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在这里,我们提出了一种用于产生和验证缺氧敏感的嵌合抗原受体 (CAR)-T 细胞的方案。该方案介绍了基于慢病毒的缺氧敏感 CAR-T 细胞的产生及其表征,包括缺氧依赖性 CAR 表达和选择性细胞毒性的验证。
广泛的研究证明了嵌合抗原受体 T (CAR-T) 细胞疗法在治疗血液系统恶性肿瘤方面的前景。然而,治疗实体瘤仍然具有挑战性,当 CAR-T 细胞攻击表达靶抗原的正常细胞时出现的安全问题就是例证。研究人员已经探索了提高 CAR-T 细胞疗法肿瘤选择性的各种方法。沿着这条路线的一个代表性策略是构建缺氧敏感的 CAR-T 细胞,其设计方法是将氧依赖性降解结构域融合到 CAR 部分,并且仅在缺氧环境 - 肿瘤微环境 (TME) 中实现高 CAR 表达。本文提出了一种产生此类 CAR-T 细胞及其功能表征的方案,包括分析响应移动培养箱建立的不同氧水平的 CAR 表达和杀伤能力变化的方法。预计构建的 CAR-T 细胞将以氧敏感方式展示 CAR 表达和细胞毒性,从而支持它们区分缺氧 TME 和常氧正常组织以进行选择性激活的能力。
嵌合抗原受体 T 细胞 (CAR-T) 疗法代表了癌症治疗的重大突破。自 2017 年美国食品药品监督管理局 (FDA) 批准首个用于治疗晚期/耐药性淋巴瘤和急性淋巴细胞白血病的 CAR-T 疗法以来,1,2,3, 10 种靶向 CD19 或 B 细胞成熟抗原 (BCMA) 的 CAR-T 疗法已在全球范围内获得批准4。然而,尽管进行了广泛的研究,但复制 CAR-T 疗法在治疗血液系统恶性肿瘤方面的显着疗效对于其在实体瘤中的应用仍然具有挑战性 5,6,7,8。
免疫抑制性肿瘤微环境 (TME) 是 CAR-T 在实体瘤环境中疗效不佳的主要因素。由于营养不足、缺氧、酸性 pH 值和代谢废物的积累,TME 会阻碍 CAR-T 细胞的活性和存活
在这项研究中,将 HER2-BBz-ODD,一种靶向 HER2 (基因 ID: 2064) 的缺氧敏感 CAR 与其常规对应物 HER2-BBz 进行了比较。两种 CAR 的示意图如图 1A 所示,其中显示 HER2-BBz-ODD 是通过将 ODD 序列添加到 CD3ξ 的 C 端而从 HER2-BBz 衍生而来的。表达两种 CAR 的慢病毒载体的构建以及通过 293T 细胞转染产生相应的慢病毒已在前面描述过 31。
将 HIF-1α 的 ODD 结构域与 CAR 部分融合是产生缺氧敏感 CAR 的主要策略。本研究中分析的缺氧敏感的 HER2 靶向 CAR 命名为 HER2-BBz-ODD,是通过将 ODD 序列整合到其常规 HER2-BBz 中来使用这种策略构建的(图 1A)。在这项研究中,我们使用慢病毒转导表达 HER2-BBz-ODD CAR 或 HER2-BBz CAR,随后检测了它们在两种细胞类型中的氧敏感性:人 PBMC 和 Jurkat T 细胞。
安全问题是任何 CAR-T 细胞疗法进入临床使用都必须解决的重要问题。利用肿瘤细胞或 TME 的独特特性已成为专注于开发选择性靶向肿瘤组织的 CAR-T 细胞的主要研究方向。在这个方向上,设计缺氧敏感的 CAR-T 是一种有吸引力的策略,正在探索几种方法,包括本研究中提出的一种方法——将 CAR 部分与天然存在的缺氧感应 ODD 蛋白结构域融合。另一种方法涉及用缺氧反应元件.......
这项工作得到了中国国家重点研发计划 (2016YFC1303402)、国家重点传染病特大专项 (2017ZX10202102, 2017ZX10304402-002-007) 和上海市卫生健康委员会总体计划 (201740194) 的支持。
....Name | Company | Catalog Number | Comments |
1.5 mL Centrifuge tube | QSP | 509-GRD-Q | Supernatants and cells cellection Protocol Step 2,3,4 |
10% ExpressCast PAGE | NCM biotech | P2012 | Immunoblotting Protocol Step 3 |
10x PBS | NCM biotech | 20220812 | Cell culture Protocol Step 4 |
10 mL pipette | Yueyibio | YB-25H | Pipetting Protocol Step 1 |
10xTRIS-Glycine-SDS electrophoresis buffer | Epizyme | 3673020 | Immunoblotting Protocol Step 3 |
15 mL Centrifuge tube | Thermo Scientific | 339650 | Supernatants and cells cellection Protocol Step 1 |
25 cm2 EasYFlask | Thermo Scientific | 156367 | Cell culture Protocol Step 3,4 |
4x Protein SDS PAGE Loading Buffer | Takara | 9173 | Immunoblotting Protocol Step 3 |
6-well flat-bottom tissue culture plates | Thermo Scientific | 140675 | T Cells culture Protocol Step 1 |
96-well black flat-bottom tissue culture plates | Greiner | 655090 | Cytotoxicity assay Protocol Step 4 |
96-well ELISA plates | Corning | 3590 | ELISA Protocol Step 5 |
96-well plate shaker | QILINBEIER | MH-2 | Shake Protocol Step 4 |
96-well U-bottom tissue culture plates | Thermo Scientific | 268200 | Supernatants cellection Protocol Step 4,5 |
anti-FLAG antibody | Sigma | F1804-50UG | Immunoblotting Protocol Step 3 |
Carbinol | Sinopharm | 10010061 | Immunoblotting Protocol Step 3 |
Carbon dioxide incubator | Thermo Scientific | 360 | Cell culture Protocol Step 1,2,3,4 |
Cell counting plate | Hausser scientific | 1492 | Cell counting Protocol Step 1,3,4 |
CELLection Pan Mouse IgG Kit | Thermo Scientific | 11531D | Mouse IgG magnetic beads Protocol Step 1 |
Centrifuge | Thermo Scientific | 75002432 | Cell culture Protocol Step 1,3,4 |
Chemiluminescence gel imaging system | BIO-RAD | 12003154 | Immunoblotting Protocol Step 3 |
Cobalt chloride solution (0.5 M) | bioleaper | BR4000203 | Hypoxic condition Protocol Step 2,3,4 |
DMEM | Corning | 10-103-CV | Cell culture Protocol Step 4 |
