We performed a single-arm, open-label, multicenter, phase I study enrolling patients from four clinical centers. Eligible participants had to be at least 18 years old and had B cell lymphoma in the refractory/relapsed status, which was defined as not reaching complete remission after four cycles of chemotherapy, including rituximab at the time of initial treatment, or two cycles of salvage treatment or having disease progression or relapse after complete remission with immunochemotherapy or hematopoietic stem cell transplantation (HSCT). Patients confirmed to be CD19 positive through immunohistochemistry or flow cytometry were included. Patients with both invasive and indolent B cell lymphoma were included in this trial, including but not limited to DLBCL, follicular lymphoma (FL), primary mediastinal large B cell lymphoma (PMBCL), and double- or triple-hit lymphoma (DHL/THL) with MYC, BCL2, and/or BCL6 rearrangements. Patients who had formerly received any gene therapy or cell therapy, those who had uncontrolled acute life-threatening infections, or those exposed to graft-versus-host disease (GVHD) requiring immunosuppressive agents were excluded. Those who used systemic steroids and immunosuppressants within 2 weeks prior to treatment were excluded.

The clinical trial followed the guidelines of the Declaration of Helsinki and was approved by the institutional review board of Zhujiang Hospital of Southern Medical University. The trial has been registered in the Chinese Clinical Trial Registry (number, ChiCTR-OOC-16007779). Written informed consent was obtained before screening.

The individualized CAR-T cells were produced as previously described (18). Briefly, peripheral blood mononuclear cells of eligible participants were obtained through apheresis and transported to Geno-Immune Medical Institute, Shenzhen, China, where the CAR-T product was manufactured. CD3-positive T cells were selected and activated using CD3/CD28 magnetic beads. Then, the T cells were lentivirally transduced with a fourth-generation anti-CD19 CAR containing various costimulatory signaling domains we synthesized a codonoptimized CD19 CAR (19z) based on anti-CD19 scFv of the mouse hybridoma FMC63 (19), and constructed a fourth generation 19z CAR, which contained a chimeric intracellular signaling element incorporating CD28 trans-membrane and cytoplasmic domain, the cytoplasmic domains of CD27 (273z, amino acid 213–260), and the CD3ζ chain intracellular domain with an inducible caspase 9 gene as previously described (20, 21). The detail of the full sequence was covered in the patent (WO2019161796 A1). After 5–7 days of expansion, 4SCAR19 T cells were harvested and transported back to the medical institution for reinfusion.

Participants received a 3-day conditioning regimen (fludarabine, 25 mg/m² for 3 days; cyclophosphamide 900 mg/m² for one day) to clear circulating endogenous lymphocytes and immunosuppressive cells. Some patients with a high tumor burden also underwent bridging chemotherapy based on previous chemotherapy regimens to decrease the probability and severity of CRS. Subjects were infused with 4SCAR19 T cells on day 0, and their clinical manifestations and serum marker levels were closely monitored. The structure of the 4SCAR19 construct and its in vivo activity are shown in Figure 1 . Participants were continuously monitored clinical responses in a preset timeline. The CAR copy number in the blood was determined by quantitative real-time polymerase chain reaction (qPCR; based on both SYBR and TaqMan probe methods) and the cytokine analysis based on cytokine bead array (CBA) as previously described (21).

Efficacy was evaluated according to the standards recommended by the International Working Group (IWG) in 1999, and the first evaluation was performed 30 days after CAR-T cell infusion. The primary endpoint was the overall response rate (ORR), defined as the proportion of patients who achieved remission. Secondary end points included duration of response (DoR), event-free survival (EFS), overall survival (OS), and incidence and severity of adverse events. Safety was evaluated according to the CTCAE V4.03 standard, CRS was graded according to a grading system known as the Lee criteria (NCI), and CAR-T-cell-related encephalopathy syndrome (CRES) was scored according to the MDACC CRES grading system (22, 23).

The 95% confidence interval (CI) of the response rate was calculated using the Clopper-Pearson method. The overall response rate of subgroups was compared by chi-square test and Fisher’s exact probability method. The differences between groups were compared by one-way ANOVA. Univariate analysis was performed by the log-rank test. Survival rates were estimated with the Kaplan–Meier method and compared by applying the log-rank test. A two-sided P value<0.05 was considered statistically significant. Analyses were performed with SPSS, version 20.

Between May 2016 and the data cutoff date, September 17, 2019, a total of 21 patients aged 31 to 77 years old with R/R NHL were enrolled and received an infusion with CD19 CAR-T cells. Eighteen (86%) patients had aggressive B cell lymphoma, and the other three patients had indolent lymphoma. Of the 12 patients with DLBCL, 1 patient had central nervous system DLBCL. Most of the patients had been heavily pretreated, with five (24%) undergoing more than five lines of antitumor chemotherapy and five patients undergoing autologous HSCT. However, the patients still had a heavy tumor burden; the longest diameter of the affected lymph nodes of 5 patients was ≥ 5 cm, and 13 patients had more than 5 involved lymph nodes. Seventeen out of 21 patients (81%) had extranodal lesions, and the most common sites for extranodal lesions were spleen (17%), gastrointestinal tract (14%), bone (14%), pleura (14%), and lungs (11%). Fourteen patients had refractory B-NHL (patients who failed to respond to the last therapy), and seven patients had relapsed B-NHL (patients who had a partial or complete response after the last line of therapy but relapsed before enrollment in this trial). The baseline characteristics of the enrolled patients are summarized in Table 1 .

