Lanitis Evripidis, Rota Giorgia, Kosti Paris, Ronet Catherine, Spill Aodrenn, Seijo Bili, Romero Pedro, Dangaj Denarda, Coukos George, Irving Melita
Department of Oncology, Ludwig Institute for Cancer Research Lausanne, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
Department of Oncology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
J Exp Med. 2021 Feb 1;218(2). doi: 10.1084/jem.20192203.
Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.
嵌合抗原受体（CAR）疗法对实体瘤的临床益处有限，但用于生成所谓第四代（4G）CAR-T细胞的联合工程策略正在朝着克服肿瘤微环境（TME）中的障碍以改善反应迈进。在很大程度上由于技术挑战，在具有免疫活性的同基因荷瘤小鼠中进行的临床前CAR疗法研究相对较少。在此，我们描述了用于高效逆转录病毒转导和扩增主要为中央记忆T细胞（TCM细胞）表型的小鼠T淋巴细胞的优化方法。我们展示了一种双顺反子逆转录病毒载体，其编码肿瘤血管靶向CAR和小鼠白细胞介素-15（mIL-15），赋予增强的效应功能、植入、肿瘤控制和TME重编程，包括NK细胞活化和M2巨噬细胞数量减少。共表达mIL-15的4G-CAR-T细胞进一步表现为抗凋亡标志物Bcl-2上调和抑制性受体PD-1的细胞表面表达降低。总体而言，这项工作为在具有免疫活性的小鼠中开发和评估4G-CAR-T细胞引入了强大的工具，这是朝着加速有效疗法进入临床迈出的重要一步。
