临床级自体 CD20 CAR T 细胞的自动化制造和鉴定用于治疗 III/IV 期黑色素瘤患者。

INTRODUCTION: Point-of-care (POC) manufacturing of chimeric antigen receptor (CAR) modified T cell has expanded rapidly over the last decade. In addition to the use of CD19 CAR T cells for hematological diseases, there is a growing interest in targeting a variety of tumor-associated epitopes. METHODS: Here, we report the manufacturing and characterization of autologous anti-CD20 CAR T cells from melanoma patients within phase I clinical trial (NCT03893019). Using a second-generation lentiviral vector for the production of the CD20 CAR T cells on the CliniMACS Prodigy®. RESULTS: We demonstrated consistency in cell composition and functionality of the products manufactured at two different production sites. The T cell purity was >98.5%, a CD4/CD8 ratio between 2.5 and 5.5 and transduction rate between 34% and 61% on day 12 (harvest). Median expansion rate was 53-fold (range, 42-65-fold) with 1.7-3.8×10 CAR T cells at harvest, a sufficient number for the planned dose escalation steps (1×10/kg, 1×10/kg, 1×10/kg BW). Complementary research of some of the products pointed out that the CAR+ cells expressed mainly central memory T-cell phenotype. All tested CAR T cell products were capable to translate into T cell activation upon engagement of CAR target cells, indicated by the increase in pro-inflammatory cytokine release and by the increase in CAR T cell amplification. Notably, there were some interindividual, cell-intrinsic differences at the level of cytokine release and amplification. CAR-mediated T cell activation depended on the level of CAR cognate antigen. DISCUSSION: In conclusion, the CliniMACS Prodigy® platform is well suited for decentralized POC manufacturing of anti-CD20 CAR T cells and may be likewise applicable for the rapid and automated manufacturing of CAR T cells directed against other targets. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/study/NCT03893019?cond=Melanoma&term=NCT03893019&rank=1, identifier NCT03893019.

简介：过去十年，嵌合抗原受体（CAR）修饰 T 细胞的即时制造（Point-of-care，POC）迅速发展。除了用于血液疾病的 CD19 CAR T 细胞，针对各种肿瘤相关表位的靶向治疗也越来越受到关注。

方法：在此，我们报告了在 I 期临床试验（NCT03893019）中从黑色素瘤患者体内制造和表征自体抗 CD20 CAR T 细胞的情况。使用第二代慢病毒载体在 CliniMACS Prodigy®上生产 CD20 CAR T 细胞。

结果：我们证明了在两个不同生产地点制造的产品在细胞组成和功能上具有一致性。第 12 天（收获）时，T 细胞纯度＞98.5%，CD4/CD8 比值在 2.5 至 5.5 之间，转导率在 34%至 61%之间。中位扩增倍数为 53 倍（范围 42-65 倍），收获时 CAR T 细胞为 1.7-3.8×10 6 个，足以满足计划的剂量递增步骤（1×10 6 /kg、1×10 6 /kg、1×10 6 /kg BW）。对一些产品的补充研究表明，CAR+细胞主要表达中央记忆 T 细胞表型。所有测试的 CAR T 细胞产品在与 CAR 靶细胞结合时都能够转化为 T 细胞激活，这表现为促炎细胞因子释放增加和 CAR T 细胞扩增增加。值得注意的是，在细胞内细胞因子释放和扩增水平上存在一些个体间的差异。CAR 介导的 T 细胞激活取决于 CAR 同源抗原的水平。

讨论：总之，CliniMACS Prodigy®平台非常适合分散式即时制造抗 CD20 CAR T 细胞，并且可能同样适用于针对其他靶标的 CAR T 细胞的快速自动化制造。

临床试验注册：https://clinicaltrials.gov/study/NCT03893019?cond=Melanoma&term=NCT03893019&rank=1，标识符 NCT03893019。