CD19/CD22 bispecific CAR-t cell therapy for relapsed/refractory large b-cell lymphoma: A prospective, single-arm, single-center, phase 2 clinical trial

Abstract Background: Chimeric antigen receptor T-cells (CAR-T) targeting CD19 have shown promising benefits to patients of relapsed or refractory large B-cell lymphoma (R/R LBCL) with limited response to conventional chemotherapy and targeted therapies, but only one-third patients got durable remission and the resistance mechanisms were complicated. CAR-T cells targeting CD22 were found to be effective in patients who failed previous anti-CD19 CAR-T cell therapy. In this prospective study, we aimed to evaluate the efficacy and safety of CD19/CD22 bispecific CAR-T cell therapy (CAR2219) in patients with R/R LBCL. Methods: Till February 28, 2025, this ongoing phase 2 clinical trial have enrolled 31 patients with R/R LBCL, and all were evaluable for efficacy and safety profiles. All patients were treated with CAR2219, which consisted of a single-chain variable fragment (scFv) derived from the anti-CD22 monoclonal antibody fused to a second scFv derived from the anti-CD19 monoclonal antibody, followed by fusion of the scFv domains to the hingeand transmembrane domain of human CD8α, and the cytoplasmic signaling domains of human 4-1BB and CD3ζ, and parallel tEGFR expression was used to detect the expression efficiency of CAR. This bispecific CAR was encoded by a third-generation self-inactivating lentiviral vector under the control of an elongation factor 1 alpha (EF1-α) promoter. Bridging therapy was allowed at the choice of the treating physicians. A 3-day lymphodepletion (cyclophosphamide: 250 mg/m2/d; fludarabine: 25 mg/m2/d) was followed by an intravenous dose of CAR2219 (2×10e6/kg). For patients who responded to CAR2219 therapy, maintenance therapy using PD-1 inhibitors, XPO1 inhibitors, BTK inhibitors, or HDAC inhibitors were also allowed. The primary endpoint was best overall response rate (ORR) as of 3 months post-CAR-T cells infusion. The secondary endpoints included best complete response rate (CRR), progression-free survival (PFS), overall survival (OS), and adverse events (AE). We defined PFS as the time from CAR-T cells infusion to disease progression or death from any cause. Results: 14 male and 17 female patients with R/R LBCL were enrolled, with a median age of 53 years old (range 22-81), and all had undergone a median of three (range 3-7) prior lines of therapy. 23/31 patients were refractory to previous last line of therapy. The median IPI score was 4 (range 1-5) at the time of CAR-T cell therapy, and 17/31 patients had more than two extranodal sites invasion. CAR2219 was successfully manufactured and infused to all 31 patients. As of June 30, 2025, with a median follow-up of 8. 1 months after CAR-T cells infusion, the best ORR was 100%, comprising a CRR of 64. 5% and a partial response (PR) rate of 35. 5%. The median PFS and OS were not reached. The Kaplan-Meier estimates for 12-month PFS and OS rates were 60. 4% 0. 441-0. 827 and 87. 1% 0. 761-0. 997, respectively. Regarding safety, the most common AEs included neutropenia (93. 5%), thrombocytopenia (77%), and anemia (71%). Cytokine release syndrome (CRS) occurred in 74% of patients, with no grade 3 or higher events. Immune effector cell-associated neurotoxicity syndrome (ICANS) occurred in 6% of patients, with only one case of grade 3. Tocilizumab and corticosteroids were effective in managing CRS and ICANS, and no treatment-related deaths were reported. In this study, peak expansion of CAR-T cells occurred between days 10 and 14 post-infusion in 90% of patients, with a median peak CAR-T cell count of 2. 35×10⁸/L. Following CAR-T therapy, 71% of patients received maintenance therapy, which primarily included PD-1 inhibitors (73%), XPO1 inhibitors (45%), BTK inhibitors (23%), and HDAC inhibitors (23%). Conclusion: CD19/CD22 bispecific CAR-T cell therapy demonstrated impressive efficacy and a favorable safety profile in patients with R/R LBCL. This trial is still ongoing. (ClinicalTrials. gov Identifier: NCT06081478)