CN116555187A - 一种Mucin1嵌合抗原受体修饰的T细胞及其应用 - Google Patents
一种Mucin1嵌合抗原受体修饰的T细胞及其应用 Download PDFInfo
- Publication number
- CN116555187A CN116555187A CN202310760077.8A CN202310760077A CN116555187A CN 116555187 A CN116555187 A CN 116555187A CN 202310760077 A CN202310760077 A CN 202310760077A CN 116555187 A CN116555187 A CN 116555187A
- Authority
- CN
- China
- Prior art keywords
- cells
- muc1
- cell
- car
- region
- 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.)
- Granted
Links
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 52
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 title claims abstract description 25
- 108010008707 Mucin-1 Proteins 0.000 title claims abstract description 21
- 102000007298 Mucin-1 Human genes 0.000 title claims abstract 11
- 239000002773 nucleotide Substances 0.000 claims abstract description 17
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 17
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 230000003834 intracellular effect Effects 0.000 claims abstract description 6
- 238000010008 shearing Methods 0.000 claims abstract description 5
- 230000000139 costimulatory effect Effects 0.000 claims abstract description 4
- 241000700605 Viruses Species 0.000 claims description 20
- 206010028980 Neoplasm Diseases 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 6
- 238000003259 recombinant expression Methods 0.000 claims description 6
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 118
- 230000002147 killing effect Effects 0.000 abstract description 16
- 238000011740 C57BL/6 mouse Methods 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 7
- 230000004614 tumor growth Effects 0.000 abstract description 7
- 230000005764 inhibitory process Effects 0.000 abstract description 6
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 102100034256 Mucin-1 Human genes 0.000 description 19
- 241000699670 Mus sp. Species 0.000 description 13
- 241000713666 Lentivirus Species 0.000 description 12
- 238000012258 culturing Methods 0.000 description 10
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 description 9
