Lymphodepletion-free jl-lightning CAR-T targeting CD19/CD22/BCMA achieved 100% CR in 3 Relapsed/Refractory B-NHL patients: A lymphodepletion regimen comparison study

Abstract Background: Broad clinical application of CAR-T therapy has been constrained by several barriers: (1) prolonged manufacturing process (≥ 2 weeks), delaying critical treatment access; (2) single-target antigen escape, enabling tumor relapse; and (3) lymphodepletion (LD)-related toxicities, excluding medical fragile patients. To overcome these limitations, we have developed novel CAR-T cells that achieved the following triple breakthroughs:Ultra-rapid 6-hour manufacturing using the JL-Lightning non-viral platformTri-specific targeting of CD19/CD22/BCMAA LD-free administration paradigm This integrated strategy enhances the efficacy by leveraging the proliferative capacity of JL-Lightning CAR-T while eliminating LD toxicities. The clinical safety and efficacy of these rapidly manufactured CAR-T cells were evaluated in patients with relapsed/refractory B-cell non-Hodgkin's lymphoma (rrB-NHL) and lymphodepletion (LD) and non-lymphodepletion (NLD) regimens were compared. Methods: This single-center, open-label exploratory study enrolled patients into two cohorts, in which patients were treated with LD ( Flu/Cy 30/300 for 3 days) or without NLD. Patients in the LD cohort received a single infusion of 5×10⁴- 2×10⁵ CAR-T cells/kg, while patients in the NLD cohort were treated with an infusion of 2×10⁵ - 5×10⁵ CAR-T cells/kg after leukapheresis. The study was designed to evaluate safety, tolerability, maximum tolerated dose (MTD), preliminary efficacy and pharmacokinetics (PK) / pharmacodynamics (PD). Results: By the data cut-off of Jul. 29, 2025, total 8 rrB-NHL patients were enrolled (LD n=5, NLD n=3). In the LD cohort, 5 patients (LBCL=4, follicular lymphoma FL=1) had a median baseline tumor volume SPD of 657.62 mm2 (range 213.28–11256.73). In the NLD cohort, the median baseline SPD was 2280.86 mm2 (range 492.28–2702.91) in 3 patients (LBCL=3). All patients achieved an objective response (ORR 100%, 8/8) with an overall complete response rate (CRR) of 87.5% (7/8). In the LD cohort, the CRR was 80.0% (4/5) with a median follow-up of 6.8 months (range 2.3-8.6). In the NLD cohort, all 3 patients achieved a complete response (CRR 100%, 3/3) with a median follow-up of 3 months (range 2.2-4.3). The median progression-free survival (PFS), overall survival (OS), and duration of response (DOR) have not yet been reached. All patients are ongoing to be closely monitored for both safety and efficacy. Regarding pharmacokinetics, tri-targeting CAR-T demonstrated robust proliferative capacity with a 1/20-1/5 of conventional CAR-T dosage. For the LD cohort, the median Cmax was 2315 cells/µL (range: 1051-10974), and Tmax was 14 days (range: 11-14). CAR-T cells were still detectable for over 6 months in some patients. For the NLD cohort, the Tmax was also 14 days (range: 11-14) and the median Cmax was 1101 cells/µL (range: 366-4543). In this study, no grade 3 or higher level of cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) occurred, and no dose-limiting toxicities (DLTs) was observed. In the LD cohort, all 5 patients experienced grade 1 CRS, and 2/5 had grade 1-2 ICANS. In the NLD cohort, 2/3 patients experienced grade 1 CRS, and 1/3 had grade 2 ICANS. In the LD cohort, grade≥3 hematologic toxicities including neutropenia (4/5), thrombocytopenia (2/5), and anemia (1/5) were observed. Two patients (2/5) experienced infection with one patient having a grade 3 event. In contrast, the NLD cohort did not report any grade≥3 hematologic toxicities or infection. Non-lymphodepletion treatment showed significant advantages in reduced toxicity and infection. Conclusions: The tri-targeting CD19/CD22/BCMA CAR-T cells rapidly manufactured within 6 hours through the JL-Lightning-CAR-T platform demonstrated high efficacy and favorable tolerability in rrB-NHL patients. The non-lymphodepletion treatment showed significant advantages in reduced toxicity and infections, while maintained robust antitumor efficacy. These findings highlight the clinical applicability of the 6-hour rapid CAR-T manufactory platform, particularly in the non-lymphodepletion setting, to improve patient outcomes for rrB-NHL and other B-cell-related diseases. (ClinicalTrials.gov ID: NCT06446128)