Molecular Characterization of Anti-Tumor Activity Mediated by Extracellular Vesicles Derived from Natural Killer Cells

自然杀伤细胞来源的细胞外囊泡介导的抗肿瘤活性的分子表征

• 批准号: 10587355
• 负责人: JACKI KORNBLUTH
• 金额: --
• 依托单位: ST. LOUIS VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587355
• 关键词: 3-Dimensional; Apoptosis; Area; Binding; Biodistribution; Biological Assay; Black American; Blood Cells; Blood Chemical Analysis; Body Weight; Bone Marrow; Bone Pain; Bortezomib; Caspase; Cell Line; Cell Therapy; Cells; Characteristics; Chemicals; Chemoresistance; Clinical; Clinical Trials; Cytolysis; Development; Diagnosis; Disease; Disease remission; Dose; Drug resistance; Fibroblasts; Fracture; Freezing; Frequencies; Genes; Goals; Half-Life; Hematologic Neoplasms; Herbicides; Human; Immunodeficient Mouse; In Vitro; Infiltration; Infusion procedures; Intravenous; Label; Laboratories; Lesion; Lytic Metastatic Lesion; Maintenance Therapy; Mediating; Membrane; Modeling; Molecular; Monitor; Multiple Myeloma; Mus; NK cell therapy; Natural Killer Cell Immunotherapy; Natural Killer Cells; Normal Cell; Organ Weight; Osteolytic; Pathway interactions; Patients; Penetration; Peripheral Blood Lymphocyte; Pharmaceutical Preparations; Preparation; Primary Neoplasm; Process; Production; Proteomics; Quality of life; Recurrence; Recurrent disease; Recurrent tumor; Refractory; Regimen; Relapse; Residual Neoplasm; Resistance; Risk; Roentgen Rays; Sampling; Stable Disease; Survival Rate; Testing; Time; Toxic effect; Tumor Burden; Veterans; Vietnam; Visual; X-Ray Computed Tomography; Xenograft Model; agent orange; bioluminescence imaging; bone; cancer cell; cancer stem cell; cancer therapy; cell killing; chemotherapy; clinical development; cohort; cytokine release syndrome; extracellular vesicles; hydrogel scaffold; improved; in vivo; in vivo evaluation; innovation; intravenous administration; intravenous injection; kidney dysfunction; kidney infection; knock-down; lenalidomide; lipidomics; loss of function; mouse model; neoplastic cell; novel therapeutics; peripheral blood; pomalidomide; prevent; relapse patients; side effect; stem cell therapy; stem-like cell; tumor; tumor hypoxia; tumor microenvironment

Multiple myeloma (MM) is the second most common hematological cancer in the U.S and increasing in frequency. Veterans who served in Vietnam where herbicides like Agent Orange were sprayed may have increased risk of developing MM. Early-stage disease is often asymptomatic, so patients are diagnosed late, with bone pain, kidney dysfunction and infections. Although there has been progress in developing new therapies for MM, it remains incurable. Patients initially achieve remission but ultimately relapse. The disease returns more quickly, tumor cells become more resistant to treatment and the patient’s quality of life declines. Natural killer (NK) cells kill MM cells in vitro and in vivo. Clinical trials using NK cell-based immunotherapy are ongoing but not widely available. Several drugs for MM (bortezomib, carfilzomib, lenalidomide) sensitize MM cells to NK-mediated lysis and/or enhance NK killing activity. However, challenges remain. Therefore, new treatments are needed to extend survival and increase durability of remission in relapsed/refractory patients and those ineligible for front-line therapy. My laboratory developed NK3.3, the only normal human NK cell line. It was cloned from peripheral blood NK cells and kills an array of tumor cells. As NK3.3 cells grow in culture, they release small membrane-bound extracellular vesicles (EVs). We demonstrated that purified NK3.3 EVs kill MM cell lines and primary patient samples, without harming normal cells. NK3.3 EVs also kill drug-resistant and cancer stem cells (CSC). There are many advantages to using NK3.3 EVs for cancer treatment. They can be generated in large quantities, are stable, and can be frozen and thawed without loss of function. EVs are resistant to the hypoxic tumor microenvironment and unlike cellular therapy, do not induce a detrimental cytokine storm. NK3.3-derived EVs may provide the advantages of NK cell therapy without the challenges of expanding cells and side effects. The goal of these studies is to establish the feasibility of using NK3.3-derived EVs for MM treatment. We developed a murine xenograft model of minimal residual disease in MM, which approximates a human clinical condition. After intravenous injection of RPM1-8226 MM cells into immunodeficient mice, tumor cells disseminate, infiltrate bones, and induce osteolytic lesions, characteristics of MM. We will test the ability of NK3.3 EVs to prevent MM recurrence after chemotherapy treatment. Aim 1: Characterize NK3.3 EVs and establish best practices for production. We will develop optimal NK3.3 culture conditions for EV production. Proteomic and lipidomic analysis will be performed on EV preparations. NK3.3 EVs will be evaluated for killing MM cell lines and patient samples and for lack of toxicity against healthy bone marrow, peripheral blood lymphocytes and fibroblasts. Aim 2: Identify the mechanism(s) of NK EV-mediated killing. NK EVs induce caspase-mediated apoptosis. However, like NK cells, NK EVs likely kill via multiple mechanisms. We will evaluate caspase- dependent and independent killing pathways in EV-treated tumor cells. We will inhibit killing pathways by gene knockdown and chemical treatment. MM cell lines and primary tumor cells will be used to determine whether drug-resistant and CSC-like MM cells are sensitive to killing by NK3.3 EVs. Aim 3: Test the in vivo efficacy of NK3.3 EVs in preventing/delaying tumor recurrence in a murine model of minimal residual disease in MM. Different concentrations of NK EVs will be administered intravenously. Tumor dissemination will be monitored by bioluminescence imaging; bone lesions by X-ray and CT scanning. Toxicity will be assessed by analysis of body and organ weights and blood chemistry. Normal and tumor-bearing mice will be infused with labeled NK EVs to monitor biodistribution and half-life. These studies are the first step towards developing NK3.3 EVs as a treatment for MM patients who desperately need new options to improve their quality of life and prolong their survival.