- 102100037850 Interferon gamma Human genes 0.000 description 9
- 108010074328 Interferon-gamma Proteins 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 206010006187 Breast cancer Diseases 0.000 description 6
- 208000026310 Breast neoplasm Diseases 0.000 description 6
- 239000012636 effector Substances 0.000 description 6
- 238000010253 intravenous injection Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 210000003462 vein Anatomy 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229960004641 rituximab Drugs 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 238000011357 CAR T-cell therapy Methods 0.000 description 2
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 2
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 2
- 101000576802 Homo sapiens Mesothelin Proteins 0.000 description 2
- 102100025096 Mesothelin Human genes 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002626 targeted therapy Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 230000004584 weight gain Effects 0.000 description 2
- 235000019786 weight gain Nutrition 0.000 description 2
- 206010000117 Abnormal behaviour Diseases 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010064912 Malignant transformation Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102000015728 Mucins Human genes 0.000 description 1
- 108010063954 Mucins Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 1
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000036212 malign transformation Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- 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
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
- C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Developmental Biology & Embryology (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供一种Mucin1嵌合抗原受体修饰的T细胞及其应用,属于遗传工程技术领域;所述Mucin1嵌合抗原受体包括导引子、单链抗体scFv‑MUC1、CD8 Hinge区、CD8跨膜区、CD226‑CD28共刺激区、CD3ζ胞内区、自剪切区T2A、***基因RQR8;所述单链抗体scFv‑MUC1的核苷酸序列如序列表中SEQ ID NO.3所示。本发明制备的CAR‑MUC1‑T细胞对MCF‑7细胞的杀伤率为86.2%,对T47D‑GL细胞的杀伤率为89.4%;本发明制备的CAR‑MUC1‑T细胞对C57BL6小鼠的肿瘤生长具有明显的抑制作用。
Description
技术领域
本发明涉及一种Mucin1嵌合抗原受体修饰的T细胞及其应用,属于遗传工程技术领域。
