Novel focused ultrasound enhanced calreticulin-nanoparticle for immune primed melanoma immunotherapy

摘要 晚期恶性黑色素瘤是一种高度致命的癌症，对化疗和放射治疗无效， 中位生存期通常不到4年。为了提高应答率，针对CTL-4的抗体， PD1、PDL1的疗效显著，在转移病例中的有效率可达50%左右。这是 很有希望，但由于缺乏免疫治疗，很大一部分患者仍然对免疫治疗没有反应 浸润性T细胞，并存在异常和抑制信号通路，钝化其表达 检查点蛋白(如CD47、PDL1)。为了促进发炎的黑色素瘤微环境，最近的研究 表明图像引导聚焦超声(FUS)可以1)调节精确机械扰动和 肿瘤温度升高诱导肿瘤抗原释放和II)上调钙网蛋白(CRT)，一种蛋白质 这是激活局部和系统抗免疫的关键。然而，确切的机制以及如何 将这种方法用于恶性黑色素瘤的临床治疗还知之甚少。 本项目的目标是将超声引导下的FUS与新型的CRT-NP脂质体相结合 转染黑色素瘤细胞以诱导CRT表达的纳米粒子制剂。我们的体外和体内数据 小鼠黑色素瘤联合CRT-NP/FUS(CFU)局部治疗可增强 CRT和调节先天(CD47)和适应性检查点蛋白(PDL1)；所有这些都显著增强 局部和全身免疫启动和抗肿瘤反应。基于这个前提，我们的中心假设 CFU靶向优化CRT/CD47/PDL1轴将提供强大的免疫启动和 产生对恶性黑色素瘤的系统免疫。 为了验证我们的假设，我们将机械地剖析和理解B16中CFU介导的免疫启动。 原位和基因工程小鼠黑色素瘤模型(目标1和2)并将该方法转化为临床 使用客户拥有的患有自发性口腔黑色素瘤的狗进行试验(AIMS 3)。具体地说，我们将评估 CFU治疗顺序、FUS暴露和CRT激活机制对小鼠的影响 并翻译此信息以提高检查点阻断在小鼠和犬肿瘤中的疗效 模特们。 我们期待这个项目中局部CFU的成功优化将使肿瘤从免疫- 抑制状态，实现一致和可预测的细胞毒性免疫细胞克隆扩增，并改善 免疫治疗的疗效与癌症的复杂性无关。如果成功，这种方法将提供一种很有前途的 治疗黑色素瘤和其他类型的实体肿瘤(如乳腺、前列腺)的新途径 克服目前的免疫治疗障碍。

Summary Malignant melanoma in advanced stages is a highly lethal form of cancer, refractory to chemo- and radio-therapy, and the median survival is typically less than 4years. To improve response rates, antibodies that target CTL-4, PD1, and PDL1 has gained prominence, and can achieve a response rate of ~50% in metastatic cases. This is promising but a large proportion of patients still do not respond to immune therapies due to the absence of infiltrating T-cells, and presence of aberrant and suppresive signaling pathways that blunt the expression of checkpoint proteins (e.g. CD47, PDL1). To promote inflamed melanoma microenvironment, recent studies indicate that image-guided focused ultrasound (FUS) can i) mediate precise mechanical perturbation and elevation of tumor temperature to induce tumor antigen release and ii) upregulate calreticulin (CRT), a protein that is key to the activation of local and systemic anti-immunity. However, the exact mechanisms and how to translate this approach for clinical treatment of malignant melanoma is poorly understood. The goal of this project is to combine ultrasound guided FUS with novel CRT-NP, a liposome-plasmid nanoparticle agent that transfects melanoma cells to induce expression of CRT. Our in vitro and in vivo data in murine melanoma suggest that combined local treatment with CRT-NP/FUS (CFUS) enhances expression of CRT and modulates innate (CD47) and adaptive checkpoint proteins (PDL1); all of which significantly enhance the local and systemic immune priming and anti-tumor responses. Based on this premise, our central hypothesis is that CFUS targeted optimization of the CRT/CD47/PDL1 axis will provide powerful immune priming and generation of systemic immunity against malignant melanoma. To test our hypothesis, we will mechanistically dissect and understand CFUS-mediated immune priming in a B16 orthotopic and genetically-engineered mouse melanoma model (Aim 1 & 2) and translate this approach to clinical use in trials using client-owned dogs with spontaneous oral melanoma (Aims 3). Specifically, we will evaluate the impact of the CFUS treatment sequence, FUS exposures, and CRT-activation mechanisms in murine melanoma and translate this information to improve efficacy of checkpoint blockage in murine and canine tumor models. We expect that the successful optimization of local CFUS in this project will liberate tumors from their immune- suppressive state, achieve consistent and predictable clonal expansion of cytotoxic immune cells, and improve immunotherapy efficacy independent of cancer complexity. If successful, this method will provide a promising new avenue for treating melanoma and other types of solid tumor (e.g., breast, prostate) by significantly overcoming current immunotherapy barriers.