肿瘤细胞治疗研究
英文原题：A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation.
本研究证明，一种新型的高内涵高通量筛选能够鉴定出增强 CAR T 细胞活性的药物。
背景：CAR-T细胞治疗是改善癌症患者结局、降低毒性的一种有前景方法。CAR-T细胞治疗CD19阳性恶性肿瘤已取得卓越成功，但多种障碍仍限制其对实体瘤患者的获益。新型制造和工程化方法有望增强CAR-T细胞对抗实体瘤的功能。然而，与单药化疗类似，CAR-T单药治疗可能无法使难治实体瘤患者达到较高治愈率。因此，药物联合CAR-T的方案可能是实现广泛临床成功所必需的。 方法：我们开发了一种基于共聚焦显微镜的新型高内涵筛选方法，评估1,114种FDA批准药物能否提高骨肉瘤细胞表面实体瘤抗原B7-H3的表达。研究采用Western blot、RT-qPCR、siRNA敲低和流式细胞术验证筛选结果并确定药物诱导B7-H3上调的机制；还通过细胞因子和细胞毒性实验，评估药物预处理是否增强B7-H3 CAR-T细胞效应功能。 结果：新型高内涵、高通量筛选发现55种药物可提高LM7骨肉瘤细胞表面B7-H3表达。其中一种药物ingenol-3-angelate（I3A）可使B7-H3表达最多提高100%，因此进入后续实验。验证实验确认，I3A诱导B7-H3表达呈双相剂量反应，且依赖细胞类型。机制研究表明，I3A通过活化蛋白激酶Cα，提高B7-H3（CD276）mRNA、总蛋白和细胞表面表达。功能实验显示，I3A诱导的B7-H3表达增强了B7-H3 CAR-T细胞的细胞因子生成和细胞毒功能。 结论：本研究证明，新型高内涵、高通量筛选可用于发现增强CAR-T细胞活性的药物。这类技术有望推动开展合理设计的药物联合CAR-T临床试验。值得注意的是，该技术还可用于多种基础和转化研究，以筛选调节细胞表面蛋白表达的药物。
BACKGROUND: CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success. METHODS: We developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function. RESULTS: Fifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays. CONCLUSIONS: This study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression.
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