Cooper L J N, Ausubel L, Gutierrez M, Stephan S, Shakeley R, Olivares S, Serrano L M, Burton L, Jensen M C V, Forman S J, DiGiusto D L
Division of Pediatric Hematology-Oncology, City of Hope National Medical Center, California 91010, USA.
Cytotherapy. 2006;8(2):105-17. doi: 10.1080/14653240600620176.
The production of therapeutic T-cell populations for adoptive immunotherapy of cancer requires extensive ex vivo cell processing, including the isolation or creation of Ag-specific T cells and their subsequent propagation to clinically relevant numbers. These procedures must be performed according to the principles of current good manufacturing practices (cGMP) for phase I clinical trials to ensure the identity, purity potency and safety of the cellular product. In this report we describe our approach to manufacturing and characterizing bulk populations of gene-modified autologous T cells for use in treating follicular lymphoma.
PBMC from healthy donors, obtained after informed consent, were stimulated in vitro with Ab to CD3epsilon (OKT3) and recombinant human IL-2 and then electroporated with plasmid DNA containing a human CD19-specific chimeric Ag receptor (CAR) gene and HSV-1 thymidine kinase (TK) gene. Stably transfected cells were selected in cytocidal concentrations of hygromycin B over multiple 14-day stimulation culture cycles and then cryopreserved. Vials of cryopreserved/selected T cells were used to initiate T-cell expansion cultures to produce cell products for clinical infusion. These cultures were characterized for phenotype, function and suitability for use in adoptive immunotherapy studies.
Our results demonstrate that bulk populations of gene-modified T cells derived from peripheral blood of healthy donors express CD19+ chimeric Ag receptor at low levels and can specifically lyse CD19+ target cells in vitro. These cells display a differentiated T-effector phenotype, are sensitive to ganciclovir-mediated killing and display a non-transformed phenotype. TCR Vbeta usage indicated that all populations tested were polyclonal. Ex vivo cell expansion from cryopreserved cell banks is sufficient to produce doses of between 5 x 10(9) and 1 x 10(10) cells/run. One of three transductions resulted in a population of cells that was not suitable for infusion but was identified during release testing. No populations displayed any evidence of bacterial, fungal or mycoplasma contamination.
We have established a manufacturing strategy that is being used to produce T cells for a phase I clinical trial for follicular lymphoma. Genetically modified T cells have been characterized by cell-surface marker phenotype, functional activity against CD19+ targets and requisite safety testing. These pre-clinical data confirm the feasibility of this approach to manufacturing T-cell products.
用于癌症过继性免疫治疗的治疗性T细胞群体的产生需要广泛的体外细胞处理，包括分离或产生抗原特异性T细胞，并随后将其扩增至临床相关数量。这些程序必须按照I期临床试验的现行药品生产质量管理规范（cGMP）原则进行，以确保细胞产品的同一性、纯度、效力和安全性。在本报告中，我们描述了用于治疗滤泡性淋巴瘤的基因修饰自体T细胞大量群体的制造和表征方法。
在获得知情同意后，从健康供体获取外周血单核细胞（PBMC），用抗CD3ε抗体（OKT3）和重组人白细胞介素-2在体外进行刺激，然后用含有人类CD19特异性嵌合抗原受体（CAR）基因和单纯疱疹病毒1型胸苷激酶（TK）基因的质粒DNA进行电穿孔。在多个14天的刺激培养周期中，在杀细胞浓度的潮霉素B中选择稳定转染的细胞，然后进行冷冻保存。使用冷冻保存/选择的T细胞小瓶启动T细胞扩增培养，以生产用于临床输注的细胞产品。对这些培养物进行表型、功能以及用于过继性免疫治疗研究的适用性方面的表征。
我们的结果表明，源自健康供体外周血的基因修饰T细胞大量群体低水平表达CD19 +嵌合抗原受体，并且在体外能够特异性裂解CD19 +靶细胞。这些细胞表现出分化的T效应细胞表型，对更昔洛韦介导的杀伤敏感，并且表现出未转化的表型。TCR Vβ使用情况表明，所有测试群体均为多克隆群体。从冷冻保存的细胞库进行体外细胞扩增足以产生每次运行5×10^9至1×10^10个细胞的剂量。三次转导中有一次产生了不适合输注的细胞群体，但在放行测试期间被识别出来。没有任何群体显示出细菌、真菌或支原体污染的迹象。
我们已经建立了一种制造策略，该策略正在用于为滤泡性淋巴瘤的I期临床试验生产T细胞。基因修饰的T细胞已经通过细胞表面标志物表型、针对CD19 +靶标的功能活性以及必要的安全性测试进行了表征。这些临床前数据证实了这种制造T细胞产品方法的可行性。
