CN116138212B - 一种制备细胞因子释放综合征小鼠模型的方法及应用 - Google Patents
一种制备细胞因子释放综合征小鼠模型的方法及应用 Download PDFInfo
- Publication number
- CN116138212B CN116138212B CN202310104028.9A CN202310104028A CN116138212B CN 116138212 B CN116138212 B CN 116138212B CN 202310104028 A CN202310104028 A CN 202310104028A CN 116138212 B CN116138212 B CN 116138212B
- Authority
- CN
- China
- Prior art keywords
- car
- cells
- mcd19
- crs
- mice
- 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
Links
- 206010052015 cytokine release syndrome Diseases 0.000 title claims abstract description 52
- 238000010172 mouse model Methods 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 14
- 241000699670 Mus sp. Species 0.000 claims abstract description 49
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 21
- 108010065805 Interleukin-12 Proteins 0.000 claims abstract description 17
- 102000013462 Interleukin-12 Human genes 0.000 claims abstract description 17
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 claims description 10
- 229960004397 cyclophosphamide Drugs 0.000 claims description 10
- 241001529936 Murinae Species 0.000 claims description 4
- 239000000427 antigen Substances 0.000 claims description 4
- 108091007433 antigens Proteins 0.000 claims description 4
- 102000036639 antigens Human genes 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 230000003092 anti-cytokine Effects 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 17
- 102100037850 Interferon gamma Human genes 0.000 abstract description 9
- 108010074328 Interferon-gamma Proteins 0.000 abstract description 9
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 abstract description 6
- 108090001005 Interleukin-6 Proteins 0.000 abstract description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 abstract description 6
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 abstract description 6
- 238000001727 in vivo Methods 0.000 abstract description 6
- 210000004185 liver Anatomy 0.000 abstract description 6
- 102000003814 Interleukin-10 Human genes 0.000 abstract description 5
- 108090000174 Interleukin-10 Proteins 0.000 abstract description 5
- 206010061902 Pancreatic neoplasm Diseases 0.000 abstract description 5
- 230000034994 death Effects 0.000 abstract description 5
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 abstract description 5
- 201000002528 pancreatic cancer Diseases 0.000 abstract description 5
- 208000008443 pancreatic carcinoma Diseases 0.000 abstract description 5
