MASTER Scaffolds for Rapid, Single-Step Manufacture and Prototyping of CAR-T cells

项目总结 尽管嵌合抗原受体(CAR)T细胞疗法在临床上取得了前所未有的成功，但其广泛的 应用受到冗长和劳动密集型的体外制造程序的限制，这导致：(I)高成本； (Ii)延迟为病情进展迅速的病人输注CAR细胞；及。(Iii)异种CAR细胞。 组成和末端分化，限制了它们的植入性和持久性。有一个明确的科学的 医学上需要改进汽车T细胞生产的方法，包括减少细胞处理的方法 倍，降低制造成本，减少汽车细胞分化。最近，我们的实验室合作 开发了一种生产汽车电池的新技术，称为MASTER(多功能海藻酸支架 细胞工程和发布)。MASTER由与αCD3结合的干燥、大孔藻酸盐材料组成 和αCD28抗体和包裹白细胞介素2的信号。掌握技术的汽车新一代 涉及将新鲜分离的未激活的患者PBMC与CAR编码的逆转录病毒载体一起播种 然后把脚手架重新植入病人体内。一旦被植入，师父就会协调汽车的每一步。 从而省去了目前αCD3/αCD28预激活的标准程序步骤， 用旋转法和白细胞介导型CAR扩增病毒转导。体外母体生成的Car 与用金标准培养的CAR细胞相比，细胞显示出细胞分化程度降低， “传统的”临床方案。体内母体生成的CAR细胞显示出远优于体内细胞的优势 持久，增强了抗肿瘤效果，并在再挑战后远远优于对肿瘤生长的预防。这个 这个系统的用途有两个：1)作为一种变革性的治疗技术，创造了增强的和负担得起的 汽车疗法用于癌症护理，以及2)作为一种研究工具，使快速开发、原型和测试 汽车治疗候选人。我们组建了一支专注的、多学科的团队，由一位 生物材料和药物输送(Brudno)，病毒工程和蛋白质生产专家(Birnbaum)，两人 临床汽车细胞生产专家(Chen，Roy)和专注于汽车细胞疗法的临床医生(Grover)。在……里面 这项建议我们寻求进一步开发和验证主脚手架和相关的制作方法 它们已准备好供研究和临床社区广泛使用，包括相关领域的研究人员 渴望在汽车领域工作，但因障碍而不愿在体内建造汽车的地区。利用 变革性的初步数据显示，货架稳定的主脚手架性能优于传统汽车 临床前淋巴瘤、原位胰腺癌、肺和卵巢转移小鼠模型中的细胞 这项提案将与广泛的捐赠者在不同的规模上验证主支架， 多个病毒载体和CAR构建并描绘了MASTER导致的表型和功能 汽车电池的生产。这些目标的成功实现将推动我们实现低成本和 可调一代CAR细胞，可用于液体和固体肿瘤，并有可能超越肿瘤学领域。

PROJECT SUMMARY Despite the unprecedented clinical success of chimeric antigen receptor (CAR) T cell therapy, its widespread application is limited by lengthy and labor-intensive ex vivo manufacturing procedures that result in: (i) high cost; (ii) delays to infuse CAR cells to patients with rapidly progressing disease; and (iii) CAR cells with heterogeneous composition and terminal differentiation, which limit their engraftment and persistence. There is a clear scientific and medical need for approaches to improve CAR T cell production, including methods to reduce cell processing times, reduce manufacturing costs, and reduce CAR cell differentiation. Recently, our labs collaboratively developed a new technology for CAR cell production called MASTER (Multifunctional Alginate Scaffolds for T cell Engineering and Release). MASTER consists of dry, macroporous alginate materials conjugated to αCD3 and αCD28 antibodies and encapsulating interleukin signaling. CAR generation with MASTER technology involves seeding freshly isolated, non-activated patient PBMCs together with CAR-encoding retroviral vectors and implanting scaffolds back into patients. Once implanted, MASTER mediates every step of the CAR production process, thereby eliminating the current standard procedural steps of αCD3/αCD28 pre-activation, viral transduction with spinoculation and interleukin-mediated CAR expansion. In vitro MASTER-generated CAR cells demonstrate reduced cellular differentiation as compared to CAR cells generated with gold-standard, “conventional” clinical protocols. In vivo MASTER-generated CAR cells demonstrate far superior in vivo cell persistence, enhanced anti-tumor efficacy and far superior prevention of tumor growth after rechallenge. The utility of this system is two-fold: 1) as a transformative therapeutic technology creating enhanced and affordable CAR therapy for cancer care and 2) as a research tool enabling rapid development, prototyping and testing of CAR therapeutic candidates. We have assembled a focused, multidisciplinary team comprised of an expert in biomaterials and drug delivery (Brudno), an expert in viral engineering and protein production (Birnbaum), two specialists in clinical CAR cell production (Chen, Roy) and a clinician focused on CAR cell therapies (Grover). In this proposal we seek to further develop and validate MASTER scaffolds and the associated methods to make them ready for widescale utilization by the research and clinical communities, including researchers in related areas eager to work in the CAR field but deterred by the barriers to test CAR construct in vivo. Leveraging transformative preliminary data that show that the shelf-stable MASTER scaffolds outperform conventional CAR cells in preclinical mouse models of lymphoma, orthotopic pancreatic cancer, and metastatic lung and ovarian tumors this proposal will validate MASTER scaffolds with a wide range of donors and at different scales, with multiple viral vectors and CAR constructs and delineate the phenotype and function resulting from MASTER production of CAR cells. The successful completion of these aims will propel our ultimate vision of low-cost and tunable generation of CAR cells for both liquid and solid tumors and potentially beyond the oncology space.