巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用
Summary
The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.
Abstract
病原体特异性T细胞的过继转移可用于预防和治疗机会性感染如巨细胞病毒（CMV）感染异基因造血干细胞移植后发生。从供者，包括第三方捐助者病毒特异性T细胞，可以传播体外 ，符合现行良好生产规范（cGMP），采用多轮抗原驱动的刺激有选择性地繁殖所需的T细胞。抗原特异性T细胞的鉴定和分离，也可基于T细胞的细胞因子的捕获系统，该系统已经被激活以分泌γ-干扰素（IFN-γ）进行。然而，广泛的人类应用的细胞因子捕获系统（CCS），以帮助恢复免疫的已受到了限制生产过程是耗时，并且需要熟练的操作人员。第二代细胞富集设备，如CliniMACS神童发展到现在使研究者以产生使用自动化，更少劳动密集的系统的病毒特异性T细胞。该装置中分离磁使用磁激活细胞分选技术，产生临床级产品未标记细胞标记的细胞，被设计为封闭系统，并且可以进行访问和操作上的台式。我们证明这个新的自动化细胞富集装置的操作来制造从CMV启动血清阳性供体获得一个稳态单采产物获得的CMV pp65抗原特异性T细胞。这些分离的T细胞可以被直接注入机构和联邦监管监督下的患者。所有的生物处理步骤包括去除红血细胞，T细胞，抗原特异性T细胞，纯化和洗涤分离的刺激完全自动化。设备，如这提高的可能性，T细胞对人类应用能奉献良好生产规范GMP外（制造）设施，而是在血库设施的工作人员可以监控自动化协议，以生产多种产品的生产。
Introduction
造血干细胞移植（HSCT）1可以与过继性T细胞治疗相结合，以提高移植物抗肿瘤效果，并提供免疫机会感染2。产生抗原特异性供体来源的T细胞输注历来所需的技能型人才和使用专门的设施，是按照GMP标准。这种T细胞的递送导致的机会性感染3的分辨率以及治疗潜在恶性肿瘤4。最近，研究人员已经证明，只有几千病毒特异性T细胞的过继转移（〜1×10 4 – 2.5×10 5细胞/千克接受者体重）可以成功地治疗同种异体HSCT后5-9机会性巨细胞病毒感染。数量有限的GMP设施，相关精湛的制造要求和电池生产相关的成本高了，但是，restricted患者获得希望的T细胞疗法10。的一种方法分离抗原特异性T细胞是基于使用一个双特异性试剂来识别CD45和IFN-γ在CCS。如图所示，这种方法可以用于产生临床级的CMV特异性T细胞采用一种自动化细胞富集的CCS装置（ 图1B）。
通过将重叠肽从CMV病毒pp65抗原用感染CMV血清阳性供体白细胞分离总有核细胞（TNC）产生CMV特异性T细胞。这些肽在人类白细胞抗原（HLA）的环境中显示的，激活该TNC内CMV启动病毒pp65特异性T细胞分泌的IFN-γ。这些T细胞可以被“捕获”和磁性分离。第一代细胞富集装置（图1A）的操作所需的在细胞培养人员熟练GMP条件下，和协调工作人员承担的多个STEPS必须产生一个“俘获”的产品。
该过程通常需要10至12小时的连续运行，因此，人员可能需要工作在两班在GMP的设施。这些约束现在由第二代装置（ 图 1B所示）的执行避免。该装置进行磁性富集，与第一代设备，但在自动化的漂白的方法的CCS的其他方面。这显著降低作为大多数步骤可以完成无人参与由工作人员在GMP的球队的负担。另外，由于装置作为一个封闭系统中，抗原特异性T细胞可以被捕获并在除参与白细胞分离的分离和制备的材料启动仪器之前的步骤台式处理。完整的仪器和第二代细胞富集设备的功能性的细节已经酒馆lished 11。
这里，我们描述的步骤，从使用自动细胞富集的CCS系统稳态单采产品丰富的CMV pp65抗原特异性T细胞。一旦分离，这些CMV特异性T细胞可以被立即注入到病人体内。
Protocol
Representative Results
Discussion
继T细胞疗法已成为一个可行的办法来治疗B细胞恶性肿瘤4。其治疗潜力是依赖于注入缺乏复制衰老2靶抗原特异性T细胞的期望数量。这可以通过用当前的良好生产规范整理抗原特异性T细胞的纯人口从扩增的T细胞的合规性来实现。两个分拣程序被广泛应用，即，荧光激活细胞分选（FACS）和磁性活化细胞分选（MACS）来产生的CMV抗原特异性T细胞因为我们最近5审查。使用一个?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Miltenyi Biotec, Germany for providing reagents and CliniMACS Prodigy equipment for evaluation studies. We thank George T. McNamara (Pediatric department, MD Anderson Cancer Center) for proof reading the manuscript. Grant support: Cancer Center Core Grant (CA16672); RO1 (CA124782, CA120956, CA141303; CA141303); R33 (CA116127); P01 (CA148600); Burroughs Wellcome Fund; Cancer Prevention and Research Institute of Texas; CLL Global Research Foundation; Estate of Noelan L. Bibler; Gillson Longenbaugh Foundation; Harry T. Mangurian, Jr., Fund for Leukemia Immunotherapy; Institute of Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lymphoma Research Foundation; MDACC’s Sister Institution Network Fund; Miller Foundation; Mr. Herb Simons; Mr. and Mrs. Joe H. Scales; Mr. Thomas Scott; National Foundation for Cancer Research; Pediatric Cancer Research Foundation; William Lawrence and Blanche Hughes Children’s Foundation.
