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

总结 生物打印植物病毒纳米颗粒用于卵巢癌的免疫治疗和复发预防 高级别浆液性卵巢癌（HGSOC）是卵巢癌中最常见和最严重的形式， HGSOC的预后很差。诱导全身抗肿瘤免疫的免疫治疗方法， 特别是预防复发的药物，是HGSOC迫切需要的。我们建议使用植物病毒样 纳米颗粒（VLP）结合缓释抗原库作为癌症疫苗方法， 缓解期的抗肿瘤免疫力下降，以阻断复发。我们的数据表明，腹膜内（IP）给药- 在卵巢癌小鼠模型中施用植物VLP调节肿瘤微环境， 免疫抑制并产生针对肿瘤抗原适应性抗肿瘤免疫和记忆。的vlp 非感染性、非细胞毒性和非细胞溶解性，但蛋白质VLP的高度重复性质 引发先天免疫激活和相关的适应性免疫应答。在此基础上，我们将开发 一种VLP生物聚合物制剂，能够在HGSOC中手术减积后进行有效的免疫治疗。我们 将掺入经辐照的肿瘤细胞作为患者特异性肿瘤抗原的来源;将细胞递送至 与VLP一起作为佐剂以启动持久的抗肿瘤免疫。建议的免疫- notherapy植入物将通过创新的3D生物打印技术生产;特别是，快速，微型 连续光学生物打印（μCOB）。该平台提供了对地形复杂性和 细胞和材料组成的支架在微米级的分辨率。我们的快速3D生物打印过程 允许多种生物相容性材料的光聚合，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 设计为使治疗剂量以编程间隔（预充/加强）释放，而不是连续缓慢释放 release.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， 优化缓慢、连续释放与分阶段释放，例如治疗性VLP的每周释放。植入物将 经过严格的质量控制和再现性测试，放行的VLP将进行结构分析 和生物测试。2)将评价免疫治疗植入物与可溶性VLP的疗效 采用小鼠卵巢癌模型（ID 8vegf/defb 29）。免疫学研究将提供深入了解 免疫治疗的机制。3)进一步探索疫苗参数和模型极低的内源性 在缓解期间的患者抗原负荷，我们将生物打印生物聚合物植入物以递送VLP和抗原（来自 在用ID 8vegf/defb 29细胞攻击之前，将细胞接种于经辐照的细胞）。对于未来的转化方法，患者肿瘤 来自外科减积和/或患者新抗原肽。临床意义很高：我们 设想对目前的治疗工作流程进行简单的修改，其中小的可降解疫苗植入物 在手术期间留在腹膜内（IP）腔中或在手术后皮下（SC）施用，或两者。

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.