Consecutive patients treated with autologous CART19 cells in a phase I/II clinical trial (NCT02537977) at the Tongji Hospital of Tongji University between January 1, 2017 and December 31, 2019 who fulfilled the following criteria were included: patients ware seronegative for HBsAg but seropositive for HBcAb at baseline; patients had undetectable serum HBV DNA (<100 IU/ml) and normal liver enzyme at baseline; patients had not received prophylactic antiviral therapy; patients had been followed-up for more than 1 month after CART19 cell infusion.

The CART19 cells in this study were manufactured according to the Good Manufacturing Practice in the Stem Cell Translational Research Center, Tongji Hospital of Tongji University. The structure of the CAR contains a murine anti-CD19 single-chain variable fragment, a 4-1BB costimulatory domain and a CD3ζ T-cell activation domain. Eligible patients for this therapy had relapsed or were refractory to their previous treatments, including autologous or allogenic hematopoietic stem cell transplantation (HSCT). They were diagnosed according to the World Health Organization classification for tumors of the hematopoietic and lymphoid tissues. Expression of CD19 on malignant B cells was confirmed by flow cytometry or immunohistochemistry. An Eastern Cooperative Oncology Group (ECOG) Performance Score of ≤2, normal organ function, measurable disease, and a life expectancy of 12 weeks or more were necessary for eligibility, whereas patients with uncontrollable infection, active graft-versus-host disease, or clinically evident neurological lesions were excluded. All patients except one older than 75 years underwent lymphodepleting chemotherapy with FC regimen (25 mg/m² fludarabine and 300 mg/m² cyclophosphamide daily for 3 days) on days -5, -4 and -3, followed by intravenous infusion of CART19 cells. Dosing of CAR-T cells was 1–3 × 10⁶ CAR-positive T cells per kilogram of body weight. Study protocols were approved by the Ethics Committee of Tongji Hospital of Tongji University and conducted in accordance with the principles of the Declaration of Helsinki. All enrolled patients provided written informed consent for the treatment and follow-up.

HBV serologic test including HBsAg, HBsAb, HBeAg, HBeAb and HBcAb, HBV DNA levels and liver function (ALT, AST, and TB levels) were screened at the time of evaluation for CAR-T cell therapy, and routinely performed at 1, 2, 3, 6 months after CART19 infusion, and every 6 months thereafter until additional therapy for disease progression or HSCT or death. Serum HBV DNA was measured by real-time viral polymerase chain reaction (PCR) in our center using an ABI 7300 real-time thermo-cycler (ABI 7300; Applied Biosystems, Foster City, CA, USA) with a lower limit of 100 IU/ml. HBV reactivation was defined as elevation of HBV DNA ≥100 IU/ml for two consecutive measurements. Hepatitis flare was defined as serum ALT level >3×upper limit of normal (ULN) or an ALT increase >100 U/L, and severe hepatitis was defined as an ALT increase >10 × ULN or TB >1.5 × ULN.

The baseline characteristics of patients collected included HBsAb, HBV DNA, age, gender, histological subtype and treatment modality (the use of anti-CD20 antibody, HSCT, etcetera). Response of subjects with ALL and non-Hodgkin lymphoma (NHL) after CAR-T cell therapy was evaluated using the National Comprehensive Cancer Network (NCCN) guidelines, version 1.2016. Progression-free survival (PFS) and overall survival (OS) were estimated as the time from first infusion to first relapse or death, respectively. The probabilities of OS and PFS were estimated by means of the Kaplan–Meier method and compared using the log-rank test.

Cytokine release syndrome (CRS) and neurologic toxicity (NT) were evaluated in accordance with criteria from the American Society for Transplantation and Cellular Therapy consensus (12). CRS and NT were considered to be severe if it was of grade 3 or higher. Other adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE V.5.0). Statistical analysis was performed using Stata version 15 SE. P value <0.05 was considered to be statistically significant. The cut-off date for data collection was May 31, 2020.

A total of 30 consecutive patients with resolved HBV infection receiving CART19 cell therapy were included in this study. Baseline characteristics of this cohort and outcomes of CART19 cell therapy are described in Table 1 and Supplementary Table 1 . The median age was 59 years (range: 14–81). 17 (57%) patients were male. There were nine (30%) patients with acute lymphoblastic leukemia (ALL), 13 (43%) with diffuse large B cell lymphoma (DLBCL), five (17%) with mantle cell lymphoma (MCL), and three (10%) with other lymphoma subtypes. Among them, nine (30%) patients were seronegative for HBsAb. No patients had any other liver disease at baseline. During their prior therapy, all lymphoma patients received rituximab and four patients underwent HSCT.

