Dual action immunostimulatory nanoparticles for treatment of aggressive cancers

项目概述：肿瘤免疫治疗是以免疫识别为前提， 杀死肿瘤细胞，因此具有根除侵袭性疾病的有希望的能力。值得注意的是， 使用免疫检查点抑制剂的免疫疗法显著延长了患者的存活。不幸的是， 很大一部分患者在5年内发生复发和转移性疾病，主要是由于 对免疫检查点抑制剂的抗性。这很大程度上源于局部免疫抑制肿瘤 微环境（TME），其富含免疫抑制性骨髓细胞。重新编程TME， 一个“热”的环境富含功能性骨髓细胞，我们设计了一个双重作用的免疫刺激剂， 纳米颗粒（双NP），其触发功能失调的肿瘤驻留骨髓细胞的高效活化。 我们的策略的前提是基于三个关键组成部分：（1）我们以前的研究表明，V域 免疫球蛋白T细胞激活抑制因子（VISTA）是一种新的髓系细胞内源性免疫检查点 蛋白质，控制抗肿瘤免疫。我们发现VISTA在免疫抑制细胞中高度表达， 骨髓细胞和阻断VISTA可以与TLR9激动剂协同作用，以重新编程免疫抑制性细胞。 骨髓细胞来增强抗肿瘤免疫力。(2)双NP与VISTA siRNA和TLR9 siRNA共负载。 激动剂CpG。在肿瘤内施用时，双NP确保了其协同免疫应答的同时摄取。 货物由相同的肿瘤驻留骨髓细胞和熟练的细胞内交付的每种货物，因此， 达到激活这些细胞的最佳效果。我们最近的研究表明， VISTA基因和TLR9的刺激导致协同的T细胞介导的肿瘤清除和治愈性肿瘤清除。 免疫应答具有针对肿瘤复发的保护性免疫记忆。(3)我们开发了一个简单的， 产生不同尺寸的可电离脂质双NP的可控方法（即，30、40或60 nm）， 一致性和一致性的程度。我们的研究表明，小的双NP实现了广泛的分布 并且在肿瘤内给药时主要被整个肿瘤体积的髓样细胞摄取。 创新：据我们所知，这是第一次将联合收割机先进的纳米颗粒设计， siRNA和TLR激动剂的递送，以及与免疫检查点蛋白特异性结合相关的基因的沉默。 到骨髓细胞 目的1：优化双核蛋白的设计，并测试其在体外和体内的重编程效果 肿瘤相关的髓样细胞。 目的2：评价双联NP的短期和长期安全性特征，并描述其作用机制 与双NP给药的剂量和频率相关的抗肿瘤免疫应答。 目的3：评价双联NP作为单药治疗和与标准品联合治疗的疗效 免疫检查点抑制剂在晚期黑色素瘤和转移性乳腺癌小鼠模型中的应用。

PROJECT SUMMARY: Cancer immunotherapy is based on the premise of immune-recognition and targeted killing of tumor cells, thus possesses the promising power to eradicate aggressive disease. Notably to date, immunotherapy with immune checkpoint inhibitors significantly prolongs the survival of patients. Unfortunately, recurrent and metastatic disease occurs to a significant portion of patients within 5 years largely due to resistance to the immune checkpoint inhibitor. This largely stems from the local immunosuppressive tumor microenvironment (TME), which is enriched with immunosuppressive myeloid cells. To reprogram the TME into a “hot” environment enriched with functional myeloid cells, we designed a dual action immunostimulatory nanoparticle (dual-NP) that triggers a highly potent activation of the dysfunctional tumor-resident myeloid cells. The premise of our strategy is based on three key components: (1) Our previous studies showed that V domain Immunoglobulin Suppressor of T cell Activation (VISTA) is a novel myeloid cell-intrinsic immune checkpoint protein, which controls antitumor immunity. We showed VISTA is highly expressed on immunosuppressive myeloid cells and blocking VISTA can synergize with TLR9 agonist to reprogram the immunosuppressive myeloid cells to boost antitumor immunity. (2) The dual-NP is co-loaded with a VISTA siRNA and the TLR9 agonist CpG. Upon intratumoral administration, the dual-NP ensures the simultaneous uptake of its synergistic cargoes by the same tumor-resident myeloid cells and proficient intracellular delivery of each cargo, thus achieving optimal effects to activate these cells. Our recent studies show that simultaneous silencing of the VISTA gene and stimulation of TLR9 leads to a synergistic T cell-mediated tumor clearance and curative responses with protective immunological memory against tumor recurrence. (3) We developed a simple and controllable method to generate ionizable lipid dual-NPs of different sizes (i.e., 30, 40 or 60 nm) with high degree of uniformity and consistency. Our studies show that small dual-NPs achieve widespread distribution and predominant uptake by myeloid cells throughout the tumor volume upon intratumoral administration. Innovation: To our knowledge, this is the first effort to combine advanced nanoparticle design, simultaneous delivery of siRNA and a TLR agonist, and silencing of a gene related to an immune checkpoint protein specific to myeloid cells. AIM 1: Optimize the design of the dual-NP and test the ex vivo and in vivo efficacy in reprograming tumorassociated myeloid cells. AIM 2: Evaluate the short and long-term safety profile of the dual-NP and characterize the mechanism of antitumor immune responses associated with dosage and frequency of dual-NP administration. AIM 3: Evaluate the therapeutic efficacy of the dual-NP as a monotherapy and in combination with standard immune checkpoint inhibitors in murine models of advanced melanoma and metastatic breast cancer.