AUTO1是一种快速离体吸附速率的CD19靶向嵌合抗原受体（CAR），已在成人淋巴母细胞性白血病中成功测试。Tscm/Tcm富集的CAR-T细胞群体提供了最佳扩张和持久性，但是在经过重度预处理的成人患者中，Tscm/Tcm细胞数量较少。为了改善这一点，我们评估了使用AKT抑制剂（VIII）来解耦T细胞扩张与分化，以富集Tscm/Tcm亚群。基于CliniMACS Prodigy平台的半自动化AUTO1制造过程中引入了VIII，无论是在小规模还是cGMP规模下。使用VIII制造的AUTO1在体外显示出Tscm/Tcm富集、扩张和细胞毒性改善，并在体内显示优越的抗肿瘤活性。此外，VIII诱导AUTO1 Th1/Th17偏向，增加多功能性，并赋予独特的代谢特征和自噬新标志，以支持增强的扩张和细胞毒性。我们展示了在ALLCAR19研究中使用VIII培养的来自B-ALL患者的AUTO1产品具有优越的表型、代谢和功能，并且基于VIII的制造方法可扩展到cGMP。最终，使用VIII生成的AUTO1可能开始克服导致CD19+复发的产品特异性因素。© 作者（或其雇主）2023年。根据CC BY-NC许可进行再使用。禁止商业再使用。由BMJ出版。

AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets.VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale.AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP.Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.