表达非分泌型的人白细胞介素15和GPC3特异性嵌合抗原受体的重组基因，其特征在于其氨基酸序列如SEQ ID NO:3所示。
4.根据权利要求3所述的一种能同时表达非分泌型的人白细胞介素15和GPC3特异性嵌合抗原受体的重组基因，其特征在于其编码基因序列如SEQ ID NO:4所示。
5.一种基因工程改造的免疫效应细胞，其特征在于表达包括权利要求1-4中任一项所述的非分泌型的人白细胞介素15或能同时表达非分泌型的人白细胞介素15和GPC3特异性嵌合抗原受体的重组基因的氨基酸或基因序列；所述的免疫效应细胞来自T淋巴细胞或NK细胞。
CN202010369235.3A 2020-05-04 2020-05-04 一种非分泌型的人白细胞介素15及应用 Active CN113603787B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010369235.3A CN113603787B (zh) 2020-05-04 2020-05-04 一种非分泌型的人白细胞介素15及应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010369235.3A CN113603787B (zh) 2020-05-04 2020-05-04 一种非分泌型的人白细胞介素15及应用

Publications (2)

Publication Number Publication Date
CN113603787A CN113603787A (zh) 2021-11-05
CN113603787B true CN113603787B (zh) 2023-05-23

Family

ID=78303080

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010369235.3A Active CN113603787B (zh) 2020-05-04 2020-05-04 一种非分泌型的人白细胞介素15及应用

Country Status (1)

Country Link
CN (1) CN113603787B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20240066127A1 (en) * 2022-04-28 2024-02-29 Immatics US, Inc. Il-12 polypeptides, il-15 polypeptides, il-18 polypeptides, cd8 polypeptides, compositions, and methods of using thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459914A (zh) * 2014-05-15 2017-02-22 新加坡国立大学 经修饰的自然杀伤细胞及其用途
CA3079076A1 (en) * 2017-10-18 2019-04-25 Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus Methods and compounds for improved immune cell therapy
EP3561053A1 (en) * 2018-04-26 2019-10-30 Baylor College of Medicine Immune effector cells and molecular adaptors with an antigen cytokine complex for effective cancer immunotherapy
EP3575325A1 (en) * 2016-12-29 2019-12-04 Timmune Biotech Inc. Multi-target chimeric antigen receptor
CN110964698A (zh) * 2019-12-25 2020-04-07 杭州中赢生物医疗科技有限公司 一种人工抗原递呈细胞及其制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3052445C (en) * 2004-07-10 2023-08-22 Kerry S. Campbell Genetically modified human natural killer cell lines

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459914A (zh) * 2014-05-15 2017-02-22 新加坡国立大学 经修饰的自然杀伤细胞及其用途
EP3575325A1 (en) * 2016-12-29 2019-12-04 Timmune Biotech Inc. Multi-target chimeric antigen receptor
CA3079076A1 (en) * 2017-10-18 2019-04-25 Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus Methods and compounds for improved immune cell therapy
EP3561053A1 (en) * 2018-04-26 2019-10-30 Baylor College of Medicine Immune effector cells and molecular adaptors with an antigen cytokine complex for effective cancer immunotherapy
CN110964698A (zh) * 2019-12-25 2020-04-07 杭州中赢生物医疗科技有限公司 一种人工抗原递呈细胞及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Expression of IL-15 in NK cells results in rapid enrichment and selective cytotoxicity of gene-modified effectors that carry a tumor-specific antigen receptor;Christiane Sahm et al;《Cancer Immunol Immunother》;20120930;第61卷(第9期);第1451~1461页 *

Also Published As

Publication number Publication date
CN113603787A (zh) 2021-11-05

Similar Documents

Publication Publication Date Title
CN110330567B (zh) 双特异性嵌合抗原受体t细胞，其制备方法和应用
JP5042826B2 (ja) 腫瘍壊死因子関連アポトーシス誘導リガンド（ｔｒａｉｌ）を産生するように遺伝子操作された腫瘍帰巣細胞
CN113512125B (zh) 一种携带stat结合基序的嵌合抗原受体分子及表达该嵌合抗原受体分子的nk细胞
CN108728459A (zh) 靶向cd19的嵌合抗原受体并联合表达il-15的方法和用途
CN109320615A (zh) 靶向新型bcma的嵌合抗原受体及其用途
WO2022127372A1 (zh) 高扩增、存续能力和杀瘤作用的CAR-iNKT及应用
CN110734498A (zh) 一种用于解除免疫抑制的融合蛋白及其应用
CN104910278A (zh) 一种用于制备cart细胞的具有高效转染能力和生物学活性的慢病毒
CN113603787B (zh) 一种非分泌型的人白细胞介素15及应用
CN111423517A (zh) 一种肿瘤细胞干性限制型car及其应用
JP2021519107A (ja) 膜結合型ＩＬ−１０を発現する遺伝子的にリプログラミングされたＴｒｅｇ
WO2022179520A1 (zh) 共表达cxcr2以及针对gpc3的star的t细胞及其应用
WO2017071173A1 (zh) 经il-12/cd62l融合蛋白改造的肿瘤治疗剂及其制法和用途
CN108456247A (zh) 靶向ny-eso-1的t细胞受体及其用途
CN111139222B (zh) 一种重组间充质干细胞及其制备方法和用途
CN111850014A (zh) 一种细胞因子增效的嵌合抗原受体及其应用
CN116836299B (zh) 嵌合抗原受体及其应用
CN108728458B (zh) 靶向mesothelin的嵌合抗原受体并联合表达IL-15的方法和用途
CN111978412A (zh) 武装靶向TGF-β的特异性嵌合抗原受体细胞及其制备方法和应用
CN114249834B (zh) 能够特异性靶向肿瘤细胞的嵌合抗原受体、其表达基因、其修饰的nk细胞及应用
CN114634580A (zh) 一种膜锚定式il-15超级复合物的研制及其在肿瘤免疫细胞治疗中的应用
CN113527494A (zh) 一种新型的抗肿瘤转换受体t细胞
CN107502596A (zh) 表达ny‑eso‑1特异性tcr的t细胞及其应用
CN111763264A (zh) 一种靶向psca的嵌合抗原受体及其应用
CN117304342B (zh) 嵌合抗原受体及其应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant