Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.
J Immunother Cancer. 2023 Feb;11(2). doi: 10.1136/jitc-2022-005691.
BACKGROUND: Limited persistence of functional CAR T cells in the immunosuppressive solid tumor microenvironment remains a major hurdle in the successful translation of CAR T cell therapy to treat solid tumors. Fine-tuning of CAR T cell activation by mutating CD3ζ chain immunoreceptor tyrosine-based activation motifs (ITAMs) in CD19-CAR T cells (containing the CD28 costimulatory domain) has proven to extend functional CAR T cell persistence in preclinical models of B cell malignancies. METHODS: In this study, two conventional second-generation MSLN-CAR T cell constructs encoding for either a CD28 co-stimulatory (M28z) or 4-1BB costimulatory (MBBz) domain and a novel mesothelin (MSLN)-directed CAR T cell construct encoding for the CD28 costimulatory domain and CD3ζ chain containing a single ITAM (M1xx) were evaluated using in vitro and in vivo preclinical models of ovarian cancer. Two ovarian cancer cell lines and two orthotopic models of ovarian cancer in NSG mice were used: SKOV-3 cells inoculated through microsurgery in the ovary and to mimic a disseminated model of advanced ovarian cancer, OVCAR-4 cells injected intraperitoneally. MSLN-CAR T cell treatment efficacy was evaluated by survival analysis and the characterization and quantification of the different MSLN-CAR T cells were performed by flow cytometry, quantitative PCR and gene expression analysis. RESULTS: M1xx CAR T cells elicited superior antitumor potency and persistence, as compared with the conventional second generation M28z and MBBz CAR T cells. Ex vivo M28z and MBBz CAR T cells displayed a more exhausted phenotype than M1xx CAR T cells as determined by co-expression of PD-1, LAG-3 and TIM-3. Furthermore, M1xx CAR T cells showed superior ex vivo IFNy, TNF and GzB production and were characterized by a self-renewal gene signature. CONCLUSIONS: Altogether, our study demonstrates the enhanced therapeutic potential of MSLN-CAR T cells expressing a mutated CD3ζ chain containing a single ITAM for the treatment of ovarian cancer. CAR T cells armored with calibrated activation potential may improve the clinical responses in solid tumors.
背景：在免疫抑制的实体肿瘤微环境中，功能性 CAR T 细胞的持久性有限，这仍然是将 CAR T 细胞疗法成功转化为治疗实体瘤的主要障碍。通过突变 CD19-CAR T 细胞（包含 CD28 共刺激结构域）中的 CD3ζ 链免疫受体酪氨酸激活基序（ITAMs）来精细调节 CAR T 细胞的激活已被证明可延长 B 细胞恶性肿瘤的临床前模型中功能性 CAR T 细胞的持久性。
方法：在这项研究中，使用两种编码 CD28 共刺激（M28z）或 4-1BB 共刺激（MBBz）结构域的常规第二代 MSLN-CAR T 细胞构建体和一种新型的间皮素（MSLN）导向的 CAR T 细胞构建体，该构建体编码 CD28 共刺激结构域和包含单个 ITAM（M1xx）的 CD3ζ 链，对卵巢癌的体外和体内临床前模型进行了评估。使用两种卵巢癌细胞系和 NSG 小鼠中的两种卵巢癌原位模型：通过卵巢内微手术接种的 SKOV-3 细胞和模拟晚期卵巢癌的播散模型，OVCAR-4 细胞腹腔内注射。通过生存分析评估 MSLN-CAR T 细胞的治疗效果，并通过流式细胞术、定量 PCR 和基因表达分析对不同的 MSLN-CAR T 细胞进行特征描述和定量。
结果：与常规第二代 M28z 和 MBBz CAR T 细胞相比，M1xx CAR T 细胞表现出更好的抗肿瘤效力和持久性。通过共表达 PD-1、LAG-3 和 TIM-3，确定 M28z 和 MBBz CAR T 细胞比 M1xx CAR T 细胞表现出更衰竭的表型。此外，M1xx CAR T 细胞表现出更好的体外 IFNy、TNF 和 GzB 产生，并具有自我更新基因特征。
结论：总之，我们的研究表明，表达突变的 CD3ζ 链的 MSLN-CAR T 细胞具有单个 ITAM，用于治疗卵巢癌，具有增强的治疗潜力。用校准的激活潜力武装的 CAR T 细胞可能会提高实体肿瘤的临床反应。
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