- 102100021943 C-C motif chemokine 2 Human genes 0.000 abstract description 4
- 101710155857 C-C motif chemokine 2 Proteins 0.000 abstract description 4
- 230000004580 weight loss Effects 0.000 abstract description 4
- -1 IL-1a Proteins 0.000 abstract description 3
- 230000012010 growth Effects 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 3
- 230000002757 inflammatory effect Effects 0.000 abstract description 3
- 206010067125 Liver injury Diseases 0.000 abstract description 2
- 208000004852 Lung Injury Diseases 0.000 abstract description 2
- 206010069363 Traumatic lung injury Diseases 0.000 abstract description 2
- 231100000753 hepatic injury Toxicity 0.000 abstract description 2
- 231100000515 lung injury Toxicity 0.000 abstract description 2
- 208000016261 weight loss Diseases 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 73
- 208000003606 Congenital Rubella Syndrome Diseases 0.000 description 31
- 102000004127 Cytokines Human genes 0.000 description 11
- 108090000695 Cytokines Proteins 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 210000001744 T-lymphocyte Anatomy 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 230000008595 infiltration Effects 0.000 description 6
- 238000001764 infiltration Methods 0.000 description 6
- 238000001802 infusion Methods 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 108091026890 Coding region Proteins 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 4
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 4
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- 241000700605 Viruses Species 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 230000017074 necrotic cell death Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 108700031361 Brachyury Proteins 0.000 description 1
- 238000011357 CAR T-cell therapy Methods 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 101001033279 Homo sapiens Interleukin-3 Proteins 0.000 description 1
- 101000716729 Homo sapiens Kit ligand Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 102000000646 Interleukin-3 Human genes 0.000 description 1
- 206010024642 Listless Diseases 0.000 description 1
- 101001044384 Mus musculus Interferon gamma Proteins 0.000 description 1
- 206010029379 Neutrophilia Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 229960001334 corticosteroids Drugs 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 210000003194 forelimb Anatomy 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000001794 hormone therapy Methods 0.000 description 1