背景技术
目前,肿瘤免疫治疗CAR-T(嵌合抗原受体T细胞免疫疗法)是继手术、化疗、放疗和靶向疗法之后出现的一种新型治疗方法,被称为治疗癌症的“第五大疗法”,近年来取得了越来越多的令人鼓舞的研究成果。
CAR应用的关键在于确定一种在肿瘤细胞表面高表达,而在正常组织中低表达或无表达的相关抗原。并且实体瘤具有高度的异质性,不同患者、同一患者不同病灶、同一病灶不同肿瘤细胞之间均存在高度的差异。这种高度的异质性致使肿瘤靶向治疗缺乏理想的通用、广谱靶点,限制了CAR-T细胞治疗实体瘤的疗效。因而,寻找有效的CAR-T细胞治疗靶点已然成为CAR-T细胞治疗的重中之重。
Mucin 1是膜结合型粘蛋白家族成员,简称MUC1,对上皮细胞更新、分化和完整性维持,癌症发生和转移等方面都起着重要作用。在正常情况下,MUC1在乳腺、胰腺、胃肠道、呼吸道及泌尿生殖道的上皮细胞的近管腔面呈低水平表达,不能被机体免疫***识别,其表达特点为极性表达、顶端分布、糖基化丰富。但MUC1在乳腺癌、卵巢癌、肺癌、***癌、结肠癌、肝癌和胰腺癌等恶性肿瘤细胞中呈异常过表达,并失去极性,遍布于整个细胞表面,可通过调控多个信号通路调节细胞恶性转化,是促进肿瘤发生与发展的重要癌基因。因此,MUC1蛋白可作为CAR-T细胞抗肿瘤治疗的一个理想靶点。
目前CAR-T疗法虽然已日渐成熟,但是始终面临着肿瘤复发、抗药的问题,并且在实际的运用中受限于MUC1表达量等原因,治疗效果与预期有差距,因此,有必要对嵌合抗原受体进行进一步改进,提高嵌合抗原受体对肿瘤抗原的靶向结合能力。
目前提供靶向性抗肿瘤T细胞的方法多种多样, CN112940137A提供一种PD-1基因敲除的靶向MUC1的CAR-T细胞制备方法,但细胞的培养周期较长且工序繁琐,该方法只涉及在临床上对癌症患者疾病控制率和安全性评价的统计。CN113248619A提供一种双靶向嵌合抗原受体,以串联方式连接MSLN、MUC1单链抗体的重链VH和轻链VL、及连接MSLN、MUC1单链抗体的第二铰链区,无法判断单靶点MUC1修饰的CAR-T的具体杀伤率。CN107227299A提供一种Anti MUC1 CAR细胞及其制备方法,并以乳腺癌细胞系MCF-7和胰腺癌细胞系BxPC-3作为靶细胞,在效靶比10:1时对靶细胞杀伤率在40%左右,杀伤率较低。
发明内容
针对现有技术存在的不足,本发明提供一种Mucin1嵌合抗原受体修饰的T细胞及其应用,实现以下发明目的:提高对靶细胞的杀伤率,提高IFN-γ的释放量,对小鼠肿瘤生长具有明显抑制作用。
为解决上述问题,本发明采取以下技术方案:
一种Mucin1嵌合抗原受体修饰的T细胞,所述Mucin1嵌合抗原受体包括导引子、单链抗体scFv-MUC1、CD8 Hinge区、CD8跨膜区、CD226-CD28共刺激区、CD3ζ胞内区、自剪切区T2A、***基因RQR8;所述单链抗体scFv-MUC1的核苷酸序列如序列表中SEQ ID NO.3所示。
所述导引子的核苷酸序列如序列表中SEQ ID NO.2所示;所述CD8 Hinge区的核苷酸序列如序列表中SEQ ID NO.4所示;所述CD8跨膜区的核苷酸序列如序列表中SEQ IDNO.5所示;所述CD226-CD28共刺激区的核苷酸序列如序列表中SEQ ID NO.6所示;所述CD3ζ胞内区的核苷酸序列如序列表中SEQ ID NO.7所示;所述自剪切区T2A的核苷酸序列如序列表中SEQ ID NO.8所示;所述***基因RQR8的核苷酸序列如序列表中SEQ ID NO.9所示。
所述Mucin1嵌合抗原受体修饰的T细胞的制备方法为将含Mucin1嵌合抗原受体的重组表达载体进行慢病毒包装,得到重组慢病毒,然后感染活化T细胞,得到Mucin1嵌合抗原受体修饰的T细胞。
所述Mucin1嵌合抗原受体修饰的T细胞在制备治疗抗肿瘤药物中的应用。
与现有技术相比,本发明取得以下有益效果:
(1)本发明制备的CAR-MUC1-T细胞与靶细胞共培养后,IFN-γ的释放量明显增多。本发明CAR-MUC1-T细胞和MCF-7细胞按照效靶比为10:1共培养24h后,IFN-γ释放量为9581pg/mL;本发明CAR-MUC1-T细胞和T47D-GL细胞按照效靶比为10:1共培养24h后,IFN-γ释放量为7836 pg/mL。
(2)本发明制备的CAR-MUC1-T细胞对MCF-7细胞的杀伤率为86.2%(效靶比为10:1),对T47D-GL细胞的杀伤率为89.4%(效靶比为10:1)。
(3)本发明制备的CAR-MUC1-T细胞对C57BL6小鼠的肿瘤生长具有明显的抑制作用,且细胞活性受***基因***的控制。
附图说明
图1为CAR-MUC1模块的连接示意图;
图2为T细胞活化指标CD69的表达率的流式图;
图3为慢病毒转染293T细胞48h后的荧光图;
图4为含pLent-EF1α- CAR-MUC1的重组慢病毒感染活化T细胞后的流式图;