Previously, our group had reported the use of CART19 cells in patients with r/r B-NHL demonstrating activity (13, 14). In this population with HBV resolved infection, comparable response and survival rates were achieved ( Figure 1 ). The overall response rate was 80%, with 70% of patients achieving complete remission (CR) (nine of nine with ALL, and 12 of 21 with NHL) and 10% (three of 21 with NHL) achieving partial remission. Among the 21 patients with NHL, the median OS was not reached and the median PFS was 8.5 months (95% confidence interval (CI), 3.8–13.2) with a median follow-up of 19.5 months (range: 3.5–38); the estimated probabilities of OS and PFS at 12-months was 59.7% (95% CI, 33.1–78.6) and 38.2% (95% CI, 16.5–59.8), respectively. Among the nine patients with ALL, the median OS was not reached and the median PFS was 12 months (95% CI, 1–26.6) with a median follow-up of 23.5 months (range: 7–40.5); the estimated probabilities of OS and PFS at 12-months was 63.5% (95% CI, 23.6–86.6) and 41.7% (95% CI, 10.9–70.8), respectively. There were three (10%) ALL patients who experienced severe CRS and no patients developed severe NT. Eight (27%) patients were treated with tocilizumab and 6 (20%) patients with corticosteroids for CRS or NT.

In this study cohort, two patients developed HBV reactivation 2 months and 14 months after CAR-T cell infusion, respectively, the latter of whom developed severe hepatitis. Neither of the two patients had received HBV vaccination and a history of active HBV infection. Both of them had antiviral prophylaxis during prior rituximab-containing chemotherapy. Details of the two patients with HBV reactivation are described in Table 2 and Figure 2 . With a median follow-up of 12 months (range: 2–37), the rate of HBV reactivation in this cohort was 6.7% (95% CI, 0.8–22.1). Specifically, none of 21 patients (0.0%) who were HBsAb positive experienced HBV reactivation versus two of nine patients (22.2%) who were HBsAb negative (p = 0.03). Although use of tocilizumab or corticosteroids has been associated with increased risk of HBV reactivation (15, 16), none of the patients who received these agents had HBV reactivation.

The first patient was a 50-year-old female who was diagnosed with DLBCL 3 years before enrollment and relapsed after two lines of therapy and auto-HSCT. At baseline, the HBV serology showed HBcAb-positive and HBsAb-negative. She achieved CR by 3 months, which lasted 9 months after CAR-T cell infusion. She did not experience CRS or NT. Quantitative PCR showed that anti-CD19 CAR transgene was detected in peripheral blood mononuclear cells from 4 days to 12 weeks after CAR-T cell infusion. Blood CD19+ B cells became undetectable after lymphodepleting chemotherapy and remained absent until 6 months after CAR-T cell infusion. The levels of serum immunoglobulins (Ig’s), including IgG, IgA, and IgM were below normal levels at baseline, and decreased even further after CAR-T cell infusion. At the 2-month follow-up evaluation, she was found to have HBV reactivation (HBV DNA 2.18 × 10³ IU/ml) without ALT/TB elevation or HBsAg seroconversion, and was successfully treated with entecavir.

The second patient was a 59-year-old male who was diagnosed with DLBCL 6 years before enrollment and had relapsed after three lines of chemotherapy and radiotherapy. The patient was also HBcAb+/HBsAb− at baseline. Grade 1 CRS occurred after CAR-T cell therapy and did not require interventions with tocilizumab or corticosteroids. He achieved CR by 3 months after CAR-T cell infusion, which is ongoing after 24 months of follow-up. qPCR detected anti-CD19 CAR transgene from 7 days after CAR-T cell infusion, which was sustained in the blood for more than 1 years. Consistently, blood CD19+ B cells were absent from 7 days after CAR-T cell infusion. The levels of serum IgG, IgA, and IgM, decreased sharply by 41, 46 and 48%, respectively, during the first 3 months after CAR-T cell infusions, and have stayed at these low levels ever since. At 2 months after the patient finished the 12-month follow-up, that is, 14 months after CAR-T cell infusion, he was admitted to hospital with fatigue, vomiting and abdominal distension. He was found to have developed HBV reactivation (HBV DNA 8.53 × 10⁵ IU/ml with HBsAg seroconversion and severe hepatitis with a significant elevation of serum ALT level of 592.1 U/L (ULN 40 U/L) and TB level of 35.4 umol/L (ULN 21 umol/L). He was treated with entecavir which resulted in a gradual reduction of HBV DNA and ALT/TB. At the last evaluation for the 24-month follow-up, HBV DNA level was 1.11 × 10³ IU/ml, and ALT/TB levels were normal. He remains on entecavir afterwards.