BackgroundCAR-T cell therapy has demonstrated significant efficacy in B-cell malignancies, but relapse due to target antigen loss remains a challenge. To address antigen escape and expression heterogeneity, we developed four CD19/CD22 CAR constructs with different configurations, designated as TanCAR-T1, TanCAR-T2, LoopCAR-T1, and LoopCAR-T2. Systematic functional screening identified LoopCAR-T1 as the optimal construct, exhibiting superior cytotoxicity, cytokine secretion, and proliferative capacity. This structure was selected for clinical investigation to evaluate its efficacy and safety in refractory/relapsed B-cell non-Hodgkin lymphoma (R/R B-NHL) treatment.MethodsThis clinical study is an open-label, multicenter investigator-initiated trial (IIT, NCT07093086). After lymphodepleting chemotherapy, patients received the infusion of CD19/22 dual-targeting CAR-T cells (dose range: 5×106to 1.0×107 CAR-T cells per kilogram of body weight). The primary objective was to evaluate safety and preliminary efficacy of the dual-targeting CD19/22 CAR-T therapy. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to ASTCT 2019 criteria. Efficacy was assessed according to Lugano 2014 criteria. Secondary objectives included pharmacodynamics and pharmacokinetic evaluation.ResultsFrom December 11, 2024, to June 30, 2025, a total of 18 patients received the novel loop-structured CD19/CD22 dual-targeting CAR-T therapy. The median age was 56 years (range: 40-75), with an equal gender distribution (9 males, 9 females). Histologically, diffuse large B-cell lymphoma (DLBCL) predominated (15 cases, 83%), with one case each of high-grade B-cell lymphoma (HGBL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL). 11 patients (61%) had Non-GCB subtype. All patients had stage III/IV disease, with 17 (94%) at stage IV, and 12 (67%) presenting B symptoms. 13 patients (72%) had an ECOG score of 1, while 5 (28%) scored ≥2. 11 patients (61%) had an IPI score ≥3, and 11 (61%) exhibited Ki-67 ≥75%. 7 patients (39%) were double-expressor, 2 (11%) had double/triple-hit genetics, and 9 (50%) had TP53 mutations. The median prior lines of therapy were 3 (range: 2-5), with 9 patients (50%) having received ≥3 lines. 3 patients (17%) had undergone autologous transplantation, and 1 (6%) had prior CD19 CAR-T treatment failure. 6 patients (89%) had extranodal involvement, including 6 (33%) with central nervous system (CNS) involvement, and 8 (44%) with ≥2 extranodal sites. 16 patients (89%) were refractory and 10 (56%) were relapsed. 15 patients (83%) received bridging therapy after leukapheresis for this dual-targeting CAR-T therapy.CRS occurred in 67% (12/18) of patients receiving CD19/22 dual-targeting CAR-T therapy, with only one case (6%) of grade 3 CRS that resolved with supportive management. Notably, no ICANS events were observed, including in all 6 patients with CNS involvement. No CAR-T-related deaths were reported.The best overall response rate (BOR) was 83% (15/18), with 67% (12/18) of patients achieving complete remission (CR) and 17% (3/18) achieving partial remission (PR). Notably, among the 6 patients with CNS involvement, the BOR remained high at 83% (5/6), with all responders (100%) achieving CR.Among all 18 patients, the median Tmax was 14 days (range: 7-21), with a median Cmax of 91,850 copies/μg DNA (range: 2,780-1,550,000) and median AUC0-28 of 738,571 (range: 32,191-17,675,117). Notably, the 3 non-responders still demonstrated robust CAR-T expansion, with a median Cmax of 210,000 copies/μg DNA (range: 2,780-370,000) and median AUC0-28 of 1,841,080 (range: 32,191-2,965,788). This observation suggests that adequate CAR-T expansion does not necessarily correlate with clinical response, highlighting potential mechanisms of resistance independent of pharmacokinetic factors.ConclusionThe novel loop-structured CD19/CD22 dual-targeting CAR-T cells demonstrate favorable safety and efficacy, offering a promising therapeutic option for refractory/relapsed B-cell non-Hodgkin lymphoma. Larger cohorts and longer follow-up are warranted to validate these findings and assess long-term outcomes.

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2025
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