肿瘤细胞治疗研究
英文原题：Liquid biopsy approach to monitor the efficacy and response to CAR-T cell therapy.
Liquid biopsy approach to monitor the efficacy and response to CAR-T cell therapy.
在同时检测患者外周血中的CAR-T细胞和B细胞恶性肿瘤时，HDSCA-HemeCAR工作流程可考虑用于风险监测和患者管理。
嵌合抗原受体 (CAR)-T 细胞已获批用于治疗血液系统恶性肿瘤。Axicabtagene ciloleucel（YESCARTA）和 brexucabtagene autoleucel（TECARTUS）是基于 FMC63 克隆表达抗 CD19 scFv 的基因修饰自体 T 细胞，在治疗 CD19+ B 细胞恶性肿瘤方面显示出令人瞩目的应答率，但在监测长期持续性以及患者体内低水平持续存在的 CAR-T 细胞的功能表征方面仍存在挑战。此外，由于 CD19 阴性驱动的复发，具备通过同时检测患者外周血中的 B 细胞恶性肿瘤和持续存在的 CAR-T 细胞来监测患者的能力，对于确保及时的治疗可选性和理解复发非常重要。
本研究展示了全面液体活检、高清单细胞检测（HDSCA）-HemeCAR 的开发和技术验证，用于（1）KTE-X19 CAR-T 细胞的识别和分析，以及（2）同时监测冷冻保存或新鲜外周血中肿瘤性 B 细胞上的 CD19 表位图谱。专有抗 CD19 CAR 试剂、健康供者转导的 CAR-T 细胞，以及由生产过程中恶性 B 细胞组分组成的患者样本，被用于检测开发。
CAR-T 检测的检测限约为 300 万个细胞中 1 个细胞，灵敏度为 91%。此外，使用基因组分析确认了 CAR 转基因的存在。本研究还报告了成功完成两项 B 细胞检测，涉及多种 CD19 变体（FMC63 和 LE-CD19）以及一个独特的第四通道生物标志物（CD20 或 CD22）。在患者样本中，我们观察到 CD19 异构体在患者内和患者间均具有高度异质性。
BACKGROUND: Chimeric antigen receptor (CAR)-T cells are approved for use in the treatment of hematological malignancies. Axicabtagene ciloleucel (YESCARTA) and brexucabtagene autoleucel (TECARTUS) genetically modified autologous T cells expressing an anti-CD19 scFv based on the FMC63 clone have shown impressive response rates for the treatment of CD19+B cell malignancies, but there remain challenges in monitoring long-term persistence as well as the functional characterization of low-level persisting CAR-T cells in patients. Furthermore, due to CD19-negative driven relapse, having the capability to monitor patients with simultaneous detection of the B cell malignancy and persisting CAR-T cells in patient peripheral blood is important for ensuring timely treatment optionality and understanding relapse. METHODS: This study demonstrates the development and technical validation of a comprehensive liquid biopsy, high-definition single cell assay (HDSCA)-HemeCAR for (1) KTE-X19 CAR-T cell identification and analysis and (2) simultaneously monitoring the CD19-epitope landscape on neoplastic B cells in cryopreserved or fresh peripheral blood. Proprietary anti-CD19 CAR reagents, healthy donor transduced CAR-T cells, and patient samples consisting of malignant B cell fractions from manufacturing were used for assay development. RESULTS: The CAR-T assay showed an approximate limit of detection at 1 cell in 3 million with a sensitivity of 91%. Genomic analysis was additionally used to confirm the presence of the CAR transgene. This study additionally reports the successful completion of two B cell assays with multiple CD19 variants (FMC63 and LE-CD19) and a unique fourth channel biomarker (CD20 or CD22). In patient samples, we observed that CD19 isoforms were highly heterogeneous both intrapatient and interpatient. CONCLUSIONS: With the simultaneous detection of the CAR-T cells and the B cell malignancy in patient peripheral blood, the HDSCA-HemeCAR workflow may be considered for risk monitoring and patient management.
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