多发性骨髓瘤（MM）是美国第二常见的血液学癌症，并且增加 频率。在越南服役的退伍军人，在那里喷洒了橙色像特工橙色的退伍军人可能有 增加了MM的风险。早期疾病通常是不对称的，因此患者被诊断出来， 骨痛，肾功能障碍和感染。尽管开发新的进展已经取得进展 MM的疗法仍然无法治愈。患者最初可以缓解，但最终可以缓解。疾病 回报率更快，肿瘤细胞变得对治疗的抵抗力越来越高，患者的生活质量下降。 天然杀伤（NK）细胞在体外和体内杀死MM细胞。使用基于NK细胞的免疫疗法的临床试验 正在进行中，但不可广泛。 MM的几种药物（Bortezomib，Carfilzomib，Lenalidomide）灵敏度 MM细胞以NK介导的裂解和/或增强NK杀伤活性。但是，仍然存在挑战。因此，新的 需要治疗以延长生存率并提高继电/难治性患者的缓解耐用性 以及那些不符合前线治疗的资格。 我的实验室开发了NK3.3，这是唯一正常的人类NK细胞系。它是从外周血克隆的 NK细胞并杀死一系列肿瘤细胞。随着NK3.3细胞在培养中的生长，它们释放了膜的小型结合 细胞外蔬菜（EV）。我们证明了纯化的NK3.3 EV杀死MM细胞系和初级患者 样品，不损害正常细胞。 NK3.3 EV还杀死了抗药性和癌症干细胞（CSC）。 使用NK3.3 EV进行癌症治疗有许多优势。它们可以很大 数量，稳定，可以冷冻和解冻而不会损失功能。电动汽车对低氧具有抵抗力 肿瘤微环境和与细胞疗法不同，不要诱导有害的细胞因子风暴。 NK3.3衍生 电动汽车可能会提供NK细胞疗法的优势，而无需扩大细胞和副作用的挑战。 这些研究的目的是确定使用NK3.3衍生的电动汽车进行MM治疗的可行性。我们 在MM中开发了最小残留疾病的鼠色谱模型，该模型近似于人类的临床 健康）状况。在静脉注射RPM1-8226 mM细胞中后，肿瘤细胞 传播，浸润的骨骼，并诱导骨化病变，MM的特征。我们将测试 NK3.3 EV可预防化学疗法治疗后MM复发。 AIM 1：表征NK3.3 EV并建立生产最佳实践。我们将发展最佳 NK3.3 EV生产的培养条件。将在EV上进行蛋白质组学和脂质组分析 准备。将评估NK3.3电动汽车的杀死MM细胞系和患者样品以及缺乏毒性 针对健康的骨髓，外周血淋巴细胞和成纤维细胞。 目标2：确定NK EV介导的杀戮的机制。 NK EV诱导caspase介导的 凋亡。但是，像NK细胞一样，NK EV可能会通过多种机制杀死。我们将评估caspase- EV处理的肿瘤细胞中的依赖和独立的杀伤途径。我们将通过基因抑制杀戮途径 敲低和化学处理。 MM细胞系和原发性肿瘤细胞将用于确定是否是否 耐药和CSC样MM细胞对被NK3.3 EV杀死敏感。 AIM 3：测试NK3.3 EV的体内效率在预防/延迟鼠类肿瘤复发中的体内效率 MM中最小残留疾病的模型。将管理不同浓度的NK EV 静脉注射。肿瘤传播将通过生物发光成像监测； X射线和骨骼病变 CT扫描。毒性将通过分析身体和器官的体重和血液化学来评估。普通的 含有标记的NK EV的含有肿瘤的小鼠将被感染，以监测生物分布和半衰期。这些 研究是开发NK3.3 EV的第一步，作为迫切需要的MM患者的治疗 改善其生活质量并延长生存的新选择。