Efficacy and Safety of Interleukin-6-Knockdown CD19-Targeted CAR T Cells(ssCART-19) for Relapsed/Refractory B-ALL

Background： Severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are significant challenges in the use of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy for patients with relapsed or refractory B-type acute lymphoblastic leukemia (r/r B-ALL). These complications limit the widespread adoption of CAR-T therapy. To address this issue, we developed a novel approach using anti-CD19 CAR T-cells incorporating a small hairpin RNA (shRNA) element to silence the interleukin-6 (IL-6) gene (ssCART-19). By reducing IL-6 expression levels in ssCART-19 cells, we aimed to decrease monocyte activation and pro-inflammatory cytokine release, thereby mitigating the incidence of severe CRS and neurotoxicity. In this phase 1/2 clinical trial conducted at two centers, we compared the efficacy and safety of ssCART-19 to classical CART-19 cells (cCART-19) in patients with r/r B-ALL (NCT03919240). Methods： Peripheral blood mononuclear cells were collected from patients through leukapheresis. ssCART-19 cells were generated by transducing anti-CD19 CAR with a shRNA-IL-6 gene silencing element into bulk peripheral T lymphocytes using a lentivirus vector. In contrast, cCART-19 cells were only transduced with an anti-CD19 CAR. Both cell types contained 4-1BB and CD3ζ co-stimulatory domains. Lymphodepletion (fludarabine at 30 mg/m 2/day and cyclophosphamide at 300 mg/m 2/day) was conducted on days -5, -4, and -3 before infusion. Each patient received a dose of 5×10 6CAR +/kg. The primary endpoint was safety, with efficacy as the secondary endpoint for both ssCART-19 and cCART-19. Results: A total of 121 patients who had received at least two prior lines of treatment were screened for this study. A total of 47 patients received ssCART-19 and 40 patients received cCART-19 were included in the analysis. The median patient age of group ssCART-19 and group cCART-19 was 33(9-64) years and 24(2-68) years. At enrollment, ssCART-19 patients and cCART-19 patients had a median percentage of BM blasts of 4.0(0.01-86.0) % and 4.0(0.02-98.0) % by flow cytometry. ssCART-19 patients and cCART-19 patients had a median of 3 (2-8) and 4（2-9）prior lines of therapy(table 1). The transfection efficiency was 44.66% (range: 13.70-84.66%) for ssCART-19 and 39.98% (range: 14.0-65.0%) for cCART-19. Adverse events within 28 days included grade 3-4 neutropenia in 14 patients (29.79%), thrombocytopenia in 17 patients (36.17%), grade 1-2 CRS in 25 patients (53.20%), grade 3-4 CRS in 7 patients (14.89%), and grade 1 ICANS in 2 patients (4.26%) in the ssCART-19 group. In the cCART-19 group, adverse events within 28 days included grade 3-4 neutropenia in 20 patients (50%), thrombocytopenia in 18 patients (45%), grade 1-2 CRS in 19 patients (47.5%), grade 3-4 CRS in 15 patients (37.5%), grade 1-2 ICANS in 4 patients (10%), and grade 3 ICANS in 2 patients (5%). The incidence of neutropenia, thrombocytopenia, grade 3-4 CRS, and ICANS in the ssCART-19 group was significantly lower than that in the cCART-19 group (p < 0.05)(Table 2). Furthermore, the peak levels of IL-6, IL-2 and TNF-α were significantly lower in the ssCART-19 group compared to the cCART-19 group (p < 0.05)(Fig.1). No significant differences were observed between the two groups in terms of the start time, peak value, duration and killing efficacy of CART cells (p > 0.05)(Fig.2). As of the data cutoff on December 25, 2020, the median follow-up time was 12 months (range: 0.5-41.33). On day 28, with 91.49% of patients achieving complete remission (CR) or CR with incomplete hematological recovery (CRi) in ssCART-19, compared to 85% in the cCART-19 group. Median overall survival (OS) was not available for ssCART-19, while it was 32.93 months for cCART-19 (p=0.575). The median progression-free survival (PFS) was 14.17 months for ssCART-19 and 15.33 months for cCART-19 (p=0.339). Although no significant differences were observed in terms of 3-year OS and PFS, the 3-month PFS was higher in the ssCART-19 group compared to the cCART-19 group (82.3% vs 66.9%, p=0.045)(Fig.3). Conclusion： Our clinical studies demonstrated the safety and high efficacy of ssCART-19 with IL-6 gene silencing in patients with relapsed/refractory B-ALL. These findings support the potential of ssCART-19 as a promising therapeutic approach for this challenging patient population.