Electronic balance | Sartorius | PRACTUM612-1CN | weigh Protocol Step 5 |
FBS | BI | 04-001-1ACS | Cell culture Protocol Step 3,4 |
GAPDH Mouse mAb | ABclonal | AC002 | Immunoblotting Protocol Step 3 |
Gel electrophoresis apparatus | BIO-RAD | 1645070 | Immunoblotting Protocol Step 3 |
GloMax Microplate Readers | Promega | GM3000 | luciferase activity measurement Protocol Step 4 |
Goat anti-Mouse IgG (H+L) | Yeasen | P1126151 | Immunoblotting Protocol Step 3 |
High speed microfreezing centrifuge | eppendorf | 5810 R | Cell culture Protocol Step 1 |
Human IFN-γ ELISA Set | BD | 555142 | ELISA Protocol Step 5 Items: Recombinant Human IFN-γ Lyophilized Standard, Detection Antibody Biotin Anti-Human IFN-γ , Capture Antibody Purified Anti-Human IFN-γ, Enzyme Reagent Streptavidin-horseradish peroxidase conjugate (SAv-HRP) |
Human IL-2 ELISA Set | BD | 555190 | ELISA Protocol Step 5 Items: Recombinant Human IL-2 Lyophilized Standard, Detection Antibody Biotin Anti-Human IL-2 , Capture Antibody Purified Anti-Human IL-2, Enzyme Reagent Streptavidin-horseradish peroxidase conjugate (SAv-HRP) |
IL-15 | R&D systems | P40933 | T Cells culture Protocol Step 1 |
IL-21 | Novoprotein | GMP-CC45 | T Cells culture Protocol Step 1 |
IL-7 | R&D systems | P13232 | T Cells culture Protocol Step 1 |
Inverted microscope | Olympus | CKX41 | Cell culture Protocol Step 1,3,4 |
Jurkat | ATCC | TIB-152 | CAR-Jurkat construction Protocol Step 3 |
LSRFortessa | BD | LSRFortessa | Flow cytometry Protocol Step 2 |
Luciferase Assay System | Promega | E1501 | luciferase reporter assay Protocol Step 4 Items: Passive lysis buffer, firefly luciferase substrate |
Microplate reader | BioTek | HTX | ELISA Protocol Step 5 |
mobile CO2/O2/N2 Incubator Chamber | China Innovation Instrument Co., Ltd. | Smartor118 | Hypoxic condition Protocol Step 2, 3, 4 |
Mouse Anti-Hexa Histidine tag | Sigma | SAB2702218 | Immunoblotting Protocol Step 3 |
NcmBlot Rapid Transfer Buffer | NCM biotech | WB4600 | Immunoblotting |
NcmECL Ultra | NCM biotech | P10300 | Immunoblotting Protocol Step 3 Items: NcmECL Ultra Luminol/Enhancer Reagent (A) ,NcmECL Ultra Stabilized Peroxide Reagent (B) |
NovoNectin -coated 48-well flat plates | Novoprotein | GMP-CH38 | CAR-T cells construction Protocol Step 1 |
OPD (o-phenylenediamine dihydrochloride) tablet set | Sigma | P9187 | Substrate Reagent Protocol Step 5 Items: OPD tablet (silver foil),urea hydrogen peroxide tablet (gold foil) |
PE-conjugated anti-DYKDDDDK | Biolegend | 637310 | Flow cytometry Protocol Step 2 |
Protamine sulfate | Sigma | P3369-1OG | Lentivirus infection Protocol Step 1 |
Protein Marker 10 Kda-250 KDa | Epizyme | WJ102 | Immunoblotting Protocol Step 3 |
Purifed NA/LE Mouse Anti-Human CD3 | BD | 566685 | T Cells culture Protocol Step 1 |
Purified NA/LE Mouse Anti-Human CD28 | BD | 555725 | T Cells culture Protocol Step 1 |
PVDF membrane | Millipore | 168627 | Immunoblotting Protocol Step 3 |
RPMI 1640 | Corning | 10-040-CVRC | Cell culture Protocol Step 3 |
Skim milk powder | Yeasen | S9129060 | Immunoblotting Protocol Step 3 |
SKOV3-Luc | ATCC | HTB-77 | Cytotoxicity assay Protocol Step 4 |
Trypsin-EDTA | NCM biotech | C125C1 | Cell culture Protocol Step 4 |
Tween 20 | Sinopharm | 30189328 | Immunoblotting Protocol Step 3 |
Water bath | keelrein | NB014467 | Heating Protocol Step 1 |
X-VIVO 15 | LONZA | 04-418Q | Serum-free lymphocyte culture medium Protocol Step 1 |
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