Dual-pronged nano-drug delivery using plant virus-like particles.

摘要 三阴性乳腺癌(TNBC)是一种侵袭性的、危及生命的疾病，预后差，预后差。 治疗选择有限。为了满足这一临床需求，我们提出了一种双管齐下的给药方法。 它将化疗和免疫药物与高度有效的药物输送平台结合在一起，本身就具有抗肿瘤作用 功效。这项拨款申请是基于我们最近的演示，即来自 植物病毒豌豆花叶病毒(CPMV)进入肿瘤微环境后，可在局部诱导出有效的病毒 和全身抗肿瘤免疫，保护动物免受转移性疾病的滋生或复发 这种疾病[自然纳米技术2016]。我们已经在多个小鼠模型中证明了治疗效果 多个解剖位置和多个品系的原发和转移肿瘤，包括小鼠 TNBC的模型。免疫激活的纳米颗粒工程设计空间和途径尚未完全确定。 理解，这一R01拨款申请的一只手臂将破译潜在的机制。第二，我们将 通过将纳米颗粒平台与 化疗和免疫药物，以加强治疗，使晚期疾病的治疗成为可能。我们 将实现以下具体目标：1)我们将解决抗肿瘤效应是否是CPMV特异性的，其中- 呃颗粒性、多价性是一个要求，以及纳米颗粒的形状和大小是否起作用。一个 将研究具有不同分子组成、大小和形状的植物来源VLP的文库，并将 使用TNBC的4T1小鼠模型进行评估。2)我们将确定细胞和信号通路在- 溶于VLP诱导的抗肿瘤免疫反应。纵向成像结合组织化学- 尝试和流式细胞术将提供对生物反应的全面洞察。以确定潜在的 信号通路，研究将在缺乏ASC、MyD88或Trif的基因敲除小鼠模型中进行 接头分子。3)作为单一疗法的免疫疗法通常只对小肿瘤有效，以及 大多数患者对单一途径的免疫治疗没有反应。治疗方案的组合，即 然而，多种免疫疗法的结合或与化疗的结合可以形成基础-- SIS代表成功。首先，我们将联合VLP和阿霉素(一种免疫原性化疗)，通过联合 给药(未结合)或联合递送(与VLP结合)。第二，我们将把VLP与 并共同传递单链RNA或CpGS，通过Toll样Re-2激活信号。 接收器。我们假设免疫刺激性VLP与化疗或免疫药物的结合 将增强疗效，同时建立系统的抗肿瘤免疫反应和记忆，这将是 通过对携带4T1原发和转移性肿瘤的小鼠的治疗进行了测试。最近对OCCO的批准- 裂解免疫治疗T-VEC突出了基于病毒的治疗的潜力。我们的应用是独特的，是- 因为我们使用植物VLP进行双管齐下的肿瘤治疗和启动抗肿瘤免疫记忆。

Summary Triple negative breast cancer (TNBC) is an aggressive life-threatening disease with poor prognosis and limited treatment options. To address this clinical need, we propose a dual-pronged drug delivery approach that combines chemo- and immune-drugs with a highly potent drug delivery platform, that itself has anti-tumor efficacy. This grant application is based on our recent demonstration that virus-like particles (VLP) from the plant virus cowpea mosaic virus (CPMV), when introduced into the tumor microenvironment, elicit potent local and systemic anti-tumor immunity and protects animals from outgrowth of metastatic disease or recurrence of the disease [Nature Nanotechnology 2016]. We have demonstrated efficacy in multiple mouse models treating primary and metastatic tumors in multiple anatomic locations and multiple mouse strains, including mouse models of TNBC. The nanoparticle engineering design space and pathway of immune-activation is not yet un- derstood, and one arm of this R01 grant application will decipher the underlying mechanism. Second, we will develop and test dual-pronged therapeutic approaches through combination of the nanoparticle platform with chemotherapies and immune-drugs to potentiate the therapy and enable treatment of late-stage disease. We will fulfill the following specific aims: 1) We will address whether the anti-tumor effect is CPMV-specific, wheth- er the particulate, multivalent nature is a requirement, and whether nanoparticle shape and size plays a role. A library of plant-derived VLPs of distinct molecular composition, size, and shape will be studied and efficacy will be assessed using the 4T1 mouse model of TNBC. 2) We will determine the cells and signaling pathways in- volved in the VLP-induced anti-tumor immune response. Longitudinal imaging in combination with histochemis- try and flow cytometry will provide comprehensive insight into the biological response. To identify the potential signaling pathways involved, studies will be performed in knockout mouse models that lack ASC, Myd88 or Trif adaptor molecules. 3) Immunotherapy as monotherapy is generally effective only against small tumors, and most patients do not respond to single approach immunotherapy. The combination of therapeutic regimes, i.e. the combination of multiple immunotherapies or combination with chemotherapy, however, could form the ba- sis for success. First, we will combine VLPs with doxorubicin (an immunogenic chemotherapy) through co- administration (unconjugated) or co-delivery (conjugated to the VLP). Second, we will combine VLPs with known `immunogenic danger signals' and co-deliver ssRNA or CpGs to activate signaling through Toll-like re- ceptors. We hypothesize that the combination of the immune-stimulatory VLP and chemo- or immune-drugs will potentiate efficacy while mounting a systemic anti-tumor immune response and memory, and this will be tested through treatment of mice bearing 4T1 primary and metastatic tumors. The recent approval of the onco- lytic immunotherapy T-VEC highlights the potential of virus-based therapeutics. Our application is distinct, be- cause we use a plant VLP for dual-pronged tumor treatment and priming of anti-tumor immune memory.