EAGER: Biomanufacturing: Creating a multifunctional nanoreagent that stimulates, genetically manipulates, and selectively expands therapeutic T cells for adoptive cell therapy

过继性免疫治疗是一种新的疾病治疗选择，基于患者源性免疫（“T”）细胞，通过基因修饰靶向癌症或感染。这种方法已经在几个医学领域确立了它的潜力。但是，尽管与化疗、放疗和手术相比，这些靶向治疗具有明显的优势，但生产基因编程淋巴细胞的复杂性和成本对它们作为标准治疗的使用构成了主要障碍。这个项目通过开发微小的“纳米颗粒”来解决这个问题，这种纳米颗粒可以刺激、基因修饰和选择性地扩大治疗淋巴细胞，只需将它们添加到培养的细胞中。纳米颗粒可以反复添加到细胞培养中，直到达到所需数量的工程淋巴细胞。通过这种方法实现靶向T细胞的大规模生产，可以转化为一种实用、低成本、广泛适用的免疫疗法来治疗患者。此外，该项目将通过参与正在进行的教学和推广计划来帮助培养未来的科学家，这些计划旨在激发学生学习生物材料新发展如何影响医学的热情。目前的淋巴细胞制造实践需要各种复杂的方案来分离、基因修饰和选择性地扩增重定向细胞，然后再将它们注入患者体内。因为这些困难的程序需要专用的设备和相当多的技术专长，在美国为每个癌症患者提供这种个性化的T细胞治疗是不切实际的。这个项目通过开发微小的“纳米颗粒”来解决这个问题，这种纳米颗粒可以刺激、基因修饰和选择性地扩大治疗淋巴细胞，只需将它们添加到培养的细胞中。该项目验证了一种假设，即经过适当改造的携带dna的纳米颗粒可以有效地将肿瘤特异性嵌合抗原受体（CAR）基因输送到培养的T细胞（CAR-T细胞）中，同时通过向细胞提供编码CAR靶向的相同表面固定抗原，诱导基因修饰淋巴细胞群的选择性生长。该假设通过两个特定目标进行验证：1)设计所提出的DNA纳米载体；2)比较使用所提出的DNA纳米载体制造的CAR-T细胞与使用病毒方法和磁珠扩增的传统方法制造的CAR-T细胞的功能和治疗效果。基于纳米颗粒的方法将能够激活、设计和繁殖T细胞，而不需要特殊的仪器、设备或培训，并且可以使用与任何临床环境兼容的自动化方案来制造，并且成本只是多步骤/多试剂方法的一小部分。纳米颗粒可以反复添加到培养物中，直到达到所需的程序化T细胞百分比。它们是可生物降解和生物相容性的，扩增后的T细胞可以很容易地通过离心从游离纳米颗粒中分离出来。该平台可以很容易地适应其他治疗细胞类型的经济商业规模制造，如自然杀伤细胞、造血干细胞、间充质干细胞或B淋巴细胞，这大大拓宽了该方法在疾病治疗中的适用性。

1644363 - StephanAdoptive immunotherapy is a new disease treatment option based on patient-derived immune ("T") cells that are genetically modified to target cancer or infections. This approach has already established its potential in several medical arenas. But despite the obvious advantages afforded by these targeted therapies compared to chemotherapy, radiotherapy, and surgery, the complexity and costs of producing genetically-programmed lymphocytes pose major obstacles to their use as standard-of-care. This project addresses the problem by developing microscopic "nanoparticles" that can stimulate, genetically modify, and selectively expand therapeutic lymphocytes simply by adding them to the cells in culture. Nanoparticles can be repeatedly added to the cell culture until the required numbers of engineered lymphocytes are achieved. Implementing the large-scale manufacture of targeted T cells afforded by this approach could translate into treating patients with an immunotherapy that is practical, low-cost, and broadly-applicable. Furthermore, the project will help develop the scientists of tomorrow through its participation in ongoing teaching and outreach programs designed to generate enthusiasm in students learning how new developments in biomaterials impact medicine. Current lymphocyte manufacturing practices require an assortment of elaborate protocols to isolate, genetically modify, and selectively expand the redirected cells before infusing them back into the patient. Because these difficult procedures entail dedicated equipment and considerable technical expertise, providing this kind of personalized T cell therapy to every cancer patient in the United States is not practical. This project addresses the problem by developing microscopic "nanoparticles" that can stimulate, genetically modify, and selectively expand therapeutic lymphocytes simply by adding them to the cells in culture. The project tests the hypothesis that appropriately engineered DNA-carrying nanoparticles can efficiently shuttle tumor-specific chimeric antigen receptor (CAR) genes into cultured T cells (CAR-T cells), and at the same time induce the selective outgrowth of the genetically-modified lymphocyte population by presenting the cells with the same surface-anchored antigens that are targeted by the encoded CAR. The hypothesis is tested via two specific aims: 1) designing the proposed DNA nanocarriers and 2) comparing the functionality and therapeutic efficacy of CAR-T cells manufactured using the proposed DNA nanocarriers versus those created by the conventional approach using viral methods and magnetic bead expansion. The nanoparticle-based methods will be able to activate, engineer, and propagate T cells without special instruments, equipment, or training and could be manufactured using automated protocols that are compatible with any clinical setting, and at a fraction of the costs involved in multistep/multi-reagent methods. Nanoparticles can be repeatedly added to the culture until the required percentage of programmed T cells is achieved. They are biodegradable and biocompatible and the expanded T cells can easily be separated from free nanoparticles by centrifugation. The platform could easily be adapted to enable economical commercial-scale manufacturing of other therapeutic cell types, such as natural killer cells, hematopoietic stem cells, mesenchymal stem cells, or B lymphocytes, which substantially broadens the applicability of the approach for the treatment of disease.