In the previous study of our center, it was found that compared with patients with low tumor burden, leukemia patients with higher tumor burden are less likely to achieve remission after 4SCAR19 infusion, and are accompanied by a higher probability of relapse/progression (18). Therefore, take the prognosis of patients into consideration, we administered bridging chemotherapy to seven patients with high tumor burden in this trial. Of all enrolled patients, 86% (18/21) received a conditioning regimen of combination fludarabine-cyclophosphamide, one patient received rituximab and fludarabine-cyclophosphamide, and the other two patients received fludarabine or cyclophosphamide alone. All 21 patients received a median dose of 8.9×10⁵/kg 4SCAR19-positive viable T cells (range, 0.3×10⁵/kg to 48.0×10⁵/kg) ( Supplemental Table S1 ). For all patients, the median time from leukapheresis to CAR-T cell infusion was 16 days.

All patients who received 4SCAR19 T cell infusion were included in the efficacy analysis, and the median follow-up was 13.7 months (range, 0.7 to 23.8) in the cohort. The best ORR was 67% (95% CI, 43 to 85), with 43% of patients achieving a complete response and 24% having a partial response. The rates of overall and complete response at month 3 were 48 and 33%, respectively, and 43 and 33% at month 6. The rates of overall and complete response were 58 and 33%, respectively, in the DLBCL group and 78 and 56% in the non-DLBCL group. Individual response is shown in the waterfall plot ( Figure 2 ). Among seven patients who received debulking therapy before conditioning, two of them achieved partial response and five of them achieved no response to debulking. One of seven patients achieved complete response, three patients achieved partial response, one patient had stable disease, and the other two patients faced progression after CAR-T cell infusion. In the subgroup analyses, there was no notable difference in the overall response rate in the demographic and prognostic subgroups ( Figure 3 ). Besides, T cell phenotypes were assessed with the CD4+ and CD8+ T cells re-infused at a ratio approximately at 1:1, and no correlation was found between T cell phenotypes and clinical responses.

The median time to first documented remission post infusion in all patients who achieved remission was 58 days (range, 29 to 63). There was no significant difference in time to remission after infusion in patients with complete remission or partial remission (P=0.509, Supplemental Table S2 ). The median DoR of patients with CR or all patients who achieved a response was 12.3 and 6.8 months, respectively, while it was 3.7 months in patients with PR. It is expected that 63% (95% CI, 23 to 86) of patients with CR and 45% (95% CI, 18 to 69) of all patients will remain relapse-free or progression-free at 12 months after remission ( Figure 4A ). Until the data cutoff date, a sustained remission of up to 20.2 months was observed after infusion, and five patients remained in remission for 5 to 20 months. After 0.4–6.8 months of remission, seven patients developed relapse or progression, but no CD19-negative relapse was found. The B cell count in these patients was close to zero at infusion time, and normal B cells did not rebound when relapsed. One patient underwent HSCT 23 days after confirmation of CR by positron emission tomography/computed tomography (PET-CT) after infusion and maintained sustained remission. Six patients who did not respond received chemotherapies and/or radiotherapy, and none of them proceeded to HSCT.

The median EFS was 6.8 months in all patients ( Figure 4B ). In addition, we found that there was a significant difference in EFS between patients with and without a response (P=0.0014, Figure 4B ), and the median EFS in patients with response was longer than that in those with no response (8.8 vs. 2.3 months). The median OS was 23.8 months (95% CI, not reached) among all 21 patients ( Figure 4C ). However, the median OS among patients who had a complete response has not been reached. The estimated rate of OS at 12 months after 4SCAR19 T cell infusion was 77% (95% CI, 50 to 91) and 86% (95% CI, 33 to 98) in all patients and patients with complete response, respectively. It is estimated that 57% (95% CI, 30 to 77) of all patients who received an infusion will survive at 22 months. As of the data cutoff date, 7 of 21 patients died of relapse or disease progression, and 1 patient died of a secondary tumor.

In the univariate analysis of OS, we found that International Prognostic Index (IPI) score, disease subtype, and response status after CAR-T cell infusion were related to the survival time of patients ( Table 2 ). The IPI score was proposed by Shi PP et al. in 1993 to assess the prognosis of NHL, and in the IPI, a higher score indicates a worse prognosis. In this trial, patients with low risk and low-medium risk survived longer than patients with intermediate risk and high risk (p=0.031, HR=7.742), suggesting that the IPI model is suitable for assessing the prognosis of patients who received CAR-T cell therapy. This analysis also showed that patients who achieved response after CAR-T cell treatment had a lower risk of death than those without response (P=0.025, HR=4.855, Figure 4C ).

