肿瘤细胞治疗研究
英文原题：Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models.
Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models.
我们的研究表明，表达含单个 ITAM 的突变 CD3 链的 MSLN-CAR T 细胞在卵巢癌治疗中具有更强的治疗潜力。
背景：免疫抑制性实体瘤微环境中功能性CAR T细胞持久性不足，仍是CAR T细胞疗法成功转化为实体瘤治疗的主要障碍。通过突变CD19-CAR T细胞（含CD28共刺激结构域）CD3链免疫受体酪氨酸活化基序（ITAM）来精细调节CAR T细胞活化，已证实可在B细胞恶性肿瘤临床前模型中延长CAR T细胞功能性持久性。方法：本研究利用卵巢癌体内外临床前模型，评估两种常规第二代MSLN-CAR T细胞构建体（分别编码CD28共刺激结构域M28z或4-1BB共刺激结构域MBBz）以及一种新型间皮素（MSLN）靶向CAR T细胞构建体M1xx（编码CD28共刺激结构域和仅含单个ITAM的CD3链）。研究使用两种卵巢癌细胞系及两种NSG小鼠原位卵巢癌模型：通过显微手术将SKOV-3细胞接种于卵巢，以及腹腔注射OVCAR-4细胞以模拟晚期卵巢癌播散。通过生存分析评估MSLN-CAR T细胞治疗效果，并采用流式细胞术、定量PCR和基因表达分析表征、定量不同MSLN-CAR T细胞。结果：与常规第二代M28z和MBBz CAR T细胞相比，M1xx CAR T细胞抗肿瘤效力和持久性更强。离体M28z和MBBz CAR T细胞比M1xx CAR T细胞呈现更明显耗竭表型，表现为PD-1、LAG-3和TIM-3共表达。此外，M1xx CAR T细胞离体IFN-γ、TNF和GzB产生能力更强，并表现出自我更新基因特征。结论：本研究总体显示，表达仅含单个ITAM突变CD3链的MSLN-CAR T细胞具有更强治疗潜力，可用于卵巢癌治疗。经校准活化能力改造的CAR T细胞，可能改善实体瘤临床反应。
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 CD3 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 CD3 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 CD3 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.
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