Materials
| CliniMACS PBS/EDTA Buffer 3 L bag | Miltenyi Biotec GmbH | 700-29 | |
| CliniMACS Prodigy Tubing Set TS 500 | Miltenyi Biotec GmbH | 130-097-182 | |
| 5 L waste bag | Miltenyi Biotec GmbH | 110-004-067 | |
| CliniMACS Cytokine Capture System (IFN-gamma) | Miltenyi Biotec GmbH | 279-01 | |
| Albumin (Human) 25% | Grifols | 58516-5216-2 | |
| Luer/Spike Interconnector | Miltenyi Biotec GmbH | 130-018-701 | |
| 0.9 % NaCl Solution (1 L) | Miltenyi Biotec GmbH | ||
| MACS GMP PepTivator HCMV pp65 | Miltenyi Biotec GmbH | 170-076-109 | |
| Water for injections | Hospira, inc, Lake Forest, IL | NDC-0409-4887-10 | |
| MILLEX GV Filter Unit 0.22 μm | Millipore | SLGV033RB | |
| TexMACS GMP Medium 2 L bag | Miltenyi Biotec GmbH | 170-076-306 | |
| Transfer Bag, 150 mL (for cellular starting material) | Miltenyi Biotec GmbH | 130-018-301 | |
| CryoMACS Freezing Bag 50 | Miltenyi Biotec GmbH | 200-074-400 | |
| 60 mL Syringes, sterile | BD, Laagstraat, Temse, Belgium | 309653 | |
| CMV sero positive apheresis product | Key Biologics, LLC, Memphis | ||
| Flow Cytometry Materials | Manufacturer | Catalog number | |
| AB Serum, GemCell | Gemini Bio-Products, West Sacramento, USA | 100-512 | |
| CD3-FITC | Miltenyi Biotec GmbH | 130-080-401 | |
| CD4-APC | Miltenyi Biotec GmbH | 130-098-033 | |
| CD8-APC-Vio770 | Miltenyi Biotec GmbH | 130-098-065 | |
| CD14-PerCP | Miltenyi Biotec GmbH | 130-098-072 | |
| CD20-PerCP | Miltenyi Biotec GmbH | 130-098-077 | |
| CD45-VioBlue | Miltenyi Biotec GmbH | 130-098-136 | |
| aIFN-γ-PE, human | Miltenyi Biotec GmbH | 130-097-940 | |
| CD3-PE | Miltenyi Biotec GmbH | 130-091-374 | |
| Propidium Iodide Solution (100 µg/mL) | Miltenyi Biotec GmbH | 130-093-233 | |
| Equipment | Manufacturer | Catalog Number | |
| CliniMACS Prodigy Device | Miltenyi Biotec GmbH | 200-075-301 | |
| Software V1.0.0.RC | |||
| MACSQuant Analyzer 10 | Miltenyi Biotec GmbH | 130-096-343 | |
| Software 2.4 | |||
| Centrifuge 5415R | Eppendorf AG | 22331 | |
| Cellometer K2 | Nexelom Bioscience, Lawrence, MA | LB-001-0016 | |
| Sterile tubing welder SCDIIB | Terumo Medical Corp., Elkton, MA | 7811 |
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