Bioprinting Plant Virus Nanoparticles for Immunotherapy and Relapse Prevention of Ovarian Cancer

摘要 生物印迹植物病毒纳米粒用于卵巢癌的免疫治疗和预防复发 高级别浆液性卵巢癌(HGSOC)是卵巢癌中最常见、最严重的一种。 合并HGSOC患者预后较差。诱导系统抗肿瘤免疫的免疫治疗方法 尤其是那些防止复发的药物，是HGSOC迫切需要的。我们建议使用类似植物病毒的 纳米粒(VLP)与缓释抗原库相结合作为癌症疫苗的方法推出系统 TEMIC在缓解期的抗肿瘤免疫以防止复发。我们的数据表明，腹膜内(IP)给药- 在卵巢癌小鼠模型中应用植物VLP调节肿瘤微环境以缓解 免疫抑制和产生适应性抗肿瘤免疫和针对肿瘤抗原的记忆。VLP 是无感染性、无细胞毒性和非细胞溶解的，但蛋白质类VLP的高度重复性 触发先天免疫激活和相关的适应性免疫反应。在此基础上，我们将发展 一种VLP生物聚合物配方，可在HGSOC手术后进行有效的免疫治疗。我们 将把照射的肿瘤细胞作为患者特异性肿瘤抗原的来源；这些细胞将被运送 与作为佐剂的VLP一起启动持久的抗肿瘤免疫。建议的IMMU- 非治疗性植入物将通过创新的3D生物打印技术生产；具体地说，是快速、微型的 连续光学生物打印(µCOB)。该平台提供了对地形复杂性和 微米级分辨率的支架的细胞和材料组成。我们的快速3D生物打印流程 允许多种生物相容材料的光聚合，并促进VLP和/或 细胞。这种方法的工程设计空间和可调性是无可挑剔的；特别是植入物将 要设计成治疗剂量按程序间隔(主要/增强)释放，而不是连续缓慢释放 放手。我们将实现三个具体目标：1)Bioprint VLP生物聚合物植入物和测试各种配置 优化缓释、持续释放与阶段性释放，例如每周释放治疗性VLP。植入物将会 经过严格的质量控制和再现性测试，发布的VLP将进行结构分析 和生物测试。2)评估免疫治疗植入物与可溶性VLP的疗效 采用小鼠卵巢癌模型(ID8Vegf/Defb29)。免疫学调查将提供对 免疫治疗的机制。3)进一步探索疫苗参数和模型极低内源性 缓解期间患者的抗原负载，我们将通过生物打印生物聚合物植入物来传递VLP和抗原(来自 照射细胞)，然后用ID8Vegf/Defb29细胞攻击。对于未来的翻译方法，患者肿瘤 从手术清除和/或患者新抗原肽将被使用。临床意义很高：我们 设想对当前的治疗工作流程进行简单的修改，其中可降解的小疫苗植入物 在手术期间留在腹膜(IP)腔内或术后皮下(SC)给药，或两者兼而有之。

Summary Bioprinting plant virus nanoparticles for immunotherapy and relapse prevention of ovarian cancer High grade serous ovarian cancer (HGSOC) is the most common and severe form of ovarian cancer and women with HGSOC have a poor prognosis. Immunotherapy approaches that induce systemic antitumor immunity, in particular those that prevent relapse, are urgently needed for HGSOC. We propose to employ plant virus-like nanoparticles (VLPs) combined with slow release antigen depots as a cancer vaccine approach to launch sys- temic antitumor immunity during remission to block relapse. Our data indicate that intraperitoneal (IP) admin- istration of plant VLPs in a mouse model of ovarian cancer modulates the tumor microenvironment to relieve immunosuppression and generate adaptive anti-tumor immunity and memory against tumor antigens. The VLPs are non-infectious, non-cytotoxic, and non-cytolytic, but the highly repetitive nature of the proteinaceous VLPs triggers innate immune activation and associated adaptive immune response. Building on this, we will develop a VLP biopolymer formulation to enable effective immunotherapy following surgical debulking in HGSOC. We will incorporate irradiated tumor cells as source for patient specific tumor antigens; the cells will be delivered together with the VLPs which act as adjuvant to launch long-lasting anti-tumor immunity. The proposed immu- notherapy implant will be produced through an innovative 3D bioprinting technique; specifically, rapid, microscale continuous optical bioprinting (µCOB). This platform offers control over both the topographical complexity and the cellular and material composition of the scaffold at micron-level resolution. Our rapid 3D bioprinting process allows for photopolymerization of multiple biocompatible materials, and facilitates incorporation of VLPs and/or cells. The engineering design space and tunability of this approach is impeccable; in particular the implant will be designed so that therapeutic doses are released in programmed intervals (prime/boost) vs. continuous slow release. We will fulfill three specific aims: 1) Bioprint VLP biopolymer implants and test various configurations to optimize slow, continuous release vs. staged, e.g. weekly release of the therapeutic VLPs. The implants will undergo rigorous quality control and reproducibility testing and released VLPs will undergo structural analysis and biological testing. 2) Evaluate efficacy of the immunotherapy implants vs. soluble VLPs will be evaluated using mouse model of ovarian cancer (ID8vegf/defb29). Immunological investigation will provide insights into the mechanism of the immunotherapy. 3) To further explore vaccine parameters and model very low endogenous patient antigen loads during remission, we will bioprint biopolymer implants to deliver VLPs and antigen (from irradiated cells) prior to challenge with ID8vegf/defb29 cells. For future translational approaches, patient tumor from surgical debulking and/or patient neoantigen peptides would be used. The clinical significance is high: we envision a simple modification to the current treatment work-flow, where small degradable vaccine implants are left in the intraperitoneal (IP) cavity during surgery or administered subcutaneously (SC) post-surgery, or both.