Nanobody-based CD19/CD20 bispecific CAR-T achieves durable remissions in r/r NHL patients

Abstract Background: While CD19-targeting chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in relapsed/refractory non-Hodgkin lymphoma (r/r NHL), high relapse rates and CD19 antigen escape remain major clinical challenges. Given that CD19 loss represents a well-characterized resistance mechanism, we developed a structure-optimized CD19/CD20 bispecific CAR-T cell (SL1716) to improve clinical outcomes and reduce relapse risk in r/r NHL patients. Objective This study aims to develop and validate a structurally optimized bispecific nanobody-based CD19/CD20 CAR-T construct (SL1716) through comprehensive in vitro functional screening, and evaluate its safety and preliminary efficacy in the patients with r/r NHL. Methods We engineered an optimized bispecific CAR construct and systematically characterized its functional advantages, including dual-target synergy, persistence, and memory phenotype. The lead construct was evaluated in an investigator-initiated trial (IIT) involving heavily pretreated r/r B-NHL patients who were refractory to or relapsed after prior therapies. The primary objective was to assess the incidence and severity of treatment-emergent adverse events (per CTCAE v5.0 criteria), with secondary objectives including overall response rate (ORR; Lugano 2014 criteria). Comprehensive assessments were conducted, including clinical responses, adverse events, and CAR-T cell expansion kinetics. Results Using our alpaca immunization and phage display platform, we identified nanobodies specific to novel epitopes on CD19 and CD20. Through systematic optimization of each CAR module, we developed SL1716 as the lead bispecific CAR-T candidate. Functional characterization demonstrated its superior synergistic efficacy, as evidenced by dual-target synergistic cytotoxicity compared to monospecific CAR-Ts, along with robust antitumor activity across various lymphoma subtypes. Notably, SL1716 indicated sustained cytotoxic potency and an enhanced memory phenotype in multi-cycle antigen rechallenge assays. Between August 2024 and May 2025, 10 eligible patients with r/r B-NHL received SL1716 infusion, including 7 (70%) with DLBCL, 2 (20%) with MCL, and 1 (10%) with PMLBCL. The cohort had a median age of 55 years (range: 19-70), with 5 female (50%) and 5 male (50%) patients. All enrolled patients had disease progression after ≥1 prior line of therapy (median 4.5 lines, range 1-11) with ECOG scores of 1-2. Disease staging revealed 6 (60%) with stage IV, 1 (10%) with stage III, and 3 (30%) with bone marrow involvement, none with CNS infiltration. Among treatment-refractory cases, 3 had prior CAR-T exposure, including 1 case of CD19/CD22 CAR-T and 2 case of CD19 CAR-T therapy, all with subsequent relapse. Following lymphodepletion, all patients received SL1716 (CD19/CD20 bispecific CAR-T) at 4×106 cells/kg, with 100% manufacturing success rate. SL1716 exhibited excellent tolerability profile, with grade Ⅰ cytokine release syndrome (CRS) observed in 9 patients (90%) and no CRS in 1 patient (10%). Importantly, no immune effector cell-associated neurotoxicity syndrome (ICANS) events were documented during the observation period. Pharmacokinetic evaluation demonstrated favorable expansion kinetics, achieving a median peak CAR copy number (Cmax) of 4.15×104 copies/μg DNA (range: 7.23×103-2.05×105) at a median time to peak (Tmax) of 12 days post-infusion (range: 7-14 days). As of the data cut-off date (July 30, 2025), the median follow-up time was 6.5 months (range 2.03–11.7). A best overall response rate (ORR) of 90% was observed, with 6 patients achieving complete remission (CR; 60%) and 3 patients achieving partial remission (PR; 30%). By the 3-month post-infusion assessment, SL1716 achieved an ORR of 87.5% among 8 evaluable patients, with 5 patients (62.5%) achieving CR and 2 patients (25%) achieving PR. One patient experienced progressive disease (PD; 12.5%). Notably, all treated patients exhibited a clinical response to SL1716, with extended follow-up data currently being collected. Conclusion SL1716, a nanobody-based CD19/CD20 bispecific CAR-T cell therapy, demonstrates a favorable safety profile and durable clinical efficacy in patients with r/r NHL. These promising results support its potential as an effective treatment option for this high-risk patient population.