von Jutrzenka-Trzebiatowski Alexandra, Gupte Rutuja, Daglar Cansu, Berndt Nicole, Arndt Claudia, Bachmann Michael, Feldmann Anja
Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Bautzner Landstrasse 400, 01328 Dresden, Germany.
Mildred Scheel Early Career Center, Faculty of Medicine Carl Gustav Carus, TUD Dresden University of Technology, 01307 Dresden, Germany.
Int J Mol Sci. 2025 May 23;26(11):5024. doi: 10.3390/ijms26115024.
The Reverse Chimeric Antigen Receptor (RevCAR) system is an adapter CAR T cell technology that allows the precise tuning of T cell activity and, thus, improved safety management. RevCAR T cells recognize and eradicate tumor cells via a bispecific adapter molecule, termed the RevCAR Target Module (RevTM). To further reduce the risk of on-target off-tumor toxicities, Dual-RevCAR T cells can be employed. These cells harbor two different RevCAR constructs, with the signaling domain of either CD3zeta or CD28. Therefore, Dual-RevCAR T cells only exert their full function when both RevCAR constructs are triggered simultaneously upon recognition of two different tumor antigens via RevTMs, enabling a precise AND-gate targeting approach and rendering them highly interesting for clinical application. For this purpose, standardized and reproducible clinical-grade cell manufacturing is required, for which the CliniMACS Prodigy can be used. Here, we present that automated processing of RevCAR and Dual-RevCAR T cells via the CliniMACS Prodigy results in potent expansion, strong transduction, and a favorable phenotype for clinical application. Moreover, obtained cell products were highly functional in a strict RevTM-dependent manner for both monospecific and AND-gate targeting, clearly underlining their high potential for clinical application against various tumor entities.
反向嵌合抗原受体（RevCAR）系统是一种衔接子CAR-T细胞技术，可实现对T细胞活性的精确调节，从而改善安全性管理。RevCAR-T细胞通过一种称为RevCAR靶向模块（RevTM）的双特异性衔接子分子识别并清除肿瘤细胞。为了进一步降低靶向非肿瘤毒性的风险，可采用双RevCAR-T细胞。这些细胞含有两种不同的RevCAR构建体，其信号结构域分别为CD3ζ或CD28。因此，双RevCAR-T细胞只有在通过RevTM识别两种不同肿瘤抗原时，两种RevCAR构建体同时被触发，才会发挥其全部功能，从而实现精确的“与”门靶向方法，使其在临床应用中极具吸引力。为此，需要标准化且可重复的临床级细胞制造，可使用CliniMACS Prodigy来实现。在此，我们展示了通过CliniMACS Prodigy对RevCAR和双RevCAR-T细胞进行自动化处理，可实现有效扩增、高效转导，并产生有利于临床应用的表型。此外，所获得的细胞产品在单特异性和“与”门靶向方面均严格依赖RevTM发挥高度功能，这清楚地表明它们在针对各种肿瘤实体的临床应用中具有很高的潜力。
