Leukemia 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.
