318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy

• Published in: Journal of clinical and translational science Vol. 6; no. s1; p. 57
• Main Authors: Hong, Yeonsun, Kim, Hye-Ran, Walling, Brandon L., Lozada, John, Amitrano, Andrea M., Sailer, Cooper J., Lim, Kihong, Mongre, Raj Kumar, Kim, Kyun-Do, Capece, Tara, Lomakina, Elena B., Reilly, Nicholas S., Waugh, Richard E., Reagan, Patrick M., Kim, Minsoo, Oakes, Patrick W., Jun, Chang-Duk
• Format: Journal Article
• Language: English
• Published: Cambridge Cambridge University Press 01-04-2022
• Subjects: Cancer; Cancer therapies; Cell culture; Cell migration; Chemokines; CXCL12 protein; Cytokines; Cytoskeleton; Intercellular adhesion molecule 1; Lymphocytes; Lymphocytes T; Lysates; Melanoma; Neurotoxicity; Population studies; Precision Medicine/Health; Protein B; Proteins; Side effects; Spectrin; Spleen; Transfection; Valued Approaches
• ISSN: 2059-8661; 2059-8661
• DOI: 10.1017/cts.2022.177

OBJECTIVES/GOALS: #NAME? METHODS/STUDY POPULATION: Cell culture & protein identification: human T cells were purified from healthy blood, then activated & cultured for 5d. CAR-T cells were collected from infusion bags of cancer patients undergoing CAR-T. Silver staining of naive & activated healthy T-cell lysates was compared; B-II spectrin was upregulated and confirmed by Western blot. Migration assays: naive & activated T-cells were imaged during migration on ICAM-1 and ICAM-1 + CXCL12 coated plates. T-cells were transfected with BII-spectrin cDNA & the chemokine dependence of migration was compared with controls. In-vivo studies: in a melanoma mouse model, BII-spectrin transfected or control T-cells were injected; tumors were followed with serial imaging. Human patient records were examined to correlate endogenous BII-spectrin levels and CAR-T response. RESULTS/ANTICIPATED RESULTS: Activated T-cells downregulate the cytoskeletal protein B-II spectrin compared to naive cells, leading to chemokine-independent migration in in vitro assays and off-target trafficking when CAR-T cells are given in vivo. Restoration of B-II spectrin levels via transfection restores chemokine-dependence of activated T-cells. In a mouse melanoma model, control mice injected with standard activated T-cells showed fewer cells in the tumor site and more cells in the off-target organs (spleen, lungs) when compared to mice injected with B-II spectrin transfected cells. Furthermore, among 3 human patients undergoing CAR-T therapy, those with higher endogenous B-II spectrin levels experienced fewer side-effects, measured by the neurotoxicity and cytokine release syndrome grades. DISCUSSION/SIGNIFICANCE: A major hurdle to widespread CAR-T therapy for cancer is significant, often fatal side-effects. Our work shows that the protein B-II spectrin is downregulated during CAR-T production, and that restoring B-II spectrin levels decreases side-effects while increasing tumor clearance--hopefully translating to better CAR-T regimens for the future.