According to the inclusion/exclusion criterion of two clinical trials (ChiCTR-ONN-16009862 and ChiCTR1800015164), we conducted a retrospective analysis of 61 eligible patients with R/R B-ALL from April 2020 and October 2023. The exclusion criteria for follow-up were as follows (1): patients progressed before infusion (2); the follow-up time less than 28 days (3); the related data lost. In the end, 16 patients were excluded and 45 patients were enrolled in this study, 17 patients of which were treated with tocilizumab infusion ( Figure 1 ).

This study protocol was reviewed and approved by the Ethics Committee of Tianjin First Central Hospital and was registered for clinical trials in the China Clinical Trial Registration Center. All participations signed written informed consent before enrollment.

The peripheral blood mononuclear cells were obtained from patients or healthy donors. CD3+ T cells were isolated by magnetic beads and cultured them in culture medium which contains CD3/CD28 stimulating beads. After activation, we transduce lentiviral vector including CD19 CAR into these cells. The CD19 CAR contained a 4-1BB costimulatory domain and a CD3-ζ signaling domain constructed as previously described (20). Finally, CAR-T cells were expanded and evaluated for transduction efficiency (approximately 50%). Prior to infusion (Day 0), all patients received lymphodepleting chemotherapies. (300mg/m²/d cyclophosphamide and 30mg/m²/d fludarabine for 3 to 2 days) (21).

CRS is the main toxicity observed in B-ALL patients treated with CAR-T cells. We graded CRS according to the ASTCT consensus (the grading criteria is detailed in Table 1 ). We classified it into mild CRS (grade 1-2 CRS) and severe CRS (grade 3-4 CRS). In addition to symptomatic treatments such as oxygen therapy, antipyretics and vasopressors, tocilizumab and corticosteroid are common drugs used in the treatment of CRS. We regarded fever that could not be explained by other causes and the increasing of cytokines as the beginning of CRS. However, the optimal timing of tocilizumab is not yet clear, and it is most commonly recommended for patients with symptoms such as refractory or persistent hypotension, hypoxia, etc. (22) In our study, tocilizumab or corticosteroid is given to patients as determined by the physicians, within 12 hours of the appearance of symptoms.

ICANS is the second observed side effect in R/R B-ALL patients treated with CD19 CAR-T. We recorded the different ICANS symptoms and the exact time of symptom appearance, then graded ICANS according to the ASTCT consensus. We also focused on the occurrence of hematological toxicity. In addition, impairment of liver and kidney function was also observed in a small percentage of patients, which were also recorded and evaluated. Finally, the infection of these patients was diagnosed based on clinical presentation and etiological examination.

Response to CAR-T cells was assessed by bone marrow morphological analysis, flow cytometry, and genetic testing on day 28. Complete response (CR) was defined as <5% bone marrow blasts in morphology regardless of cell count recovery. And it was further classified into MRD+CR or MRD-CR based on minimal residual disease (MRD). The efficacy of tocilizumab or corticosteroid was assessed by serial monitoring of temperature, ferritin, C-reactive protein (CRP) and cytokines (including IL-2, IL-6, IL-10, TNF-α (tumor necrosis factor-α), etc). The end of CRS was considered to be the disappearance of fever and a decrease in cytokines. Finally, we evaluated the progression-free survival (PFS) and overall survival (OS) of these two groups of patients (toci group and non-toci group).

The characteristics of patients were analyzed using descriptive statistics. All measurement data were described in terms of median and range and compared by using t tests or Mann-Whitney tests. Enumeration data were described as frequencies (%) and compared using chi-squared tests or Fisher’s exact tests. Statistical analyses were performed using the SPSS v26.0 software (Chicago, IL, USA) and the GraphPad Prism v9 software (GraphPad, La Jolla, CA, USA).

From April 2020 to October 2023, a total of 61 patients with R/R B-ALL were proposed to receive CAR-T cells. Among them,1 case experienced disease progression before receiving CAR-T therapy, eventually 60 patients received CAR-T therapy. Among these 60 cases, excluding 4 cases with follow-up time less than 28 days, and 11 cases with relevant data loss, finally 45 cases were included in our retrospective study. Baseline characteristics of all 45 patients and the subgroup analysis of 2 groups (toci group and non-toci group) were summarized ( Table 2 ). Of the total 45 patients, 53.3% (24/45) were male, the median age was 36 years (range 8–67), and 84.8% (38/45) were high-risk phenotype or genotypes. The median number of therapy lines before CAR-T cell infusion was 2 (range, 1–3). As shown in the table, the two groups were very similar in terms of gender, genotypic risk level, previous treatments and dose of CAR-T cells. The differences were not statistically significant. However, the toci group had a higher tumor load, CRS grade and dose of corticosteroid with significant statistical difference. We also performed a logistic regression analysis of these potential factors ( Supplementary Table S4 ). We found that none of the factors were statistically significant. In this study, there was insufficient evidence of an association between these factors and the use of tocilizumab in treatment.

