Programming multi-pronged immune response to glioblastoma with IL-13Ra2/TGF-b CAR-T cell therapy.

使用 IL-13Ra2/TGF-b CAR-T 细胞疗法对胶质母细胞瘤进行多管齐下的免疫反应。

• 批准号: 10586951
• 负责人: Yvonne Yu-Hsuan Chen
• 金额: $ 61.3万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586951
• 关键词: Alpha Interleukin 2 Receptor; Antigens; Autopsy; Biopsy; Bone Marrow; Brain; C57BL/6 Mouse; CAR T cell therapy; Cell physiology; Cells; Cessation of life; Clinic; Clinical; Clinical Treatment; Cytometry; Data; Dominant-Negative Mutation; Engineering; Environment; Epitope spreading; Evaluation; Feedback; Flow Cytometry; Genetic Heterogeneity; Glioblastoma; Glioma; Heterogeneity; Human; IL13RA1 gene; Immune; Immune response; Immune system; Immunity; Immunocompetent; Immunofluorescence Immunologic; Immunohistochemistry; Immunosuppression; Immunosuppressive Agents; Immunotherapy; Infiltration; Inflammatory; Intravenous; Libraries; Lymphocyte; Malignant neoplasm of brain; Medical; Mesenchymal; Modeling; Monitor; Mus; Myelogenous; Myeloid Cells; Myeloid-derived suppressor cells; Neoplasm Metastasis; Newly Diagnosed; Patients; Phase I Clinical Trials; Phenotype; Play; Primary Brain Neoplasms; Proliferating; Reporting; Resected; Route; Safety; Signal Transduction; Specificity; Spleen; Stimulant; Surface Antigens; Survival Rate; T-Lymphocyte; Testing; Time; Toxic effect; Transforming Growth Factor Beta 2; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Tumor Promotion; Tumor-Infiltrating Lymphocytes; Tumor-associated macrophages; Tumor-infiltrating immune cells; Work; Xenograft Model; Xenograft procedure; beta Chain Antigen T Cell Receptor; chimeric antigen receptor; chimeric antigen receptor T cells; clinical translation; cytokine; effective therapy; engineered T cells; immunoregulation; improved; in vivo; mouse model; neoplastic cell; novel; pre-clinical; preservation; pressure; recruit; response; single-cell RNA sequencing; targeted treatment; translational potential; treatment group; tumor; tumor growth; tumor microenvironment; tumor progression

ABSTRACT Glioblastoma multiforme (GBM) is the most common type of primary brain tumor, with a five-year survival rate of only 5.5%. Chimeric antigen receptor (CAR)-T cell therapy has shown safety but limited efficacy in the treatment of patients with GBM to date. GBM is characterized by dramatic antigen heterogeneity, thus immunotherapy targeting any single antigen is unlikely to achieve complete and durable response. In addition, GBM cells and surrounding tumor stroma overproduce transforming growth factor beta (TGF-β), which not only promotes tumor growth and metastasis, but also actively modulate the immune response by suppressing T-cell function and recruiting suppressive myeloid cells. Our analysis of patient GBM biopsies further confirmed that tumor-infiltrating myeloid cells (TIMs) express both TGF-β and TGF-β receptors, indicating the presence of an immunosuppressive feedback loop in the GBM tumor microenvironment (TME). Here, we aim to develop a multi- pronged immunotherapy featuring bispecific CAR-T cells that can not only attack GBM directly, but also modify the TME to overcome immunosuppression and induce multiclonal immune responses against GBM. Our group has developed a bispecific CAR that can directly target GBM cells through recognition of IL- 13Rα2, a clinically validated GBM-associated surface antigen, while simultaneously converting TGF-β into a stimulant for the engineered T cells. We have demonstrated that bispecific IL-13Rα2/TGF-β CAR-T cells are superior to single-input IL-13Rα2 CAR-T cells in both human GBM xenograft and immunocompetent mouse models. Here, we aim to demonstrate the safety and efficacy of IL-13Rα2/TGF-β CAR-T cells for clinical translation. In addition to demonstrating clearance of IL-13Rα2+ GBM, we will explore the potential for IL- 13Rα2/TGF-β CAR-T cells to induce endogenous immune response against IL-13Rα2– tumor cells using mouse models of heterogenous GBM. We will perform in-depth analyses by flow cytometry, multiplexed immuno- fluorescence, cytometry by time of flight (CyTOF), and single-cell RNA sequencing to understand the impact of bispecific CAR-T cells on the immune composition and function in the GBM TME. We will also perform T-cell receptor beta (TCRβ) sequencing on tumor-infiltrating lymphocytes to quantify the potential for epitope spreading. We will perform rigorous safety evaluations in immunocompetent mouse models, applying worst-case scenario pressure tests to explore the toxicity profile of IL-13Rα2/TGF-β CAR-T cells. Finally, we will verify efficacy of IL-13Rα2/TGF-β CAR-T cell therapy in heterogeneous human gliomas ex vivo & in vivo, using both fresh patient GBM biopsies containing TIMs and GliomaPDOX models—i.e., mice bearing patient-derived GBM tumors that have never been passaged ex vivo and retain the genetic heterogeneity seen in human patients. Successful completion of this project will generate a comprehensive set of preclinical data in support of a phase- 1 clinical trial to treat GBM patients with this novel, multi-pronged immunotherapy option.

