Presentation description

B-cell Acute Lymphoblastic Leukemia (B-ALL) is a blood cancer marked by the uncontrolled growth of immature B cells, which commonly express the surface antigen CD19. While standard chemotherapy has improved survival rates, outcomes for relapsed or refractory cases remain poor. (Chimeric antigen receptor) CAR-T cell therapy, a treatment that involves genetically engineering a patient's T cells to recognize and kill cancer cells, has shown great promise for B-ALL. Notably, Kymriah, the first FDA-approved CAR-T product, achieved high remission rates in patients with otherwise treatment-resistant disease.
Our lab is focusing on engineering the next generation of CAR T cell therapies to further improve outcomes, such as developing a synNOTCH CAR T for B-ALL. In order to detect low expression of the synNOTCH receptor, an N-terminal c-myc tag is used during flow cytometry. To aid in this effort, we built a positive control CAR by reconstructing the Kymriah anti-CD19 4-1BBζ second-generation CAR to include the same N-terminal c-myc tag used in our synNOTCH designs. This allows direct comparison of expression and detection across systems using flow cytometry.
To engineer the tagged anti-CD19 CAR, we subcloned the antiCD19 scFv, appended the N terminal c-myc tag, and assembled it into a 4-1BBζ CAR lentiviral vector backbone to enable stable transduction into human T cells. Standard molecular biology techniques such as restriction enzyme digestion, PCR, ligation, and bacterial transformation, were used to assemble the construct. Flow cytometry confirmed robust detection of the c-myc tag in T cells, demonstrating the construct's value as a positive control for synNOTCH experiments. Additionally, the CAR retained strong cytotoxic activity in killing CD19+ cancer cells in vitro.
This work reinforces core principles of CAR design and molecular cloning, while providing us with critical skills and experience to advance engineered T cell therapies for hematologic cancers.