肿瘤细胞治疗研究
英文原题：Efficient manufacturing of CAR-T cells from whole blood: a scalable approach to reduce costs and enhance accessibility in cancer therapy.
可从全血中成功制备具有治疗相关剂量的 CD19/CD22 CAR-T 细胞。
背景：CAR-T 细胞显著推动了白血病和淋巴瘤等癌症的治疗进展。传统上通过白细胞单采从患者采集T细胞，该过程费用高、可能具有侵入性，并需要专用设备和训练有素的人员。全血采集在技术上简单得多，但全血起始材料未广泛用于临床CAR-T制备，部分原因是缺乏适用于良好生产规范（GMP）环境的生产流程。无需白细胞单采而直接从全血采集细胞起始材料，可简化制备流程、降低成本，进而提升CAR-T治疗可及性。 方法：研究采集8名健康供者及1名儿童B细胞急性淋巴细胞白血病（B-ALL）患者的全血样本。使用Sepax C-Pro（Cytiva）仪器，通过密度梯度分离提取单个核细胞（MNC）。随后采用符合GMP的7天流程制备CAR-T细胞：使用抗CD3和IL-2活化T细胞，以编码CD19/CD22双特异性CAR的GMP级慢病毒载体转导，并在透气细胞培养袋中扩增。通过流式细胞术、细胞因子释放及细胞毒性实验评估所得CAR-T细胞的表型和功能。 结果：健康供者全血平均体积为77.7 mL（范围29–96 mL），处理后平均分离获得4,220万个CD3 T细胞（范围730万–6,300万）。解冻后以100万–1,000万个起始CD3+ T细胞开始CAR-T培养，第7天T细胞中位数达到1.05亿个（范围6,100万–1.88亿）。平均转导效率为66±11%，平均获得7,740万个转导CAR-T细胞（范围3,080万–1.435亿）。使用一名近期接受化疗的复发B-ALL患者全血样本（28 mL）时也取得相似结果。 结论：可成功使用全血制备具有治疗意义剂量的CD19/CD22 CAR-T细胞。平均80 mL全血所产CAR-T细胞足以按1×10⁶个/kg剂量为一名50 kg儿童制备单次治疗剂量；体型更大的患者则可通过增加采血量简单扩大规模。该方法还证明了一种具有成本效益的T细胞活化和扩增方式。结合全血采集更简单这一优势，该方法尤其有望提高中低收入国家的CAR-T可及性。通过降低成本和劳动需求，这一策略可能显著扩大CAR-T疗法的全球可及范围。
BACKGROUND: Chimeric antigen receptor T (CAR-T) cells have significantly advanced the treatment of cancers such as leukemia and lymphoma. Traditionally, T cells are collected from patients through leukapheresis, an expensive and potentially invasive process that requires specialized equipment and trained personnel. Although whole blood collections are much more technically straightforward, whole blood starting material has not been widely utilized for clinical CAR-T cell manufacturing, in part due to lack of manufacturing processes designed for use in a good manufacturing practice (GMP) environment. Collecting cellular starting material from whole blood without leukapheresis could reduce manufacturing complexity and cost, thereby improving accessibility to CAR-T cell therapy. METHODS: Whole blood samples were collected from eight healthy donors and one pediatric B-cell acute lymphoblastic leukemia (B-ALL) patient. These samples were processed using the Sepax C-Pro (Cytiva) instrument to isolate mononuclear cells (MNCs) via density gradient separation. CAR-T cells were then manufactured from the isolated MNCs using a GMP-compliant 7-day protocol, whereby T cells were activated with anti-CD3 and IL-2, transduced with GMP lentiviral vector encoding a CD19/CD22 bispecific CAR, and expanded in gas permeable cell culture bags. The resulting CAR-T cells were then evaluated for their phenotypic and functional properties using flow cytometry, cytokine release and cytotoxicity assays. RESULTS: From an average 77.7 mL of whole blood from healthy donors (range = 29-96 mL), we isolated an average of 42.2 10 6 CD3 T cells (range 7.3-63.0) postprocessing. CAR-T cell cultures were initiated from thaw using 1-10 10 6 starting CD3 + T cells, yielding a median T cell number of 105 10 6 cells on day 7 (range 61-188 10 6 ). We observed 66 11% mean transduction efficiency and produced a mean of 77.4 10 6 transduced CAR-T cells (range 30.8-143.5 10 6 ). Similar results were obtained when using a blood sample (28mL) obtained from a patient with relapsed B-ALL who had received recent chemotherapy. CONCLUSIONS: Therapeutically relevant doses of CD19/CD22 CAR-T cells can be successfully manufactured from whole blood. On average, 80 mL of whole blood yields enough CAR-T cells to create a single dose for a pediatric patient (50 kg) at a dosage of 1 10 6 CAR-T cells/kg. For larger patients, scaling up is straightforward by collecting a larger blood volume. This method also demonstrates a cost-effective approach to T cell activation and expansion which, alongside a more straightforward collection of whole blood, makes it more widely accessible especially for middle- and low-income countries. By reducing costs and labor, this strategy has the potential to significantly expand global access to CAR-T cell therapy.
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