制造用于采用免疫疗法的奇美抗原受体（CAR）T细胞
Summary
我们描述了一种可靠地生成嵌合抗原受体（CAR）T细胞的方法，并测试其体外和体内的分化和功能。
Abstract
采用免疫疗法对治疗癌症和传染病有希望。我们描述了一种用嵌合抗原受体（CAR）转导原发人类T细胞并扩展其后代前体的简单方法。我们包括检测，以测量CAR表达以及分化，增殖能力和细胞解细胞活性。我们描述检测，以测量在CAR T细胞中产生效应细胞因子和炎症细胞因子分泌物。该方法为采用免疫治疗的临床前模型培养CAR T细胞提供了可靠、全面的方法。
Introduction
奇美抗原受体（CARs）提供了一种有希望的方法，可以针对不同的肿瘤抗原重定向T细胞。CARs 是结合抗原靶点的合成受体。虽然其精确组成是可变的，但 CA 通常包含 3 个不同的域。细胞外域直接结合到靶抗原，通常由通过细胞外铰链与CAR相连的单链抗体片段组成。第二个域，通常来自T细胞受体（TCR）复合物的CD3*链，促进CAR参与后的T细胞激活。包括第三个成本领域，以增强T细胞功能，移植，代谢和持久性。CAR T细胞治疗在各种造血恶性肿瘤中的成功，包括B细胞急性淋巴细胞白血病（ALL）、慢性淋巴细胞白血病（CLL）和多发性骨髓瘤，凸显了这种方法1、2、3、4、5、6的治疗前景。最近美国食品和药物管理局 （FDA） 批准两种 CD19 特异性 CAR T 细胞疗法，针对儿科和年轻成人的 tisagenlecleucel 和用于扩散大 B 细胞淋巴瘤的 axicabagene 胆珠，强化了 CAR T 细胞治疗的转化价值。
基于CAR T的方法包括从外周血分离T细胞、活化、基因改造和外体扩张。分化是调节CAR T细胞疗效的重要参数。因此，在外体培养过程中限制T细胞分化可增强注入产物的移植、扩张和坚持能力，在采用转移2、7、8、9之后提供长期免疫监测。T细胞由几个不同的子集组成，包括：幼稚的T细胞（Tn）、中央记忆（Tcm）、效应器记忆（Tem）、效应器分化（T）和干细胞记忆（Tscm）。效应者分化T细胞具有强效细胞解细胞能力;然而，他们是短暂的和移植差10，11，12。相反，具有较少分化表型的T细胞，包括幼稚的T细胞和Tcm在采用细胞转移13、14、15、16、17、18之后表现出优异的增生和增殖能力。预制造产品中收集的T细胞的组成可能因患者而异，并与CAR T细胞的治疗潜力相关。在起始阿普西斯产品中，具有天真型免疫表型的T细胞比例与移植和临床反应高度相关19。
培养持续时间是影响CAR T细胞分化的重要参数，为采用转移做准备。我们最近开发了一种方法，使用缩写培养范式20生成高质量的CAR T细胞。利用我们的方法，我们表明，有限的培养导致CAR T细胞具有优越的效应功能和持久性后，在白血病的异种移植模型的收养转移。在这里，我们介绍可靠生成CART19细胞的方法（用于表达CD33和4-1BB信令域的抗CD19 scFv的自体T细胞），并包括分析的详细说明，在采用转移之前提供对CAR T生物活性和有效性的洞察。
Protocol
所有动物研究都得到宾夕法尼亚大学动物护理和使用委员会的批准。 1. T细胞激活、转导和扩张 与抗CD3/CD28磁珠（例如，dynabet）混合，在6孔细胞培养皿中，以每T细胞3个珠的比例激活新鲜或冷冻保存的初级人类T细胞。X-VIVO 15培养基中的培养T细胞辅以5%正常人AB血清、2mM L-谷氨酰胺、20 mM HEPES和IL2（100单位/mL）。在扩张过程中将T细胞的浓度保持在106T细胞/mL。培?…
Representative Results
使用上述方法，我们刺激和扩展T细胞3或9天（图1A，B）。我们还通过测量细胞表面表达的独特糖蛋白的丰度，分析了它们的分化特征，如图1C中概述的浇注策略所示。在外体培养过程中，我们显示出一个逐渐向效应器分化的转变（图1D）。我们评估了CAR T细胞对抗原的反应作用和增殖能力。我?…
Discussion
在这里，我们描述了测量CAR T细胞的功能和功效的方法，这些细胞在整个外体培养过程中以不同的间隔收获。我们的方法提供全面的检测分析，旨在评估增殖能力以及体外效应器功能。我们描述了如何通过CAR刺激测量CAR T细胞活性，并使用在对数扩张阶段的第3天和第9天收获的CAR T细胞进行详细白血病异种移植模型。
在比较在外体扩张期间在不同时间点采集的CAR T细胞的功效存?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了诺华制药公司通过与宾夕法尼亚大学（迈克尔·米隆）的研究联盟以及圣巴尔德里克基金会学者奖（萨巴·加塞米）提供的资金的支持。
Materials
