优化用于增强适应性和临床可扩展性的非病毒睡眠美后生成的CARCIK细胞的GMP级生产。

Optimized GMP-grade production of non-viral Sleeping Beauty-generated CARCIK cells for enhanced fitness and clinical scalability.

作者信息

Pisani Ilaria, Melita Giusi, de Souza Patricia Borges, Galimberti Stefania, Savino Angela Maria, Sarno Jolanda, Landoni Beatrice, Crippa Stefano, Gotti Elisa, Cuofano Carolina, Pedrini Olga, Capelli Chiara, Matera Giada, Belotti Daniela, Cesana Stefania, Cabiati Benedetta, Quaroni Michele, Colombo Valentina, Mazza Massimiliano, Vergani Barbara, Gaimari Anna, Nicolini Fabio, Tazzari Marcella, Bocchini Martine, Serafini Marta, Rambaldi Alessandro, Rambaldi Benedetta, Dastoli Giuseppe, Biondi Andrea, Gaipa Giuseppe, Introna Martino, Golay Josée, Tettamanti Sarah

机构信息

Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy.

Advanced Cellular Therapies and Rare Tumors Unit, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori" S.r.l., Meldola, Italy.

出版信息

J Transl Med. 2025 May 19;23(1):559. doi: 10.1186/s12967-025-06416-3.

DOI:10.1186/s12967-025-06416-3

PMID:40390044

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12087026/

Abstract

BACKGROUND

Strict adherence to GMP guidelines and regulatory compliance is crucial when transitioning from research to clinical-grade production of ATMPs like CAR T cells. The success of CAR T cell therapy in treating hematological malignancies highlights the need for closed or automated systems to ensure quality and efficacy. Recent evidence also suggests that ex vivo culture conditions can significantly impact CAR T cell functionality.

METHODS

We present our optimized methodology for expanding Sleeping Beauty transposon-engineered Chimeric Antigen Receptor-Cytokine-Induced Killer (CARCIK) cells using G-Rex devices and evaluate its impact on CARCIK cell phenotype and T cell fitness.

RESULTS

Building on our previously validated protocol, we introduced key simplifications to optimize the CARCIK differentiation process. Delaying the nucleofection step eliminated the need for feeder cells while maintaining efficient CAR expression and high cell viability. Transitioning from T-flasks to G-Rex bioreactors reduced operator hands-on time from 21 to 28 days to 14-17 days and resulted in a less differentiated CARCIK cell product. Metabolic and transcriptional analyses showed that the novel protocol improves CARCIK cell fitness and in vivo efficacy against B-cell lymphoma. The novel method was validated in Good Manufacturing Practices (GMP) conditions at our two Cell Factories and yielded enough numbers of CARCIK-CD19 cells for clinical use.

CONCLUSIONS

Optimizing non-viral CARCIK cell production using G-Rex bioreactors and refined timing adjustments has streamlined the workflow, enhanced cell fitness, and resulted in a highly effective therapeutic product with demonstrated in vivo efficacy in mice. These improvements reduced manipulation and contamination risks, while optimizing logistics and space efficiency, facilitating allogeneic CARCIK generation for a current phase I/II clinical trial (NCT05869279) in patients with R/R CD19 + non-Hodgkin Lymphoma (B-cell NHL) and Chronic Lymphocytic Leukemia (CLL), confirming the approach's scalability and clinical potential.

摘要

背景

从研究阶段过渡到临床级别的嵌合抗原受体T细胞（CAR T细胞）等先进疗法医学产品（ATMP）的生产时，严格遵守药品生产质量管理规范（GMP）指南和法规要求至关重要。CAR T细胞疗法在治疗血液系统恶性肿瘤方面的成功凸显了采用封闭或自动化系统以确保质量和疗效的必要性。近期证据还表明，体外培养条件会显著影响CAR T细胞的功能。

方法

我们展示了使用G-Rex装置扩增经睡美人转座子工程改造的嵌合抗原受体-细胞因子诱导杀伤细胞（CARCIK）的优化方法，并评估其对CARCIK细胞表型和T细胞适应性的影响。

结果

基于我们之前验证过的方案，我们引入了关键的简化步骤以优化CARCIK细胞的分化过程。推迟核转染步骤无需饲养细胞，同时保持高效的CAR表达和高细胞活力。从T型烧瓶过渡到G-Rex生物反应器，将操作人员的实际操作时间从21至28天减少到14至17天，并产生分化程度较低的CARCIK细胞产品。代谢和转录分析表明，新方案提高了CARCIK细胞的适应性以及对B细胞淋巴瘤的体内疗效。该新方法在我们的两个细胞工厂的药品生产质量管理规范（GMP）条件下得到验证，并产生了足够数量的用于临床的CARCIK-CD19细胞。

结论

使用G-Rex生物反应器优化非病毒CARCIK细胞生产并进行精细的时间调整，简化了工作流程，增强了细胞适应性，并产生了一种在小鼠体内具有显著疗效的高效治疗产品。这些改进降低了操作和污染风险，同时优化了物流和空间效率，便于为复发/难治性CD19+非霍奇金淋巴瘤（B细胞NHL）和慢性淋巴细胞白血病（CLL）患者开展的当前I/II期临床试验（NCT05869279）生成同种异体CARCIK细胞，证实了该方法的可扩展性和临床潜力。