Abstract
The focus of this research project was to develop assays for investigating the retention of CAR-T cells in diffuse large B-cell lymphoma patients. Diffuse large B-cell lymphoma (DLBCL) is a type of cancer found in blood-forming tissue. Around 40% of DLBCL patients experience relapse or refractory disease (R/R) using the current standard treatment. In recent years, many clinical trials have begun investigating the use of CAR-T cell therapies on cancer patients suffering from R/R disease. This project continued the investigation efforts conducted by the UC Davis Alpha Stem Cell Clinic to increase the affordability of CAR-T cell therapies to DLBCL patients. For these treatments to be approved by the FDA, and later become accessible to the public, the retention of the CAR-T cell treatment within the patient’s body must first be identified. Our primary goal was to develop assays for determining the retention of CAR-T cells in DLBCL patients after treatment. To indirectly determine the retention of CAR-T cells over time, we checked for the presence of the CAR transgene in blood samples using real-time PCR (RT-PCR). A standard curve of CAR transgene detection using RT-PCR analysis was created, showing the frequency of the CAR transgene present in the samples. Our results indicated that this method does not have the sensitivity to distinguish between a lack of the CAR transgene present and very low amounts of the CAR transgene present. To directly determine the retention of CAR-T cells over time, we measured the percentage of CAR-T cells present in blood samples using flow cytometry. Like the RT-PCR analysis, a standard curve of CAR-T cell detection using flow cytometry was created, showing the frequency of CAR-T cells present in the samples. Once again, our results indicated that this method does not have the sensitivity to detect CAR-T cells in a sample mixed with unmodified peripheral blood mononuclear cells. To determine the amount of various T-cell subsets present in a blood sample before and after CAR-T cell therapy, we quantified the amount of various T-cell subsets present in the blood sample using flow cytometry. Further optimization of the 11-color flow cytometry panel is required to allow for the differentiation of naïve T-cells, effector T-cells, and memory T-cells between CD8+ and CD4+ subpopulations. In recent years, CAR-T cell therapies have shown to be successful for DLBCL patients. Patients with R/R aggressive B cell lymphomas have shown a 40%-54% complete response rate to CAR-T cell therapy. However, with steep prices, this form of treatment remains inaccessible to many. The work to release a more affordable CAR-T cell therapy for patients with DLBCL must continue.