图5为含pLent-EF1α- CAR-MUC1-2的重组慢病毒感染活化T细胞后的流式图;
图6为含pLent-EF1α-空载质粒的重组慢病毒感染活化T细胞后的流式图;
图7为CAR-MUC1-T细胞、CAR-MUC1-2-T细胞、空载T细胞分别和靶细胞共培养24h后, IFN-γ释放量的柱状图;
图8为CAR-MUC1-T细胞、CAR-MUC1-2-T细胞、空载T细胞对靶细胞的体外杀伤率的柱状图;
图9为小鼠体内注射CAR-MUC1-T细胞、CAR-MUC1-2-T细胞、空载T细胞、活化T细胞后的体重变化图;
图10为CAR-T细胞对C57BL6小鼠乳腺癌肿瘤生长的抑制效率图。
具体实施方式
实施例1 重组表达载体pLent-EF1α-CAR-MUC1的构建
CAR-MUC1模块示意见图1(完整核酸序列见附录SEQ ID NO.1);
CAR-MUC1各模块序列:
(1)导引子Leader (SEQ ID NO.2)
(2)单链抗体scFv-MUC1(SEQ ID NO.3)
(3)CD8 Hinge区(SEQ ID NO.4)
(4)CD8跨膜区 (SEQ ID NO.5)
(5)CD226-CD28共刺激区(SEQ ID NO.6)
(6)CD3ζ胞内区(SEQ ID NO.7)
(7)自剪切区T2A(SEQ ID NO.8)
(8)***基因RQR8 (SEQ ID NO.9)
分别按照上述从(1)-(8)的顺序委托山东弘诺生物科技有限公司合成其整个表达框,***pLent-EF1α载体(购自Vigene公司) BamHI-NotI位点,转化到E.coli(Top10),经测序正确后,使用OMEGA公司的质粒提取试剂盒提取质粒,获得重组表达载体pLent-EF1α-CAR-MUC1浓度为1.1µg/µL。
同时采用CN106220736A中公开的Anti-MUC-1-scFV作为单链抗体(SEQ IDNO.10),替代上述SEQ ID NO.3,其余模块均采用上述本发明的序列,构建包含CN106220736A中公开的Anti-MUC-1-scFV序列的重组表达载体,命名为pLent-EF1α-CAR-MUC1-2,浓度为1.0µg/µL。
构建Plent-EF1α-空载质粒:浓度为1.0µg/µL。
实施例2 pLent-EF1α-CAR-MUC1修饰T细胞的制备
1. 活化T细胞的制备
取75mL患者自体外周血,用Ficoll-Paque淋巴细胞分离液分离外周血单个核细胞。分离后的细胞用BD公司提供的CD8+分选试剂分选出CD8+T细胞,进行细胞计数,按1×106个细胞/mL进行接种,加入含有终浓度为1500IU/mL IL-2的KBM551细胞培养基(购自Corning,货号:88-551-CM);然后加入与细胞相同数量的CD3CD28磁珠进行活化T细胞,活化24h后取出磁珠,得到活化T细胞,利用流式细胞仪检测CD69的表达率, CD69的表达率为73.2%,见图2。
2. 慢病毒包装
复苏293T细胞,培养3天,根据细胞密度进行传代,传1代后,细胞融合度达到80%时进行转染。六孔板中按6×105个细胞/孔进行接种,每孔加入2mLDMEM培养基(购自Gibco公司,货号11960-044), 为第二天的转染做准备。转染前将六孔板换成新鲜DMEM培养基,37℃温箱中培养1h。
转染试剂的准备:在5mL离心管中,分别配制A管与B管试剂(Tube A and Tube B);A管与B管的配制见表1。
表1
配好后,放置5min,然后将A管缓慢加入B管,混合均匀,室温放置20min,形成脂质体-DNA混合物;将混合物加入培养瓶中,轻微混匀,置于37℃,5%CO2培养箱中培养。
48小时后,显微镜下观察293T细胞转染后的形态变化(图3)。72小时后将含有病毒的细胞培养上清收集到离心管中,3500rpm/min离心10min,去除细胞碎片,4.5μm滤器过滤后以70000g离心力,4℃离心2h,将沉淀用100μL的PBS重悬,分装后放入-80℃保存,得到病毒液,同时测定病毒滴度。本发明中含有pLent-EF1α-CAR-MUC1的病毒液的病毒滴度为2.18×108TU/mL,含pLent-EF1α-CAR-MUC1-2的病毒液的病毒滴度为1.95×108TU/mL,含有pLent-EF1α-空载质粒的病毒液的病毒滴度为2.26×108TU/mL。
3.慢病毒感染活化的T细胞