- 102000055151 human KITLG Human genes 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000007233 immunological mechanism Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011221 initial treatment Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 208000017971 listlessness Diseases 0.000 description 1
- 230000000998 lymphohematopoietic effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108010056030 retronectin Proteins 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000004926 tubular epithelial cell Anatomy 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000002477 vacuolizing effect Effects 0.000 description 1
- 230000009723 vascular congestion Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/20—Animal model comprising regulated expression system
- A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提出了一种制备细胞因子释放综合征小鼠模型的方法及应用。本发明尝试通过回输IL‑12 CAR‑T细胞诱发小鼠CRS,建立一种新的CRS模型。结果发现mCD19/IL‑12‑CAR‑T细胞体外和体内均分泌高水平IL‑12,低剂量1×106个mCD19/IL‑12‑CAR‑T细胞回输小鼠未诱发严重CRS,但可以抑制胰腺癌实体瘤的生长,高剂量回输mCD19/IL‑12‑CAR‑T细胞后,出现激发小鼠体内多种CRS相关炎性因子IL‑6、IL‑1a、IFN‑γ、IL‑10、TNF‑α、MCP‑1等迅速升高,体重减轻、肝脏肺脏等组织损伤,最终导致死亡等一系列典型CRS表现,从而建立一种新的CRS小鼠模型。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种制备细胞因子释放综合征小鼠模型的方法及应用。
背景技术
嵌合抗原受体T细胞(chimeric antigen receptor T cell,CAR-T)疗法开辟了肿瘤免疫治疗的新领域,针对B细胞表面标志物CD19的CAR-T细胞临床试验已显示出对多种血液系统恶性肿瘤的显著疗效。虽然CAR-T临床表现积极结果,但同时也带来许多不良反应。其中细胞因子释放综合征(Cytokine Release Syndrome,CRS)是最常见的一种严重并发症,由于CAR-T细胞治疗过程中活化的免疫细胞释放过量细胞因子而引起的超常免疫反应,表现为大量细胞因子短暂地、显著地升高,严重的CRS可危及患者生命。建立合适的动物模型是设计预防或减轻CRS策略的有效手段。负荷肿瘤的NSG小鼠通常用作CAR-T治疗的临床前模型,以及SCID-beige小鼠模型、CD20 CAR-T引起的免疫激活级联反应的恒河猴模型等。但建立这些模型价格昂贵,实验成本很高,不便于大规模开展相关的实验研究。
为了更好研究CRS发生的免疫机制及对治疗药物的疗效评估,需要建立技术方法简单、易于重复、便于大规模推广的的CRS小鼠模型。已报道的常用CAR-T相关CRS小鼠模型有两种,但是均重复性、稳定性较差。一种为SCID-beige小鼠腹腔内注射3×106个Raji-GFP-Flu白血病细胞,肿瘤生长3周后通过生物发光成像评估肿瘤负担,再腹腔注射3×107个CD19.28z CAR-T细胞,CAR-T输注2~3天后,小鼠发生CRS,血清细胞因子升高,并最终死亡。但是这些小鼠不同个体肿瘤负荷偏差较大,导致小鼠死亡时间不均一,CRS治疗观察窗较窄,不利于实验研究。此外该模型采用人源CAR-T细胞,使得IFN-γ、IL-6等重要细胞因子及受体无法跨物种识别,这限制了该模型对人体CRS反应的模拟程度。另一种为通过肝内注射将人脐带血造血干细胞和祖细胞移植到亚致死剂量照射的新生NSG小鼠,或者表达人类干细胞因子、粒细胞-巨噬细胞集落刺激因子和IL-3的NSG-SGM3小鼠,重建淋巴造血系统,得到人源化NSG或者SGM3小鼠。这种模型对人CRS模拟程度更高,但小鼠品系很难获得,国内缺乏正式引进,造价昂贵,不利于实验的常规广泛开展。目前这两种小鼠模型已被多次报道应用,能较好反应临床CAR-T CRS的症状,另外也有研究用LPS诱发小鼠的CRS,但不能完全反应CAR-T CRS的病理特征和免疫学机制。目前缺乏造价低廉、稳定、能降低小鼠肿瘤负担的CAR-T CRS模型。
发明内容
发明目的:本发明的发明目的为提供一种体内细胞因子释放综合征小鼠模型及其构建方法,该模型消除小鼠肿瘤负担,简化实验过程,提高实验的稳定性,便于推广应用。
为了解决上述技术问题,本发明公开了一种制备细胞因子释放综合征小鼠模型的方法,其通过将2×106~1×107个CAR-T细胞通过尾静脉回输至小鼠体内得到,其中,所述CAR-T细胞表达识别鼠源抗原的CAR和外源性的IL12。