All 45 patients received CD19 CAR-T cells. Of the total 45 patients, 88.9% (N=40) achieved MRD-CR, 8.9% (N=4) achieved MRD+CR, and 2.2% (N=1) achieved partial remission (PR) ( Figure 2A ). After infusion, 91.1% (41/45) of patients had various grades of CRS, including 7 patients in grade 1, 19 patients in grade 2, and 15 patients in grade 3. There were no grade 4 CRS events. The median duration of CRS in patients in toci group was 6.1 (range, 2-14) days. In comparison, the median duration in non-toci group was 5.5 (range, 0-12) days. And ICANS occurred in two patients (4.5%). One patient developed grade 3 ICANS characterized by syncope and vision decrease that lasted 7 days.

Tocilizumab was applied to 17 patients, including 8 patients with grade 2 CRS and 9 patients with grade 3 CRS. The administration of tocilizumab was mostly within 12 hours after the onset of CRS-related symptoms, and a repeated dose of tocilizumab was given if the symptoms were not significantly relieved 8 hours after infusion. One to six doses of tocilizumab were used to suppress CRS with median dose of 480 (range, 160–800) mg. The dosing range for tocilizumab was 24-48 hours. Corticosteroids were used in 25 patients, and the cumulative methylprednisolone-equivalent corticosteroid dose was 100 mg (range, 7.5–302.5). A total of 10 patients in the non-toci group were treated with corticosteroid only, including 1 patient with grade 1 CRS, 3 patients with grade 2 CRS and 6 patients with grade 3 CRS. There were no cases of ICANS, nor any instances of grade 4 CRS or higher. In combination with the clinical symptoms, tocilizumab was not chosen due to the physician’s decision and the financial constraints of a part of the patients.

Clinical symptoms of CRS in patients after CAR-T cells infusion include unexplained fever, hypotension and hypoxemia. These symptoms are the main basis for grading according to ASTCT criteria. Accordingly, the clinical presentation of the patients was documented and the duration of CRS was calculated, with particular attention to the toci group. For grade 3 CRS, we found that the duration in the toci group was significantly shorter than non-toci group, and the difference was statistically significant. But there was no significant difference in duration between the two groups in grade 1-2 CRS ( Figure 2B ).

The serum cytokine levels were correlated with CRS levels, so we continuously monitored changes of serum cytokine levels for 17 patients in the toci group. ( Figure 3 ) 58.8% (10/17) patients exhibited a reduction in cytokine levels following tocilizumab treatment. Meanwhile, their clinical symptoms associated with CRS were markedly alleviated. We consider that this illustrates a substantial therapeutic role of tocilizumab in these patients.

Nevertheless, 41.2% (7/17) patients still showed elevated serum level of IL-6 after application of tocilizumab. Concurrently, their clinical manifestations showed no significant improvement. This may be the result of a number of factors. First, serum level of IL-6 is affected by CAR-T cell expansion. Pt 4 and Pt 11 applied tocilizumab at day 6、9、11 and day 6、8, respectively, while they had persistent expansion of CAR-T cell until day 15. Secondly, the acute graft-versus-host disease after infusion of donor-derived CAR-T cell also has some effects. Pt 5 developed acute intestinal GVHD and her symptoms resolved after treatment with ruxolitinib. Subsequently her serum level of IL-6 also exhibited a reduction. Finally, infection is also a very important influencing factor. Pt 3、6、9、10 showed different sites of infection, such as pneumonia, cholecystitis, etc. After adjustment for antibiotics and supportive care, Pt 、9、10 were stable after infection control, while Pt 6 died of respiratory failure due to severe lung infection.

Overall, the majority of 7 patients had a transient and mild increase in serum levels of IL-6. No patients developed ICANS due to elevated serum IL-6 levels. And it is not clear how tocilizumab affects the levels of other cytokines.

Although tocilizumab is effective in relieving symptoms of severe CRS, the effect of tocilizumab on CAR-T cell expansion and efficacy is controversial. In our study, 94.12%(n=16)of the toci group and 85.71%(n=24)of the non toci group obtained MRD-CR. There was no statistically significant difference in the CR rate between toci and non-toci groups (p=0.616). In addition, there was no statistical difference in PFS (p=0.350) and OS (p=0.273) between the two groups ( Figure 4 ). Meanwhile, tocilizumab didn’t affect the expansion of CAR-T cells ( Supplementary Figure S1 ).

Moreover, tocilizumab does not increase the incidence of side effects. There were 52.9%(n=9)of patients in toci group and 35.7%(n=10)of patients in non-toci group developed serious infections. The differences were not statistically significant (p=0.353) ( Figure 2C ). In terms of hematological toxicity, we also compared the dosage of granulocyte colony stimulating factor (G-CSF) used between two groups to further verify the safety of tocilizumab, but the results still showed no statistically significant difference (p=0.227) ( Figure 2D ). In the Supplementary Materials , we also demonstrated that tocilizumab has no significant effect on liver and kidney function ( Supplementary Figure S2 ).