Production and Evaluation of a Vault-CCL21 Nanocapsule as a Lung Cancer Treatment

描述（由申请人提供）：项目概述：肺癌是美国癌症死亡的主要原因，每年的死亡人数超过乳腺癌、前列腺癌、结肠癌、肝癌、肾癌和皮肤癌的总和。从经济角度来看，在美国，2000年肺癌死亡造成的生命损失价值超过2400亿美元，估计到2020年将增加到4330亿美元以上。由于肺癌带来的巨大医疗保健和经济负担，改善患者预后的新治疗策略将产生非常重大的影响。 加州大学洛杉矶分校（Lenny罗马实验室）的合作者已经鉴定并表征了一种独特的核糖核蛋白纳米颗粒结构，该结构高度稳定，并且在所有高等真核生物中普遍存在。主要穹隆蛋白（MVP）是核心纳米颗粒成分，它很容易被改造以允许连接其他试剂，包括有前途的癌症治疗剂，如趋化因子配体21（CCL 21）。CCL 21是一种淋巴样趋化因子，是成熟树突状细胞（DC）、初始和记忆T细胞的化学引诱物。临床前研究已经证明，肿瘤内施用CCL 21基因修饰的树突状细胞导致肿瘤根除。 拱顶已在切萨皮克PERL，Inc.使用PERLXpress蛋白表达平台将C-PERL蛋白表达至非常高的水平。独特而强大的系统使用重组杆状病毒在全昆虫中在自动化平台中表达，以产生高蛋白质产量，具有成本效益。初步数据显示MVP易于表达并正确组装形成纳米颗粒。此外，已经证明了模型蛋白（pCherry）的包装。. 我们预期，持续的开发将表明，CCL 21-Vaults可以通过表达和包装组成蛋白质，然后通过严格的纯化方法以产生高产率的高质量纳米胶囊而容易地以高质量制备。我们假设，肿瘤内施用源自杆状病毒感染的整个昆虫的重组CCL 21-穹窿体将避免自体DC制备，最大限度地减少批次间的差异，并允许可比性和标准化，使得颗粒可用作晚期NSCLC的现成试剂。具体目标1：工程化，表达和纯化Vault-CCL 21纳米帽蛋白复合物，用于使用标准蛋白质分析工具以及电子显微镜进行初始生化表征。继续纯化开发以优化产量并确保最高质量和纯度。具体目标二：进行体外分析，包括趋化性和相关试验，以评估和比较CCL 21-vault的生化特征，并与先前在SF 9细胞中表达的检测的CCL 21-vault进行比较。 公共卫生相关性：该项目与肺癌导致的常见死亡和残疾原因的治疗相关。该研究将支持使用天然存在的人穹窿蛋白的重组版本与CCL 21抗肿瘤剂组合的候选疗法的持续开发。这些组合技术可能会产生一种靶向肺癌的药物，证明了一种大规模，低成本，可重复的治疗方法。这是一种潜在的破坏性方法，可以彻底改变肺癌领域，并可能应用于其他癌症。

DESCRIPTION (provided by applicant): Project Summary: Lung cancer is the leading cause of cancer death in the United States and is responsible for more deaths each year than breast, prostate, colon, hepatic, renal and skin cancers combined. Viewed in economic terms, in the United States, the value of life lost from lung cancer deaths in the year 2000 was more than $240 billion, which is estimated to rise to more than $433 billion in 2020. Because of the immense health care and economic burden imposed by lung cancer, new therapy strategies that improve patient outcomes will lead to very a significant impact. Collaborators at UCLA (Laboratory of Lenny Rome) have identified and characterized a unique ribonucleoprotein nanoparticle structure that is highly stable, and is found ubiquitously in all higher eukaryotes. Major Vault Protein (MVP) is the core nanoparticle component, and it is readily engineered to permit attachment of other agents, including promising cancer therapeutics like chemokine ligand 21 (CCL21). CCL21 is a lymphoid chemokine that is chemoattractant for mature dendritic cells (DCs), naive and memory T cells. Preclinical studies have demonstrated that intratumoral administration of CCL21 gene-modified dendritic cells led to tumor eradication. Vaults have been expressed at Chesapeake PERL, Inc. (C-PERL) to very high levels using the PERLXpress protein expression platform. The unique and powerful system uses recombinant baculovirus expression in whole insects in an automated platform to generate high protein yields cost effectively. Preliminary data shows that MVP is readily expressed and correctly assembled to form nanoparticles. Further, packaging of a model protein (pCherry) has been demonstrated. . We expect that continued development will show that CCL21-Vaults can be readily prepared in high quality by expression, and packaging of the constituent proteins, followed by stringent purification methods to generate high yields of high quality nanocapsule. We hypothesize that intratumoral administration of recombinant CCL21-vaults derived from baculovirus infection of whole insects will circumvent autologous DC preparation, minimize batch to batch variability and allow for comparability and standardization so that the particle can be used as an off-the-shelf reagent for advanced NSCLC. Specific Aim 1: Engineer, express and purify Vault-CCL21 nanocapsule protein complexes for initial biochemical characterization using standard protein analytical tools as well as electron microscopy. Continue purification development to optimize yield and ensure highest quality and purity. Specific Aim 2: Perform in vitro analyses, including chemotactic and related assays to assess and compare the biochemical profile for CCL21-vault and compare to previously tested CCL21-vault expressed in SF9 cells. PUBLIC HEALTH RELEVANCE: This project is relevant to treatment of common causes of death and disability due to lung cancer. The research will support continued development of a candidate therapy using a recombinant version of naturally occurring human vault protein in combination with CCL21 antitumor agent. The combination technologies may result in a drug that targets lung cancer, demonstrating a large scale, low cost, reproducible therapy. This is a potentially disruptive approach that can revolutionize the lung cancer field, and may be applied to other cancers.