Enhanced efficacy and safety of optimized tandem CD19/CD20 CAR T-cell Therapy in refractory/relapsed B-cell lymphoma

Abstract Background Relapse after CD19-directed CAR T-cell therapy remains a challenge in relapsed/refractory (r/r) B-cell lymphoma (BCL). While tandem CD19/CD20 CAR-T cells show promise, we observed limitations in the functionality and persistence of a second-generation construct (CAR2019). To address this, we engineered an optimized tandem CAR (CAR2019(AS)) by inserting specific protease cleavage sites (AS) between the scFv and hinge regions. This study evaluated the preclinical and clinical safety and efficacy of CAR2019(AS). Methods Preclinically, CAR2019(AS) and CAR2019 were compared for cytotoxicity against Raji cells, tumor control in Raji-LUC-bearing NSG mice, and persistence during repeated tumor stimulation. Clinically, a multicenter, phase I trial (NCT05388695) enrolled 53 r/r BCL patients across three centers. Patients received CAR2019(AS) at dose levels of 2×106 (n=8), 4×106 (n=25), or 6×106 (n=23) cells/kg. Results Preclinically, CAR2019(AS) demonstrated superior cytotoxicity, tumor control in vivo, and resistance to exhaustion, maintaining potent activity and eradicating tumors over 5 rounds of stimulation, unlike CAR2019 which failed by round 4. Clinically, 52 patients were evaluable for efficacy (median age 47 years; 96% stage III/IV; 89% ≥3 prior lines). Best overall response rate (ORR) was 84.6% (44/52), with a complete response rate (CRR) of 53.8% (28/52). With a median follow-up of 13.9 months, median progression-free survival (PFS) and overall survival (OS) were not reached. Estimated 2-year PFS and OS rates were 69.2% (95% CI, 53.8-80.4%) and 76.2% (95% CI, 61.8-85.8%), respectively. Exploratory analysis suggested a higher CRR with ASCT consolidation post-CAR2019(AS) (65.2% vs 44.8%). Grade ≥3 cytokine release syndrome and neurotoxicity occurred in 2% and 0% of patients, respectively. Common grade ≥3 adverse events within 1 month were neutropenia (100%), thrombocytopenia (62%), and infection (25%). CAR2019(AS) exhibited robust expansion (median peak: 48486.9 copies/μg gDNA on day 10). Conclusions Our novel incorporation of protease cleavage sites (AS) significantly enhances tandem CD19/CD20 CAR-T functionality and exhaustion resistance. CAR2019(AS) achieves exceptional clinical efficacy with promising survival in r/r BCL, establishing a potent dual-targeting strategy to overcome CD19 monotherapy limitations.