其中,其中所述鼠源抗原为mCD19。
所述CAR的结构为抗mCD19 scFv-BBz。
具体地,抗mCD19 scFv的序列如SEQ ID NO:1所示;所述IL12的序列如SEQ ID NO:4所示,BBz结构包括mCD8a TM-m41BB ICOS-mCD3ζ,所述BBZ的序列如Sequence ID No.2所示。
其中CAR-T细胞的施用剂量为2×106~1×107个细胞。
优选地,向小鼠施用CAR-T细胞之前,先向小鼠注射环磷酰胺。
具体地,向普通小鼠体内腹腔注射环磷酰胺两天后通过尾静脉注射回输2×106~1×107个mCD19/IL-12-CAR-T细胞得到。
其中所述环磷酰胺的剂量为150-200mg/kg,施用CAR-T细胞和注射环磷酰胺之间间隔2-4天。
其中所述小鼠为C57BL/6J小鼠。
通过上述方法构建得到的细胞因子释放综合征小鼠模型也在本发明的保护范围值呢。
本发明进一步提出了上述细胞因子释放综合征小鼠模型在制备筛选抗细胞因子释放综合征药物的产品中的应用。
有益效果:与先有技术相比,本申请具有如下优点:
(1)本申请的模型使用普通品系C57BL/6J小鼠及通过尾静脉回输体外培养的小鼠CAR-T细胞即可建立,CRS的产生不依赖于接种肿瘤的大小和类型,避免了小鼠在实验期间因肿瘤生长速度的差异及负担过重而导致的不均一死亡,CRS发生时间主要在CAR-T回输后的1~6天,小鼠表现出典型CRS症状,如体重减轻,血清细胞因子显著升高等,符合临床CAR-T诱发CRS表征,此模型更稳定,制作周期短,降低了实验成本,重复性高;
(2)目前CRS的治疗首选使用激素治疗,抗IL-6受体拮抗剂托珠单抗联合或者不联合皮质类固醇的给药是主要的治疗方法。托珠单抗干预的最佳时机尚不清楚,并且是正在进行的临床试验的主要解决问题,本发明为CRS治疗药物的有效性、安全性及药物筛选提供了合适的工具模型,为CRS治疗药物的有效性、安全性及药物筛选提供了合适的工具模型,保证其模型的重复性和高效性,具有广泛的应用前景。
附图说明
图1为mCD19-CAR-T和mCD19/IL-12-CAR-T分子构建结构示意图;
图2为mCD19-CAR-T、mCD19/IL-12-CAR-T细胞阳性表达率示意图;
图3为ELISA检测mCD19-CAR-T、mCD19/IL-12-CAR-T细胞IL-12、IFN-γ的分泌的结果;
图4为mCD19/IL-12-CAR-T细胞对小鼠胰腺癌Panc02-CD19细胞移植瘤的生长的影响;
图5为高剂量mCD19/IL-12-CAR-T细胞对小鼠体质量(A)与生存曲线(B)的影响;
图6为输注高剂量mCD19/IL-12-CAR-T细胞后对小鼠血清中细胞因子水平的影响(H-E染色,200×);
图7为输注高剂量mCD19/IL-12-CAR-T细胞后对小鼠肝、脾、肺和肾组织病理学改变的影响。
具体实施方式
下面结合附图和具体实施方式对本发明做更进一步的具体说明,本发明的上述和/或其他方面的优点将
会变得更加清楚。
下述实施例中使用的小鼠为普通品系C57BL/6J雌性小鼠,年龄6-8周左右。
实施例1mCD19-CAR-T、mCD19/IL-12-CAR-T细胞的构建及活性测试。
(1)质粒构建
依次连接CD19 scFV、BBZ的编码序列片段,并在两端加入XhoI/EcoRI酶切位点。将片段克隆入MSCV载体,获得MSCV-mCD19-CAR质粒,用于mCD19-CAR-T细胞的制备。T2A和IL-12的编码片段克隆入MSCV-mCD19-CAR载体,获得MSCV-mCD19-CAR-IL-12质粒,用于mCD19/IL-12-CAR-T细胞的制备。
其中,CD19 scFV的编码序列如Sequence ID No.1所示,BBZ的编码序列如Sequence ID No.2所示;T2A的编码序列如Sequence ID No.3所示;IL-12的编码片段如IL-12的Sequence ID No.4所示。
实验结果如图1所示,成功构建了靶向CD19的经典第二代CAR分子结构,依次连接T2A和IL-12的编码序列片段。
(2)逆转录病毒包装
将MSCV-mCD19-CAR、MSCV-mCD19-CAR-IL-12质粒与辅助质粒pCL-Eco(上海禾午生物科技有限公司)通过X-treme GENE HP DNA试剂(Roche,货号06366236001)共转染进293T细胞中,于37℃,5% CO2条件培养过夜。在转染后72h收集上清,2000g,4℃离心10min,获得逆转录病毒上清液。
(3)mCD19-CAR-T细胞、mCD19/IL-12-CAR-T细胞的制备及鉴定
从C57BL/6J小鼠脾脏分离T淋巴细胞,并用DynaBeads CD3/CD28 CTSTM(Gibco,货号40203D)激活1天,在第2天将T细胞转导至带有预先用RetroNectin(Takara)包被过夜的24孔板中,分别加入MSCV-mCD19-CAR病毒、MSCV-mCD19-CAR-IL-12病毒、完全培养基(阴性对照T细胞,NT),2000g,32℃离心2h后继续扩增培养。隔天更换新鲜培养基,维持细胞密度在1×106/ml,病毒感染3天后,即可获得mCD19-CAR-T、mCD19/IL-12-CAR-T细胞。通过GoatAnti-Rat IgG(H&L)Biotin(BioVision,货号6910-250)作为一抗,APC Streptavidin(BDPharmingen,货号554067)作为二抗染色检查阳性率。
