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

总结 晚期恶性黑色素瘤是一种高度致命的癌症，化疗和放疗难以治愈， 中位生存期一般少于4年。为了提高应答率，靶向CTL-4， PD 1和PDL 1已经获得了显著性，并且在转移性病例中可以达到约50%的响应率。这是 有希望，但由于缺乏免疫治疗，大部分患者仍然对免疫治疗没有反应。 浸润性T细胞，以及存在异常和抑制性信号传导途径， 检查点蛋白（例如CD 47、PDL 1）。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i）调节精确机械扰动， 升高肿瘤温度以诱导肿瘤抗原释放和ii）上调钙网蛋白（CRT），一种蛋白质 这是激活局部和全身抗免疫的关键。然而，确切的机制和如何 将这种方法转化为恶性黑色素瘤的临床治疗知之甚少。 本项目的目标是将联合收割机超声引导FUS与新型CRT-NP（一种脂质体-质粒）结合 本发明涉及一种纳米颗粒剂，其抑制黑素瘤细胞以诱导CRT表达。我们的体外和体内数据， 鼠黑色素瘤表明，CRT-NP/FUS（CFUS）联合局部治疗可增强 CRT和调节先天性（CD 47）和适应性检查点蛋白（PDL 1）;所有这些都显著增强 局部和全身免疫引发和抗肿瘤反应。基于这个前提，我们的中心假设 CRT/CD 47/PDL 1轴的CFUS靶向优化将提供强大的免疫引发， 产生针对恶性黑色素瘤的全身免疫。 为了验证我们的假设，我们将机械地解剖和理解B16细胞中CFUS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），并将这种方法转化为临床 在试验中使用客户拥有的自发性口腔黑色素瘤犬（目的3）。具体来说，我们将评估 CFUS处理顺序、FUS暴露和CRT激活机制对小鼠的影响 黑色素瘤，并将此信息转化为提高小鼠和犬肿瘤中检查点阻断的功效 模型 我们期望在该项目中局部CFUS的成功优化将使肿瘤从它们的免疫系统中解放出来， 抑制状态，实现细胞毒性免疫细胞的一致和可预测的克隆扩增，并改善 免疫治疗功效独立于癌症复杂性。如果成功，这种方法将提供一种有前途的 用于治疗黑素瘤和其它类型的实体瘤（例如，乳腺、前列腺）， 克服目前免疫治疗的障碍。

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.