摘要 多形性胶质母细胞瘤（GBM）是最常见的原发性脑肿瘤类型，具有五年生存率 只有5.5%。嵌合抗原受体（CAR）-T细胞疗法已显示出安全性，但在免疫缺陷综合征中的疗效有限。 迄今为止，治疗GBM的患者。GBM的特征在于显著的抗原异质性，因此 靶向任何单一抗原的免疫疗法不太可能实现完全和持久的应答。此外，本发明还提供了一种方法， GBM细胞和周围肿瘤间质过度产生转化生长因子β（TGF-β），这不仅 促进肿瘤生长和转移，但也通过抑制T细胞活化来积极调节免疫应答。 功能和募集抑制性骨髓细胞。我们对患者GBM活检的分析进一步证实， 肿瘤浸润性髓样细胞（TIM）表达TGF-β和TGF-β受体，表明存在TGF-β受体。 GBM肿瘤微环境（TME）中的免疫抑制反馈回路。在这里，我们的目标是开发一个多- 双特异性CAR-T细胞不仅可以直接攻击GBM， TME克服免疫抑制并诱导针对GBM的多克隆免疫应答。 我们的小组已经开发了一种双特异性CAR，它可以通过识别IL-10直接靶向GBM细胞。 13 R α2，一种临床验证的GBM相关表面抗原，同时将TGF-β转化为 刺激工程化T细胞。我们已经证明，双特异性IL-13 R α2/TGF-β CAR-T细胞是 在人GBM异种移植物和免疫活性小鼠中均上级单输入IL-13 R α2 CAR-T细胞 模型在此，我们旨在证明IL-13 R α2/TGF-β CAR-T细胞用于临床治疗的安全性和有效性。 翻译.除了证明IL-13 R α2+ GBM的清除外，我们还将探索IL-13 R α2+ GBM的潜力。 使用小鼠13 R α2/TGF-β CAR-T细胞诱导针对IL-13 R α2-肿瘤细胞的内源性免疫应答 异质性GBM模型。我们将通过流式细胞术、多重免疫分析、 荧光、飞行时间流式细胞术（CyTOF）和单细胞RNA测序，以了解 在一些实施方案中，双特异性CAR-T细胞对GBM TME中的免疫组成和功能的影响。我们还将进行T细胞 受体β（TCRβ）测序，以量化表位扩散的可能性。 我们将在免疫活性小鼠模型中进行严格的安全性评价， 情景压力测试，以探索IL-13 R α2/TGF-β CAR-T细胞的毒性特征。最后，我们将验证 IL-13 R α2/TGF-β CAR-T细胞疗法在离体和体内异质性人胶质瘤中的疗效，使用两者 含有TIM和胶质瘤PDOX模型的新鲜患者GBM活组织检查-即，携带患者源性GBM的小鼠 从未离体传代并保留在人类患者中观察到的遗传异质性的肿瘤。 该项目的成功完成将产生一套全面的临床前数据，以支持一个阶段- 1项临床试验，用这种新型的多管齐下的免疫治疗选择治疗GBM患者。