| Anti CD3/CD28 dynabeads | Thermo Fisher | 40203D | |
| APC Mouse Anti-Human CD8 | BD Biosciences | 555369 | RRID:AB_398595 |
| APC-H7 Mouse anti-Human CD8 Antibody | BD Biosciences | 560179 | RRID:AB_1645481 |
| BD FACS Lysing Solution 10X Concentrate | BD Biosciences | 349202 | |
| BD Trucount Absolute Counting Tubes | BD Biosciences | 340334 | |
| Brilliant Violet 510 anti-human CD4 Antibody | BioLegend | 317444 | RRID:AB_2561866 |
| Brilliant Violet 605 anti-human CD3 Antibody | BioLegend | 317322 | RRID:AB_2561911 |
| CellTrace CFSE Cell Proliferation Kit | Life Technolohgies | C34554 | |
| CountBright Absolute Counting Beads, | Invitrogen | C36950 | |
| FITC anti-Human CD197 (CCR7) Antibody | BD Pharmingen | 561271 | RRID:AB_10561679 |
| FITC Mouse Anti-Human CD4 | BD Biosciences | 555346 | RRID:AB_395751 |
| HEPES | Gibco | 15630-080 | |
| Human AB serum | Valley Biomedical | HP1022 | |
| Human IL-2 IS, premium grade | Miltenyi | 130-097-744 | |
| L-glutamine | Gibco | 28030-081 | |
| Liquid scintillation counter, MicroBeta trilux | Perkin Elmer | ||
| LIVE/DEAD Fixable Violet | Molecular Probes | L34964 | |
| Multisizer Coulter Counter | Beckman Coulter | ||
| Na251CrO4 | Perkin Elmer | NEZ030S001MC | |
| Pacific Blue anti-human CD14 Antibody | BioLegend | 325616 | RRID:AB_830689 |
| Pacific Blue anti-human CD19 Antibody | BioLegend | 302223 | |
| PE anti-human CD45RO Antibody | BD Biosciences | 555493 | RRID:AB_395884 |
| PE/Cy5 anti-human CD95 (Fas) Antibody | BioLegend | 305610 | RRID:AB_493652 |
| PE/Cy7 anti-human CD27 Antibody | Beckman Coulter | A54823 | |
| Phenol red-free medium | Gibco | 10373-017 | |
| UltraPure SDS Solution, 10% | Invitrogen | 15553027 | |
| Via-Probe | BD Biosciences | 555815 | |
| X-VIVO 15 | Gibco | 04-418Q | |
| XenoLight D-Luciferin – K+ Salt | Perkin Elmer | 122799 |
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Cite This Article
Ghassemi, S., Milone, M. C. Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy. J. Vis. Exp. (154), e59949, doi:10.3791/59949 (2019).