EAGER: Biomanufacturing: Optimizing the Isolation, Transfection, and Expansion of CAR-T Cells with Modified PES Membranes

1645225 -埃尔默白血病每年导致数千人死亡。一种名为CAR-T细胞疗法的有前途的新疗法已被证明可以有效治疗急性淋巴细胞白血病（ALL）。 在CAR-T细胞疗法中，T细胞（一种特殊类型的白色血细胞）从患者体内提取并重新编程。然后将它们重新注射到患者体内，以寻找并摧毁癌细胞。 虽然这种治疗已被证明是非常有效的，但用于提取、重编程和培养T细胞的技术非常昂贵和耗时。 该项目的目标是开发新技术，简化CAR-T细胞治疗的每一步。 第一个目标是开发新的膜，以帮助从患者的血液中分离T细胞。 然后，将优化一种有效的基因工程技术来重编程分离的T细胞。 最后，将开发加速工程化T细胞生长速度的方法。 优化这些步骤将显著降低CAR-T细胞治疗所需的成本和时间，从而使这种强大的新疗法能够帮助更多的癌症患者。 为该项目购买的设备将用于本科和研究生课程的演示，作为讨论CAR-T疗法的学习模块的一部分。 每个实验都将被记录下来，并用于制作教学视频，这些视频将在网上发布，并用于培训新生掌握先进的实验室技术。用嵌合抗原受体（CAR）重编程T细胞，使它们能够找到并摧毁癌细胞。该技术已被用于有效消除白血病患者体内的癌细胞。 然而，目前分离、转化和扩增CAR-T细胞的方法过于昂贵和耗时。该项目的目标是简化和简化T细胞生物制造的每一步。 通过开发具有固定抗体和/或其他配体的新型聚醚砜膜，可以选择性捕获幼稚和中央记忆T细胞（因为这些表型对于免疫治疗更有效），T细胞分离将得到简化。将修饰膜以分离转染的T细胞并活化T细胞。 T细胞转染将通过优化CRISPR/Cas介导的转染策略来增强，该策略不需要电穿孔并且可以在生物反应器中原位完成。CRISPR/Cas将用于整合和共表达CAR与膜结合的抗生物素蛋白，用于转染子分离。 将通过改变WAVE生物反应器中的培养条件并使用具有固定化抗CD 3和抗CD 28抗体的灌注过滤器来优化T细胞扩增以用于T细胞活化。 还将调整培养条件（细胞因子组成/浓度）以使具有幼稚/中枢记忆表型的T细胞的数量最大化（即，使效应T细胞表型最小化）。 总之，这些步骤将产生一种新型的T细胞生物制造系统，该系统将允许用户在具有附接的膜盒的生物反应器内容易地分离、分离和扩增T细胞。 该系统将被设计为比基于珠的技术具有更低的成本和更短的扩展时间。 它的封闭性和即插即用的墨盒连接可能最终允许它在医院内使用，从而避免运输，存储和其他后勤问题。 除了CAR-T细胞疗法的改进外，该项目还可能产生其他一些新的见解和进展。 例如，T细胞的转染将通过共施用先天免疫系统中几种不同蛋白质的抑制剂来增强。 这些研究的结果可以揭示哪些蛋白质对T细胞具有特异性，以及为什么CRISPR/Cas基因组编辑的效率在T细胞中相对较低。 此外，WAVE-ATF系统将用于研究剪切水平如何影响T细胞活化。 这项工作还将确定生物反应器中的培养条件是否影响T细胞分化为幼稚、中央记忆或效应记忆T细胞，这可用于最大限度地减少分化为免疫治疗不太理想的效应记忆T细胞亚群。

1645225 - ElmerLeukemia causes thousands of deaths every year. A promising new treatment called CAR-T cell therapy has been shown to effectively treat acute lymphoblastic leukemia (ALL). In CAR-T cell therapy, T cells (a special type of white blood cell) are extracted from the patient and reprogrammed. They are then re-injected into the patient to seek out and destroy cancer cells. While this treatment has been shown to be highly effective, the techniques used to extract, reprogram, and grow the T cells are prohibitively expensive and time consuming. The goal of this project is to develop new technologies that will streamline every step in CAR-T cell therapy. The first objective is to develop novel membranes to help isolate T cells from a patient's blood. Then, an efficient genetic engineering technique to reprogram the isolated T cells will be optimized. Finally, methods will be developed to accelerate the growth rate of the engineered T cells. Optimizing these steps will significantly decrease costs and time required for CAR-T cell therapy, thereby enabling this powerful new treatment to help more cancer patients. The equipment purchased for the project will be used in demonstrations for undergraduate and graduate courses as part of learning modules that discuss CAR-T therapy. Each of the experiments conducted will be recorded and used to create instructional videos that will be posted online and used to train new students on advanced laboratory techniques.Reprogramming T cells with chimeric antigen receptors (CAR) enables them to find and destroy cancer cells. This technique has been used to effectively eliminate cancer cells in leukemia patients. However, current methods to isolate, transform, and expand the CAR-T cells are prohibitively expensive and time consuming. The goal of this project is to streamline and simplify every step in T cell biomanufacturing. T cell isolation will be streamlined by developing novel polyethersulfone membranes with immobilized antibodies and/or other ligands that selectively capture naïve and central memory T cells (since these phenotypes are more effective for immunotherapy). Membranes will be modified to isolate transfected T cells and to activate T cells. T cell transfection will be enhanced by optimizing a CRISPR/Cas-mediated transfection strategy that does not require electroporation and can be done in situ in the bioreactor. CRISPR/Cas will be used to integrate and co-express the CAR with membrane-bound avidin for transfectant isolation. T cell expansion will be optimized by varying culture conditions in a WAVE bioreactor and using a perfusion filter with immobilized anti-CD3 and anti-CD28 antibodies for T cell activation. Culture conditions (cytokine composition/concentration) will also be adjusted to maximize the number of T cells with the naïve/central memory phenotype (i.e. minimizing the effector T cell phenotype). Together, these steps will yield a novel T-cell biomanufacturing system that will allow the user to easily isolate, transfect, and expand T cells inside a bioreactor with attached membrane cartridges. This system will be designed to have lower costs and shorter expansion times than bead-based technologies. Its closed nature and plug-and-play cartridge connections may eventually allow it to be used inside a hospital, thereby avoiding transportation, storage, and other logistical issues. In addition to the improvement of CAR-T cell therapy, several other new insights and advances may also come out of the project. For example, the transfection of T cells will be enhanced by co-administering inhibitors for several different proteins in the innate immune system. The results of these studies may reveal which of those proteins are specific to T cells and why the efficiency of CRISPR/Cas genome editing is relatively low in T cells. In addition, the WAVE-ATF system will be used to investigate how shear levels affect T cell activation. This work will also determine whether culture conditions in the bioreactor influence differentiation of T cells to naïve, central memory or effector memory T cells, which could be used to minimize differentiation to the effector memory T cell subset that is less desirable for immunotherapy.