实验结果如图2所示,上述分子包装逆转录病毒,使用逆转录病毒转染小鼠T细胞,得到mCD19-CAR-T、mCD19/IL-12-CAR-T细胞。3d后,通过流式细胞术采用山羊抗鼠IgG抗体检测病毒转染效率,mCD19-CAR-T、mCD19/IL-12-CAR-T细胞的转染效率分别为65.1%、56.9%。经过7d扩增,细胞增殖达200倍左右。
(4)ELISA检测mCD19-CAR-T、mCD19/IL-12-CAR-T的IL-12、IFN-γ分泌水平
IL-12检测:收集CAR-T细胞上清液,利用Mouse IL-12DuoSet ELISAKit试剂盒(R&D Systems,货号DY419)检测。
IFN-γ检测:以Panc02细胞或Panc02-mCD19细胞为靶细胞,mCD19-CAR-T、mCD19/IL-12-CAR-T、NT细胞为效应细胞,按5:1效靶比将这两种细胞共培养24h后,收集培养物上清液,利用Mouse IFN-gamma DuoSet ELISAKit试剂盒(R&D Systems,货号DY485)检测。
实验结果如图3所示,mCD19/IL-12-CAR-T细胞分泌IL-12的水平显著高于NT细胞和mCD19-CAR-T细胞(P<0.01,图3A)。将各组CAR-T细胞与非靶细胞Panc02共培养,与NT组和mCD19-CAR-T组比较,mCD19/IL-12-CAR-T组细胞IFN-γ的分泌水平显著升高(P<0.01);与靶细胞Panc02-CD19共培养后,mCD19/IL-12-CAR-T组细胞分泌IFN-γ水平显著升高(P<0.01,图3B)。实验结果表明,mCD19/IL-12-CAR-T细胞对肿瘤细胞的特异性杀伤作用。
实施例2体内小鼠肿瘤模型的建立。
在小鼠左前肢腋下部位经皮下接种5×105个Panc02-mCD19胰腺癌细胞,将小鼠按照随机数字表法随机分为3组,每组6只,待肿瘤体积生长至100mm3时,分别向各组小鼠尾静脉注射1×106个NT细胞、1×106个mCD19-CAR-T细胞、1×106个mCD19/IL-12-CAR-T细胞,在输注CAR-T细胞前48h,腹腔注射200mg/kg环磷酰胺预处理小鼠。每天均用卡尺分别测量和计算小鼠肿瘤体积,每天监测小鼠的体重变化,直至实验结束。
实验结果如图4所示,成功建立小鼠胰腺癌Panc02-CD19细胞移植瘤模型,与NT细胞和mCD19-CAR-T细胞处理组小鼠相比,mCD19/IL-12-CAR-T细胞处理组小鼠肿瘤体积明显减小(P<0.01,图4A);小鼠体质量在CAR-T细胞输注1~2周时有下降趋势,但未出现脑炎表现,小鼠未发生死亡,且在1周左右恢复正常,mCD19/IL-12-CAR-T细胞处理组小鼠体质量与NT细胞和mCD19-CAR-T细胞处理组比较差异无统计学意义(P>0.05,图4B)。实验结果表明,低剂量回输的mCD19/IL-12-CAR-T细胞对小鼠有一过性的毒副反应,但尚未发生严重CRS,表达IL-12可以增强CAR-T细胞的肿瘤抑制效果。
实施例3体内小鼠CRS模型的建立。
(1)模型建立
将普通品系C57BL/6J正常小鼠按照随机数字表法随机分为3组,每组6只,均腹腔注射200mg/kg环磷酰胺进行预处理,第3天分别尾静脉注射直接回输2×106个NT细胞、2×106个mCD19-CAR-T细胞、2×106个mCD19/IL-12-CAR-T细胞,每天监测小鼠日常活动,体重减轻及死亡率。第6天通过眼眶出血后收集血样,并在室温下凝结30min,离心分离血清并储存在-80℃。第10天小鼠脱颈椎处死,取小鼠肝、脾、肺、肾等组织,多聚甲醛固定并石蜡包埋,用于组织学分析。
实验结果如图5所示。使用200mg/kg环磷酰胺预处理小鼠2d后,mCD19/IL-12-CAR-T组小鼠出现发抖、不适、精神萎靡和起毛,表现出严重的体质量减轻,并最终导致死亡(图5A);mCD19-CAR-T组小鼠未出现体质量减轻,状态与NT组相比无明显差异,小鼠均存活。mCD19/IL-12-CAR-T组小鼠在回输第6天开始出现死亡,到第10天其死亡率达80%(图5B)。
(2)小鼠血清细胞因子测定:
采用基于复合微球的流式细胞仪免疫测定试剂盒(MU Inflam Panel(13-plex)w/Vbp Multi-Analyte Flow Assay Kit,Biolegend),检测小鼠血清中IL-6、MCP-1、IL-1、IL-10、TNF-α、IFN-γ等13种细胞因子的水平。
实验结果如图6所示,在CAR-T细胞回输4d后,流式细胞术检测结果,与NT组和mCD19-CAR-T组相比,mCD19/IL-12-CAR-T组小鼠血清中IFN-γ、MCP-1、IL-6、TNF-α、IL-12、IL-1和IL-10含量均显著升高(均P<0.01)。实验结果表明,输注高剂量的mCD19/IL-12-CAR-T细胞后,导致小鼠出现全身性CRS。
(3)组织病理学检查:
获得小鼠组织在室温下用4%多聚甲醛固定24h,然后将收集的组织样本包埋在石蜡中,以4μm的厚度连续切片,并用苏木精和伊红(HE)染色进行形态学分析和病理学检查。
实验结果如图7所示,在CAR-T细胞回输8d后,mCD19/IL-12-CAR-T组小鼠肝可见弥漫性肝细胞气球样变性,胞质空泡化,多处可见坏死灶,伴淋巴细胞浸润和血管淤血;脾组织中可见大面积淤血、淋巴细胞坏死,伴大量中性粒细胞浸润;肺可见弥漫性肺泡壁增厚,伴大量淋巴细胞以及中性粒细胞浸润,大量血管周围水肿;肾间质多处可见淤血。mCD19-CAR-T组肾小管上皮细胞、肝细胞出现少量水样变性,脾中性粒细胞增多;肺伴随淋巴细胞浸润,病变程度明显低于mCD19/IL-12-CAR-T组,NT组未见明显病变。实验结果表明,输注高剂量mCD19/IL-12-CAR-T细胞后,致使实验组小鼠肝、脾、肺和肾组织发生炎性细胞浸润甚至坏死、淤血等病理学改变。
