肿瘤细胞治疗研究
英文原题：Enrichment of CD4(+) and CD8(+) T lymphocytes with a column-free flow-based device for clinical cell manufacturing.
我们的数据显示，CD4/CD8富集迅速且稳定，富集时间缩短至1小时或更短。
近年来，过继性T细胞免疫疗法已被开发用于治疗多种血液系统恶性肿瘤，包括复发或难治性非霍奇金淋巴瘤、B细胞白血病和多发性骨髓瘤。大多数商业批准的过继性T细胞疗法由基于嵌合抗原受体（CAR）的T细胞组成，这些T细胞是患者自身的T细胞，经过工程改造以识别特定表面抗原，如CD19或CD20。未筛选的外周血单个核细胞（PBMCs）最近已被用于几种生产方案，但绝大多数方案仍使用CD4/CD8筛选的T细胞。这些CAR-T产品生产的第一步涉及同时筛选/纯化CD4+和CD8+（或CD4/CD8阳性）T细胞。临床生产中选择CD4/CD8亚群的典型方法涉及免疫磁珠标记，然后使用基于静态柱的方法选择阳性标记的细胞，这种方法容易发生细胞堵塞事件，在封闭系统中通常需要约2至3小时。在此，我们使用了一种新的无柱、基于流式、完全封闭的系统，适用于临床细胞生产，能够以快速方式分离高纯度的CD4/CD8细胞，可适应不同容量而不影响细胞回收率。这种新方法允许显著更快的细胞筛选，保持用于下游CAR-T细胞生产的细胞质量。我们报告了使用新的MARS Bar富集平台的成功验证运行结果，该平台使用人单采术来源的白细胞在筛选缓冲液中或直接在T细胞培养基中分离CD4/CD8，用于后续CAR-T细胞生产。我们的数据显示，CD4/CD8富集快速且稳健，富集时间缩短至1小时或更短。基于CD3+表达，新鲜和冻融T细胞的总体纯度分别为95.51±1.23%和93.13±0.30%。新鲜和冻融细胞的回收率分别为64.68±14.05%和57.06±6.28%。随后，我们进一步测试了使用LOVO Automated Cell Processing System进行细胞洗涤/体积缩减后的MARS Bar富集平台，从而使CD3+纯度一致性更高，细胞回收率提高至68.50±3.54%。富集后的细胞以高活力为特征，即与LOVO设备一起使用时，新鲜leukopaks的活力为90.5±0.05%。总之，使用MARS Bar平台的新方法使得人们能够通过在临床生产环境中使用独立仪器以及cGMP级试剂和缓冲液，来定制和标准化选择过程。
In recent years, adoptive T cell-based immunotherapies have been developed to treat a wide range of hematologic malignancies, including relapsed or refractory non-Hodgkin lymphoma, B-cell leukemia, and multiple myeloma. Most of the commercially approved adoptive T cell therapies are composed of chimeric antigen receptor (CAR)-based T cells, which are a patient's own T cells engineered for recognition of a specific surface antigen, such as CD19 or CD20. Unselected peripheral blood mononuclear cells (PBMCs) have recently been used in several manufacturing protocols, but the vast majority of protocols still use CD4/CD8-selected T cells. The first step in manufacture of these CAR-T products involves simultaneous selection/purification of CD4 + and CD8 + (or CD4/CD8 positive) T cells. The typical approach for selection of CD4/CD8 subsets for clinical manufacturing involves immunomagnetic labeling followed by selection of positively labeled cells using static column-based approaches that are prone to cell clogging events and typically take approximately 2 to 3 hours in a closed system. Here, we used a new column-free, flow-based, fully closed system suitable for clinical cell manufacturing for isolation of CD4/CD8 cells with high purity in a rapid fashion that could accommodate varying capacities without compromising cell recovery. This new approach allows markedly faster cell selection, preserving the quality of the cells that are used for downstream CAR-T cell manufacture. We report the results of our successful validation runs using the new MARS Bar enrichment platform using human apheresis-derived leukocytes for CD4/CD8 isolation in a selection buffer or directly in T cell culture media for subsequent CAR-T cell production. Our data show a rapid and robust CD4/CD8 enrichment with an enrichment time shortened to 1 hour or less. Overall purity (based on CD3 + expression) of the cells was 95.51 1.23% and 93.13 0.30% for fresh and thawed T cells, respectively. Cell recoveries were 64.68 14.05% and 57.06 6.28% for fresh and thawed cells, respectively. We then further tested the MARS Bar enrichment platform after cell wash/volume reduction using the LOVO Automated Cell Processing System, leading to a higher consistency in CD3 + purity and increased cell recovery of 68.50 3.54%. Enriched cells were characterized by high viability, ie. 90.5 0.05% for fresh leukopaks when used together with the LOVO device. Altogether, the new approach using the MARS Bar platform allows one to customize and standardize the selection process by using a stand-alone instrument in a clinical manufacturing setting together with cGMP grade reagents and buffers.
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