SBIR Phase I: Endogenously secreted bispecific natural killer cell engagers (BIKEs) for therapy of solid tumors

这个小型企业创新研究（SBIR）一期项目提供了一种新颖的肌肉内基因传递平台，支持任何治疗性蛋白质的持续表达，这种能力尚未实现商业化。提供持续表达和内源性分泌双特异性自然杀伤细胞接触器（BiKE）疗法的能力有望通过提供绕过当前实体瘤免疫治疗的解决方案来改变范式。这种方法比目前最先进的方法侵入性小，可以消除持续/频繁重复输注治疗药物的需要，并避免在给药期间住院的需要。该方法的价格较低，与其他免疫疗法相比，可能将治疗成本降低数万至数十万美元，并且适用于资源匮乏的环境，显著提高了治疗的可获得性。这种概念验证疗法将针对肝细胞癌，一种占肝癌90%的实体肿瘤。该平台具有广泛的应用，支持提供任何基因治疗应用（例如，单基因疾病），这些应用可以受益于分泌蛋白的系统表达，包括双特异性抗体T细胞参与器、治疗性抗体和候选疫苗（例如，内源性治疗性抗体生产、DNA疫苗的递送和治疗性蛋白的表达，以治疗单基因罕见疾病）。该平台的预期影响包括改善治疗效果和改善患者的生活质量。该项目旨在推进一种安全、高效的肌肉内基因传递系统的开发，用于可重复使用的基因传递，并展示该平台在体内表达内源性分泌的双特异性自然杀伤细胞参与体（BIKEs）的能力，以治疗肝细胞癌（HCC），一种实体肿瘤。迄今为止，基因疗法治疗癌症的方法一直是昂贵的、劳动密集型的，而且疗效有限。与注射裸DNA相比，该平台有望将基因传递能力提高1000倍以上，并使基因产物能够有效地分泌到血液中，从而实现全身表达。具体目的是建立一个细胞表达系统，用于体外生产、纯化和功能验证重组BiKE，并用商品化的慢病毒转导生物发光肝癌细胞系，共表达RedFLuc和分泌GLuc，以更灵敏地检测肿瘤存活。该项目还将在人源化的原位肝细胞癌（HCC）小鼠模型中验证BiKE表达结构的有效性。在肌内注射后3-60天，通过酶联免疫吸附试验（ELISA）评估，在血清水平≥100-500 ng/ml时，可分泌的BiKE持续全身表达≥1个月，从而建立概念验证。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This Small Business Innovation Research (SBIR) Phase I project provides a novel, intramuscular gene delivery platform that supports sustained expression of any therapeutic protein, a capability yet to be commercially realized. The ability to provide sustained expression and endogenous secretion of bispecific natural killer cell engager (BiKE) therapeutics is expected to be paradigm shifting by providing a solution that bypasses current immunotherapy treatments for solid tumors. This approach, which is less invasive than the current state-of-the art, could eliminate the need for continuous/frequent repeated infusions of therapeutics and would circumvent the need for hospitalization during administration. The approach has a lower price point, potentially reducing the cost of therapy by tens to hundreds of thousands of dollars compared to other immunotherapies, and is amenable to low resource settings, significantly increasing the availability of treatment. The proof-of-concept therapeutic will target hepatocellular carcinoma, a solid tumor that accounts for 90% of liver cancers. The platform has broad applications, supporting delivery of any gene therapy application (e.g., monogenic disease) that can benefit from systemic expression of a secreted protein, including bi-specific antibody T cell engagers, therapeutic antibodies, and vaccine candidates (e.g., endogenous therapeutic antibody production, delivery of DNA vaccines, and expression of therapeutic proteins to treat monogenic rare diseases). Anticipated impacts of the platform include improved treatment efficacy and improved patient quality of life. This project seeks to advance the development of a safe, efficient intramuscular gene delivery system for redosable gene delivery as well as the demonstration of the platform’s ability to express endogenously secreted bispecific natural killer cell engagers (BIKEs) in vivo for treatment of hepatocellular carcinoma (HCC), a solid tumor. To date, gene therapy approaches to cancer treatment have been costly, labor intensive, and limited in efficacy. This platform is expected to enhance gene delivery by over 1,000-fold compared to the injection of naked DNA and to enable efficient secretion of the gene product into the blood stream, thereby allowing for systemic expression. Specific aims are to establish a cell expression system for production, purification, and functional validation of the recombinant BiKE in vitro and to make bioluminescent hepatoma cell lines transduced with a commercialized lentivirus co-expressing RedFLuc and secreted GLuc for more sensitive detection of tumor survival. The project will also validate the efficacy of the BiKE expression construct in a humanized, orthotopic hepatocellular carcinoma (HCC) mouse model. Proof of concept will be established with the demonstration of sustained systemic expression of the secretable BiKE for ≥ 1 month at serum levels of ≥100-500 ng/ml, as evaluated by enzyme-linked immunosorbent assay (ELISA) assays at days 3-60 post-intramuscular delivery.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.