Targeting Caspase-1 to Reduce CAR T-Cell Apoptosis and Tumor Cell Pyroptosis to Enhance the Therapeutic Index of CAR T-Cell Therapy in Hematological Malignancies
Year: 2025
Type: article
Abstract: Chimeric antigen receptor (CAR) T-cell therapy has transformed hematological cancer treatment but faces limits due to durable response rates and toxicities like cytokine release syndrome (CRS). We reported that modulating CD19 antigen with monoclonal antibodies reduced CAR T apoptosis and tumor pyroptosis , improving safety and efficacy (PMID: 37879074 ). We hypothesize that reducing tumor pyroptosis is critical to enhancing CAR T safety and durability. To test this hypothesis, we explored caspase inhibition to regulate tumor pyroptosis . Assessing Casp1, Casp3, Casp4, Casp7, and Casp11 inhibitors with CAR T, we found Casp1/4 inhibition didn't affect cytotoxicity, leading us to focus on Casp1/4 inhibition to reduce CAR T apoptosis and tumor pyroptosis . We evaluated CART19 proliferation after CD19 + JeKo stimulation with Casp1/4 inhibitor VX765 . VX765-treated CAR T showed increased proliferation at day 5 (p=0.03), reduced apoptosis, lower pyroptosis, and higher tumor apoptosis (Figure 1A). VX765-treated CAR T had elevated Bcl2 expression (p=0.03). Tumor pyroptosis, measured by HMGB-1, was significantly reduced by VX765 (Figure 1B). We then assessed the impact of Casp1/4 inhibition in an in vitro model of CAR T exhaustion. We stimulated CAR T with CD19 + JeKo every other day for 2 weeks and then assessed CCR7 and CD45RA expression on CAR T. VX765-treated CAR T showed higher central memory and naïve and lower terminally differentiated effector memory phenotypes (Figure 2A-B). We next studied the effect of Casp1/4 inhibition in vivo using an immunocompetent mouse model to mimic CAR T-cell toxicities, including CRS. We generated murine CART19 (mCART19). C57B6 mice were engrafted with luciferase + CD19 + TBL12 lymphoma cells . Mice were treated 12 days later with cyclophosphamide (300 mg/kg, ip) and randomized to receive: 1) untransduced T cell (UTD), 2) mCART19 + vehcile, or 3) mCART19 + VX765 (25 mg/kg). VX765 was given before CAR T injection and on day 2 post-injection. Mice were monitored for CAR T toxicity and tumor burden via weekly bioluminescent imaging. Vehicle-treated mice lost weight significantly (p=0.02), while VX765-treated mice maintained weight and showed no CRS signs. Flow cytometry revealed reduced CAR T apoptosis (p=0.04) and tumor pyroptosis (p=0.03) in VX765-treated mice. Western blot showed lower cleaved GSDME and GSDMD in these mice, with prolonged CAR T persistence and durable anti-tumor activity. In summary, Casp1/4 inhibition significantly reduced CAR T apoptosis, improved memory phenotype, and increased delayed CAR T proliferation. VX765 also reduced tumor pyroptosis and CRS incidence without impacting CAR T anti-tumor activity in vivo . Given the favorable preclinical outcomes and existing clinical data on Casp1/4 inhibition, this approach holds substantial translational potential for enhancing CAR T-cell therapy in large B-cell lymphoma.
Authors: R. Leo Sakemura, Wannakorn Khopanlert, Truc N Huynh, Brooke L Kimball, Grace DeFranco, Sophia Goldberg, Dominic Skeele, Jennifer Feigin, Olivia Sirpilla, Carli M. Stewart, Kun Yun, Claudia Manriquez Roman, Ismail Can, Omar Gutierrez Ruiz, Ekene J. Ogbodo, Long Mai, Hong Xia, Mehrdad Hefazi, Elizabeth L. Siegler, Saad S. Kenderian
Institution: Mayo Clinic
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $3,650
OpenAlex ID: W4407977415