No significant differences were found between patients with DLBCL and patients with non-DLBCL regarding DoR (P=0.745, Figure 5A ) or EFS (P=0.532, Figure 5B ), but the non-DLBCL group outperformed the DLBCL group in OS (P=0.021, HR=0.116, Figure 5C ), which was mentioned in the section about the univariate analysis. The median OS of the non-DLBCL group has not been reach, while it was 12.4 months (95% CI, 8.9 to 15.9 months) in the DLBCL group. Eighty-six percent (95% CI, 33 to 98) of patients in the non-DLBCL group are estimated to survive after 22 months, while 35% (95% CI, 9 to 64) of patients in the DLBCL group are estimated to survive after 22 months.

4SCAR19 T cell expansion was monitored after infusion on a regular schedule. Until the data cutoff date, all the expansion data were collected and varied among the patients ( Figure 6A ). CAR-T cells were still detectable in vivo 160 days after infusion in a patient with partial response. The median survival time of CAR-T cells in patients with response was longer than that in patients without response (51 versus 28 days); however, there was no significant difference between the two groups (P=0.06, Figure 6B ), which may be related to the small sample size. Similar results were observed in the peak of the ratio of CAR-T cells to mononuclear cells (0.74 versus 0.9%, P=0.084, Figure 6C ) and the time to maximal CAR level in peripheral blood (14 versus 14 days, P=0.156, Figure 6D ) in patients with a response versus in those without a response, which indicated that CAR-T cell expansion in vivo had little effect on clinical outcome. There was no significant difference in CAR-T cell dose and clinical response (P=0.332), and no correlation between CAR-T cell dose and amplification peak was observed (P=0.339, data not shown). Seventy-six percent (16/21) of patients maintained a complete response or partial response after CAR-T cells became undetectable.

In our study, diverse adverse events were observed in all patients receiving 4SCAR19 T cell infusion. The most common adverse events of any grade were reduced white blood cell count (in 100% of patients), decreased neutrophil count (100%), anemia (90%), decreased platelet count (62%), and infection (33%) ( Table 3 ). The most common adverse events of grade 3 or 4 included decreased neutrophil count (in 76% of patients), decreased white blood cell count (71%), decreased platelet count (29%), anemia (24%), febrile neutropenia (19%), and pulmonary infection (10%). After symptomatic treatment, the patient’s symptoms were alleviated. There were no deaths due to infusion of CAR-T cells.

Cytokine release syndrome (CRS) was observed in 3 (14%) patients on days 2, 6, and 7, respectively, accompanied by increased serum levels of C-reactive protein (CRP) and interleukin-6 (IL-6), and lasted for 2 to 10 days. All cases were grade 1, with symptoms of pyrexia, fatigue, diarrhea, etc. Patient symptoms were ameliorated after antipyretic therapy, empirical antibiotic treatment, and supporting therapy, and serum levels of CRP and IL-6 also gradually decreased. None of the patients received tocilizumab or glucocorticoids. Unfortunately, none of these three patients achieved remission (two had stable disease, and one had disease progression).

One patient developed grade 3 CAR-T cell-related encephalopathy syndrome (CRES) on day 33 after infusion, with clinical manifestations of cognitive impairment, aphonia, unresponsiveness, mild irritability and headache, febrile neutropenia, hypogammaglobulinemia (decreased by 0.71-fold), and upper respiratory tract infection, but not accompanied by abnormal elevations of CRP or IL-6. The patient did not have central nervous system diseases before CAR-T cell infusion. After dehydration and intravenous dexamethasone treatment, the CRES was resolved. The patient had a poor prognosis, with disease progression occurring 29 days after the partial response.

In this study, both CRS and CRES occurred during the proliferation phase of CAR-T cells, suggesting that their occurrence may be related to the activation and proliferation of CAR-T cells in vivo ( Supplemental Figure S1 ). There was a significant difference in the peak serum concentration of CRP (P=0.032), pretreatment LDH (P=0.02), and CAR-T cell infusion dose (P=0.039) between patients with and without CRS/CRES ( Supplemental Figure S2 ). However, no significant difference was found in the peak serum concentration of IL-6 (P=0.107) or the peak of CAR-T cell expansion in vivo (P=0.654) between the two groups.

Cytopenia was the most common adverse event in this study, and all patients experienced a reduction in at least one line of cellular elements in the circulating blood. Sixty-two percent of patients received recombinant human granulocyte colony stimulating factor (rhG-CSF), and three patients were given platelet and/or erythrocyte transfusion as supplementation. Some patients experienced prolonged cytopenia for more than 28 days or 3 months ( Supplemental Table S3 ), and one patient had pancytopenia lasting longer than 28 days.

According to the detection of lymphocyte subsets after CAR-T cell infusion, all 19 patients tested experienced B cell dysplasia, which lasted for a maximum of 251 days. Nine of these patients (47%) developed hypogammaglobulinemia from 2 to 35 days after CAR-T cell infusion, with a minimum reduction of 0.57 times (relative to the lower limit of normal), and the hypogammaglobulinemia could be cured after intravenous injection of human immunoglobulin. Upper respiratory tract infection occurred in three of these nine patients. These results show that B cell dysplasia can be controlled clinically, and patients can well tolerate the long-term loss of B cells.