从-80℃拿出上述制备的三种病毒液,解冻后加入含有终浓度为1500IU/mL IL-2的KBM551培养基,将病毒滴度稀释到3×107TU/mL,得到稀释后的病毒液。用稀释后的病毒液重悬1×106个活化T细胞,使病毒颗粒数与活化T细胞的数量比例为3:1,得到病毒和细胞悬液。将病毒和细胞悬液加入到6孔板中,每孔2mL,37℃,5%的CO2培养箱中培养48h,收集细胞,400g、5min离心,弃上清,对细胞计数,按照1×106个细胞/mL密度接种,加入含有终浓度为1500IU/mL IL-2的KBM551培养基,每隔3天倍比加液,37℃、5%的CO2培养箱中培养13天使细胞扩增至足够的用量,得到含pLent-EF1α-CAR-MUC1的重组慢病毒感染后的T细胞,简称为CAR-MUC1-T细胞;含pLent-EF1α-CAR-MUC1-2的重组慢病毒感染后的T细胞,简称为CAR-MUC1-2-T细胞;含pLent-EF1α-空载质粒的重组慢病毒感染后的T细胞,简称为空载T细胞。通过流式细胞术检测嵌合抗原受体表达。以活化T细胞作为阴性对照,本发明中含pLent-EF1α- CAR-MUC1的重组慢病毒对活化T细胞的感染率为56.9%,含pLent-EF1α- CAR-MUC1-2的重组慢病毒对活化T细胞的感染率为50.7%,含pLent-EF1α-空载质粒的重组慢病毒对活化T细胞的感染率为57.4%(见图4-6)。
实施例3 三种T细胞的体外IFN-γ释放实验
以乳腺癌细胞系MCF-7、T47D-GL分别作为靶细胞,效应细胞为CAR-MUC1-T细胞、CAR-MUC1-2-T细胞、空载T细胞, 效靶比分别为1:1、5:1、10:1,靶细胞数量为1×105/孔,根据不同效靶比对应效应细胞,每孔加入200μL含有10vol% FBS的DMEM培养基,各组均设3个复孔,取3个复孔的平均值,效应细胞和靶细胞共同培养24h后,收集细胞上清,用ELISA试剂盒检测IFN-γ的含量。
结果如表2、图7所示,以MCF-7和T47D-GL作为靶细胞,本发明的CAR-MUC1-T细胞相较于CAR-MUC1-2-T细胞来说,IFN-γ的释放量明显增多,杀伤力更强,并呈现效靶比梯度依赖性即效靶比越高细胞毒性作用越强。
表2 三种T细胞和靶细胞共同培养24h后IFN-γ释放量(pg/mL)
。
实施例4三种T细胞的体外杀伤实验
将MCF-7细胞和T47D-GL细胞作为靶细胞,CAR-MUC1-T细胞、CAR-MUC1-2-T细胞和空载T细胞作为效应细胞进行杀伤活性测定。按照效应细胞(1×105/孔)与靶细胞(1×104/孔)数量比例为10:1加入48孔培养板中,每孔加入200μL含有10vol% FBS的DMEM培养基,置于5% CO2、37℃培养箱培养,24h后每孔加入20µL CCK-8,继续孵育2h后,酶标仪检测450nm波长,读取OD值。
具体分组如下:
实验组A:CAR-MUC1-T细胞和MCF-7细胞共培养;
实验组B:CAR-MUC1-T细胞和T47D-GL细胞共培养;
实验组C:CAR-MUC1-2-T细胞和MCF-7细胞共培养;
实验组D:CAR-MUC1-2-T细胞和T47D-GL细胞共培养;
对照组E:空载T细胞和MCF-7细胞共培养;
对照组F:空载T细胞和T47D-GL细胞共培养;
空白组G:MCF-7细胞、T47D-GL细胞。
按以下公式计算细胞杀伤率:杀伤率(%)=[1-(空白组OD值-效应细胞组OD值)/空白组OD值]×100%。
结果表明(见图8),实验A-D,以及对照E-F的杀伤率依次为86.2%、89.4%、45.3%、46.1%和15.3%、16.8%, CAR-MUC1-T细胞对两种靶细胞的杀伤效率明显高于CAR-MUC1-2-T细胞,两者均高于对照组。因此,本发明制备的CAR-MUC1-T细胞,能够增强细胞的杀伤能力。
实施例5 CAR-T细胞的体内毒性实验
6-8周的C57BL6小鼠(购自南京君科生物工程有限公司),分成5组,每组10只,验证CAR-T细胞的体内毒性实验。实验组分别为:
a.对照组,尾部静脉注射同等体积的生理盐水;
b.实验一组,尾部静脉注射2×107个细胞/只(活化T细胞);
c.实验二组,尾部静脉注射2×107个细胞/只(空载T细胞);
d.实验三组,尾部静脉注射2×107个细胞/只(CAR-MUC1-T细胞);
e.实验四组,尾部静脉注射2×107个细胞/只(CAR-MUC1-2-T细胞)。
注射后每天观察小鼠的行为表现,每周对小鼠称重一次,利用动物活体成像***监测小鼠体内CAR-T细胞是否存在。45天后,解剖小鼠,对小鼠的主要组织如脑、心脏、肺、肝、结肠和肾脏进行病理观察。
实验过程中,并未观察到小鼠有异常的行为表现,如图9所示,小鼠的体重增加没有明显差异,且血液中检测到CAR-T细胞持续存在35天。解剖小鼠后对主要组织的病理观察,CAR-T小鼠并没有发现组织的病变。
表3 各实验组培养45天后小鼠体重增重变化
。
实施例6 CAR-T细胞对C57BL6小鼠乳腺癌肿瘤生长抑制作用
6-8周的雄性C57BL6小鼠(购自南京君科生物工程有限公司)于动物房饲养(室温23±2℃,湿度50%±10%),收集对数期的MCF-7细胞,磷酸盐缓冲液(PBS)稀释至2×106个/mL。无菌条件下,小鼠左侧腋下接种0.2mL MCF-7细胞悬浮液,观察两周,待腋下出现米粒大小较硬的结节作为建模成功的标准。
C57BL6乳腺癌模型小鼠(游标卡尺量取皮下肿瘤组织块的大小为90-100mm3)随机分成6组,每组10只,开始注射治疗实验。实验组分别为:
