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

总结 三阴性乳腺癌（TNBC）是一种侵袭性的危及生命的疾病，预后不良， 有限的治疗选择。为了满足这种临床需求，我们提出了一种双管齐下的药物递送方法 它将化疗和免疫药物与一个高效的药物输送平台相结合， 功效这项拨款申请是基于我们最近的证明，即病毒样颗粒（VLP）从 植物病毒豇豆花叶病毒（CPMV），当被引入肿瘤微环境中时， 和全身抗肿瘤免疫，并保护动物免受转移性疾病的生长或肿瘤复发。 [Nature Nanotechnology 2016]。我们已经在多种小鼠模型中证明了治疗 多个解剖位置和多个小鼠品系中的原发性和转移性肿瘤，包括小鼠 TNBC的模式。免疫激活的纳米粒子工程设计空间和途径尚未确定， 理解，和这个R 01赠款申请的一个手臂将破译潜在的机制。二是 开发和测试双管齐下的治疗方法，通过纳米颗粒平台与 化学疗法和免疫药物，以加强治疗并能够治疗晚期疾病。我们 将实现以下具体目标：1）我们将解决抗肿瘤作用是否是CPMV特异性的， 对于微粒，要求多价性质，以及纳米颗粒的形状和大小是否起作用。一 将研究具有不同分子组成、大小和形状的植物来源的VLP文库， 使用TNBC的4 T1小鼠模型进行评估。2)我们将确定细胞和信号通路- 参与VLP诱导的抗肿瘤免疫应答。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了解。识别潜在 涉及的信号通路，将在缺乏ASC、Myd 88或Trif的敲除小鼠模型中进行研究 衔接分子3)免疫疗法作为单一疗法通常仅对小肿瘤有效， 大多数患者对单一途径免疫疗法没有反应。治疗方案，即 然而，多种免疫疗法的组合或与化疗的组合可以形成基础， 妹妹成功。首先，我们将联合收割机VLP与阿霉素（一种免疫原性化疗药物）联合， 施用（未缀合）或共递送（缀合至VLP）。其次，我们将联合收割机与 已知的“免疫原性危险信号”和共同递送ssRNA或CpG以通过Toll样重链激活信号传导， 感受器我们假设免疫刺激性VLP和化疗或免疫药物的组合 将增强疗效，同时建立系统性抗肿瘤免疫应答和记忆，这将是 通过治疗携带4 T1原发性和转移性肿瘤的小鼠进行测试。最近，关于《关于... 裂解性免疫疗法T-VEC突出了基于病毒的疗法的潜力。我们的应用程序是不同的，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.