IL-12是一种具有强大肿瘤抑制活性的促炎细胞因子,可诱导T细胞、NK细胞释放IFN-γ、TNF-α等细胞因子,导致巨噬细胞、树突状细胞、内皮细胞及其他免疫细胞的活化,这些细胞进一步释放过量的炎性细胞因子如IL-6、IL-1a、IL-10、TNF-α等,引起连锁反应,形成激活炎症的循环,在CAR-T相关CRS中起重要的启动激发作用。利用小鼠尾静脉高剂量回输IL-12CAR-T细胞的方式,诱发CRS,建立一种新的CRS小鼠模型。
本发明尝试通过回输IL-12CAR-T细胞诱发小鼠CRS,建立一种新的CRS模型。结果发现mCD19/IL-12-CAR-T细胞体外和体内均分泌高水平IL-12,低剂量1×106个mCD19/IL-12-CAR-T细胞回输小鼠未诱发严重CRS,但可以抑制胰腺癌实体瘤的生长,高剂量回输mCD19/IL-12-CAR-T细胞后,出现激发小鼠体内多种CRS相关炎性因子IL-6、IL-1a、IFN-γ、IL-10、TNF-α、MCP-1等迅速升高,体重减轻、肝脏肺脏等组织损伤,最终导致死亡等一系列典型CRS表现,从而建立一种新的CRS小鼠模型。
本发明提供了一种构建体内细胞因子释放综合征小鼠模型的思路及方法,具体实现该技术方案的方法和途径很多,以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (2)
1.一种制备细胞因子释放综合征小鼠模型的方法,其特征在于,包含以下步骤:
向普通品系C57BL/6J小鼠通过尾静脉回输2×106 ~1×107个 CAR-T细胞得到,其中,所述CAR-T细胞表达识别鼠源抗原的CAR和外源性的IL12,向小鼠施用CAR-T细胞之前,先向小鼠注射环磷酰胺,其中所述环磷酰胺的剂量为150-200 mg/kg,施用CAR-T细胞和注射环磷酰胺之间间隔2-4天;所述鼠源抗原为mCD19;所述CAR的结构为抗mCD19 scFv-BBz抗mCD19scFv的序列如SEQ ID NO:1所示;所述IL12的序列如SEQ ID NO:4所示。
2.权利要求1所述的细胞因子释放综合征小鼠模型的方法在制备筛选抗细胞因子释放综合征药物的产品中的应用。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2023100412674 | 2023-01-11 | ||
CN202310041267 | 2023-01-11 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116138212A CN116138212A (zh) | 2023-05-23 |
CN116138212B true CN116138212B (zh) | 2024-03-05 |
Family
ID=86355792
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310104028.9A Active CN116138212B (zh) | 2023-01-11 | 2023-04-10 | 一种制备细胞因子释放综合征小鼠模型的方法及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116138212B (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106459989A (zh) * | 2013-12-19 | 2017-02-22 | 诺华股份有限公司 | 人间皮素嵌合抗原受体及其用途 |
CN107072184A (zh) * | 2014-09-19 | 2017-08-18 | 瑞泽恩制药公司 | 嵌合抗原受体 |
CN107530376A (zh) * | 2015-02-18 | 2018-01-02 | 恩立夫克治疗有限责任公司 | 用于癌症治疗的联合免疫疗法和细胞因子控制疗法 |
CN111050545A (zh) * | 2017-06-29 | 2020-04-21 | 朱诺治疗学股份有限公司 | 评估与免疫疗法相关的毒性的小鼠模型 |
CN111601817A (zh) * | 2017-11-14 | 2020-08-28 | 纪念斯隆-凯特琳癌症中心 | 分泌il-33的免疫应答细胞及其用途 |
-
2023
- 2023-04-10 CN CN202310104028.9A patent/CN116138212B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106459989A (zh) * | 2013-12-19 | 2017-02-22 | 诺华股份有限公司 | 人间皮素嵌合抗原受体及其用途 |
CN107072184A (zh) * | 2014-09-19 | 2017-08-18 | 瑞泽恩制药公司 | 嵌合抗原受体 |
CN107530376A (zh) * | 2015-02-18 | 2018-01-02 | 恩立夫克治疗有限责任公司 | 用于癌症治疗的联合免疫疗法和细胞因子控制疗法 |
CN111050545A (zh) * | 2017-06-29 | 2020-04-21 | 朱诺治疗学股份有限公司 | 评估与免疫疗法相关的毒性的小鼠模型 |
CN111601817A (zh) * | 2017-11-14 | 2020-08-28 | 纪念斯隆-凯特琳癌症中心 | 分泌il-33的免疫应答细胞及其用途 |
Non-Patent Citations (4)
Title |
---|
CAR-T细胞治疗产品非临床药效学研究关注点;侯田田;黄瑛;霍艳;;中国药事(第09期);全文 * |
CAR-T细胞的作用机制及其在B淋巴细胞肿瘤治疗中的研究进展;李晓清;杜新;刘焕勋;陈伟红;古庆利;胡春宏;;标记免疫分析与临床(第06期);全文 * |