a.对照组,尾部静脉注射同等体积的生理盐水;
b.治疗一组,尾部静脉注射2×106个细胞/只(活化T细胞);
c.治疗二组,尾部静脉注射2×106个细胞/只(空载T细胞);
d.治疗三组,尾部静脉注射2×106个细胞/只(CAR-MUC1-T细胞);
e.治疗四组,尾部静脉注射2×106个细胞/只(CAR-MUC1-2-T细胞);
f.治疗五组,尾部静脉注射2×106个细胞/只(CAR-MUC1-T细胞),24h后注射利妥昔单抗,每天一次。
每周免疫上述各组小鼠一次,连续免疫两周,以首次注射当天为0天记录,每三天通过游标卡尺测量各个治疗组小鼠皮下肿瘤组织块大小,用肿块均值绘制肿瘤生长曲线图,5周后解剖小鼠,将肿瘤组织利用石蜡包埋,切片。
肿瘤体积增长结果如表4 所示。结果表明本发明制备的CAR-MUC1-T细胞对C57BL6小鼠肿瘤生长的抑制作用最好(图10),相较于CAR-MUC1-2-T细胞细胞,可以明显抑制肿瘤的增长,注射利妥昔单抗后CAR-T细胞对肿瘤基本不起作用,说明利妥昔单抗可以启动本发明中CAR-MUC1-T细胞的******RQR8,细胞活性受***基因***的控制。
表4 治疗过程中小鼠肿瘤体积的统计(mm3)
。
Claims (4)
1.一种Mucin1嵌合抗原受体修饰的T细胞,其特征在于:所述Mucin1嵌合抗原受体包括导引子、单链抗体scFv-MUC1、CD8 Hinge区、CD8跨膜区、CD226-CD28共刺激区、CD3ζ胞内区、自剪切区T2A、***基因RQR8;所述单链抗体scFv-MUC1的核苷酸序列如序列表中SEQ IDNO.3所示。
2.根据权利要求1所述的一种Mucin1嵌合抗原受体修饰的T细胞,其特征在于:所述导引子的核苷酸序列如序列表中SEQ ID NO.2所示;所述CD8 Hinge区的核苷酸序列如序列表中SEQ ID NO.4所示;所述CD8跨膜区的核苷酸序列如序列表中SEQ ID NO.5所示;所述CD226-CD28共刺激区的核苷酸序列如序列表中SEQ ID NO.6所示;所述CD3ζ胞内区的核苷酸序列如序列表中SEQ ID NO.7所示;所述自剪切区T2A的核苷酸序列如序列表中SEQ IDNO.8所示;所述***基因RQR8的核苷酸序列如序列表中SEQ ID NO.9所示。
3.根据权利要求1所述的一种Mucin1嵌合抗原受体修饰的T细胞,其特征在于:所述Mucin1嵌合抗原受体修饰的T细胞的制备方法为将含Mucin1嵌合抗原受体的重组表达载体进行慢病毒包装,得到重组慢病毒,然后感染活化T细胞,得到Mucin1嵌合抗原受体修饰的T细胞。
4.权利要求1所述的Mucin1嵌合抗原受体修饰的T细胞在制备治疗抗肿瘤药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310760077.8A CN116555187B (zh) | 2023-06-27 | 2023-06-27 | 一种Mucin1嵌合抗原受体修饰的T细胞及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310760077.8A CN116555187B (zh) | 2023-06-27 | 2023-06-27 | 一种Mucin1嵌合抗原受体修饰的T细胞及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116555187A true CN116555187A (zh) | 2023-08-08 |
CN116555187B CN116555187B (zh) | 2023-09-08 |
Family
ID=87491810
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310760077.8A Active CN116555187B (zh) | 2023-06-27 | 2023-06-27 | 一种Mucin1嵌合抗原受体修饰的T细胞及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116555187B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117143825A (zh) * | 2023-11-01 | 2023-12-01 | 上海兴瑞一达生物科技有限公司 | 一种msln嵌合抗原受体修饰的t细胞及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103880956A (zh) * | 2014-03-10 | 2014-06-25 | 中国人民解放军第四军医大学 | 抗muc1单克隆抗体及其轻链和重链可变区 |
-
2023