可诱导表达IL-12的GPC3靶向性CAR-T细胞在免疫健全小鼠中的抗乳腺癌作用;刘莹;李宗海;蒋华;;肿瘤(第07期);全文 * |
靶向实体瘤微环境以提高CAR-T细胞疗效的新策略研究;顾杰艺;张二浩;徐寒梅;;药物生物技术(第02期);全文 * |
Also Published As
Publication number | Publication date |
---|---|
CN116138212A (zh) | 2023-05-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6920644B2 (ja) | 幹細胞の免疫制御作用を調節する方法 | |
Lynch et al. | Interleukin 7 promotes long-term in vitro growth of antitumor cytotoxic T lymphocytes with immunotherapeutic efficacy in vivo. | |
CN106659742B (zh) | 表达免疫应答刺激细胞因子以吸引和/或激活免疫细胞的基因修饰间充质干细胞 | |
TWI830771B (zh) | 包含核酸及car修飾的免疫細胞的治療劑及其應用 | |
JP3524918B2 (ja) | 癌のリンホカイン遺伝子療法 | |
CN102321577B (zh) | 抗肿瘤过继免疫细胞制备方法及制得的免疫细胞 | |
DK2534242T3 (en) | Improved composition for the inhibition of tumor cell proliferation | |
US9694059B2 (en) | Ex vivo, fast and efficient process to obtain activated antigen-presenting cells that are useful for therapies against cancer and immune system-related diseases | |
WO2018137643A1 (zh) | 溶瘤病毒作为用于治疗肿瘤和/或癌症的免疫刺激剂的应用 | |
US20200216806A1 (en) | Allogeneic Dendritic Cells For Use In Cancer Treatment | |
Van den Bergh et al. | Characterization of interleukin-15-transpresenting dendritic cells for clinical use | |
CN116138212B (zh) | 一种制备细胞因子释放综合征小鼠模型的方法及应用 | |
US8101195B2 (en) | Artificial lymph node for treating cancer | |
CN111499766B (zh) | 针对慢性淋巴细胞白血病的免疫效应细胞、其制备方法和应用 | |
WO1995009235A9 (en) | Immunodeficient mouse models of pathogenesis of human disease and efficacy and toxicity of disease treatments | |
WO1995009235A1 (en) | Immunodeficient mouse models of pathogenesis of human disease and efficacy and toxicity of disease treatments | |
Zhao et al. | The Role of Mesenchymal Stem Cells in Cancer Immunotherapy | |
KR101470908B1 (ko) | 항암 면역 치료제 | |
Chan | Investigating Components of the Innate Immune System Following Administration of a Dendritic Cell-Based Vaccine | |
CN114836428B (zh) | 一种tigit基因干扰的嵌合抗原受体nk细胞及其应用 | |
Lattime et al. | In Situ Gene Insertion for Immunotherapy Using Vaccinia Virus Vectors | |
Schirrmaker | Improvements of survival in nine phase II clinical studies with different types of cancer upon anti-tumor vaccination with an autologous tumor cell vaccine modified by virus infection to introduce danger signals | |
CN115651913A (zh) | 具有抗肿瘤活性的细胞及抑制肿瘤生长的方法 | |
Osada et al. | Natural killer cell activation and dendritic cell-based vaccines | |
CN115975050A (zh) | 嵌合的人源t细胞受体、核酸、载体、细胞和药物组合物 |
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 |