- 2023-06-27 CN CN202310760077.8A patent/CN116555187B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103880956A (zh) * | 2014-03-10 | 2014-06-25 | 中国人民解放军第四军医大学 | 抗muc1单克隆抗体及其轻链和重链可变区 |
Non-Patent Citations (2)
Title |
---|
AGNIESZKA GORNOWICZ ET AL.: "Mucin1 as a molecular target of a novel diisoquinoline derivative combined with anti-MUC1 antibody in AGS gastric cancer cells.", 《MOLECULES》, pages 1 - 21 * |
孔北华等: "单链抗体导向卵巢上皮癌进行靶向基因转移的实验研究", 《现代妇产科进展》, pages 324 - 327 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117143825A (zh) * | 2023-11-01 | 2023-12-01 | 上海兴瑞一达生物科技有限公司 | 一种msln嵌合抗原受体修饰的t细胞及其应用 |
CN117143825B (zh) * | 2023-11-01 | 2024-01-23 | 上海兴瑞一达生物科技有限公司 | 一种msln嵌合抗原受体修饰的t细胞及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN116555187B (zh) | 2023-09-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111315873B (zh) | 分离的重组溶瘤痘病毒、药物组合物及其在***和/或癌症的药物中的用途 | |
KR101419711B1 (ko) | 항원제시세포의 활성화 처리방법 | |
US20210154231A1 (en) | Method for producing t cells modified by chimeric antigen receptor | |
CN116555187B (zh) | 一种Mucin1嵌合抗原受体修饰的T细胞及其应用 | |
CN110317822B (zh) | Trop2嵌合抗原受体、其t细胞及其制备方法和用途 | |
CN113214408B (zh) | 一种嵌合抗原受体巨噬细胞及其制备方法和用途 | |
CN110055269B (zh) | 人间皮素嵌合抗原受体、其t细胞及其制备方法和用途 | |
CN106574241A (zh) | 癌症免疫疗法组合物和方法 | |
US20190307794A1 (en) | Method for inducing transdifferentiation of immune cells based on exosomes | |
CN116769723B (zh) | 一种gd2嵌合抗原受体修饰的t细胞及其应用 | |
WO2020259151A1 (zh) | 一种ctl细胞的制备方法及应用 | |
JP2023502191A (ja) | プラスミドの組合せおよび改変免疫細胞の調製におけるその適用 | |
JP2024501127A (ja) | 腫瘍浸潤リンパ球培地及びその使用 | |
CN104001185A (zh) | 一种cea阳性肿瘤特异性树突状细胞疫苗的制备方法 | |
WO2023005486A1 (zh) | 一种ebv复合抗原、树突状细胞疫苗及其应用 | |
CN110139875B (zh) | Col14a1衍生的肿瘤抗原多肽及其应用 | |
CN111733154B (zh) | 磁场处理的免疫细胞及其用途 | |
CN113854234A (zh) | 一种小鼠胰腺癌模型及其构建方法和应用 | |
CN117143825B (zh) | 一种msln嵌合抗原受体修饰的t细胞及其应用 | |
CN108034669B (zh) | 一种抗vegf基因、及利用其修饰的t细胞、制备方法和应用 | |
CN116904400B (zh) | 可利霉素在体外car/tcr-t细胞产品制备过程优化中的应用 | |
CN115969971B (zh) | 组合物在制备***的药物中的应用 | |
CN116555188B (zh) | 一种抗pd-1双靶点的car-nk细胞及其应用 | |
CN116445416B (zh) | 一种基因修饰的car-nk细胞及其制备方法和应用 | |
CN115925974B (zh) | 一种针对实体瘤的通用性的ips来源的car-nk细胞的制备方法 |
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 |